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Molecular Determinants of the Regioselectivity of Toluene/ o-Xylene Monooxygenase from Pseudomonas sp. Strain OX1

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Eugenio Notomista, Valeria Cafaro, Giuseppe Bozza and Alberto Di Donato Appl. Environ. Microbiol. 2009, 75(3):823. DOI: 10.1128/AEM.01951-08. Published Ahead of Print 12 December 2008.

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2009, p. 823–836 0099-2240/09/$08.00⫹0 doi:10.1128/AEM.01951-08 Copyright © 2009, American Society for Microbiology. All Rights Reserved.

Vol. 75, No. 3

Molecular Determinants of the Regioselectivity of Toluene/o-Xylene Monooxygenase from Pseudomonas sp. Strain OX1䌤† Eugenio Notomista,1,2‡* Valeria Cafaro,1‡ Giuseppe Bozza,1 and Alberto Di Donato1,3 Dipartimento di Biologia Strutturale e Funzionale, Universita ` di Napoli Federico II, Complesso Universitario di Monte S. Angelo, Via Cinthia 4, 80126 Naples, Italy,1 and Facolta ` di Scienze Biotecnologiche, Universita ` di Napoli Federico II,2 and 3 CEINGE-Biotecnologie Avanzate S.c.ar.l., Naples, Italy

Bacterial multicomponent monooxygenases (BMMs) are a heterogeneous family of di-iron monooxygenases which share the very interesting ability to hydroxylate aliphatic and/or aromatic hydrocarbons. Each BMM possesses defined substrate specificity and regioselectivity which match the metabolic requirements of the strain from which it has been isolated. Pseudomonas sp. strain OX1, a strain able to metabolize o-, m-, and p-cresols, produces the BMM toluene/o-xylene monooxygenase (ToMO), which converts toluene to a mixture of o-, m-, and p-cresol isomers. In order to investigate the molecular determinants of ToMO regioselectivity, we prepared and characterized 15 single-mutant and 3 double-mutant forms of the ToMO active site pocket. Using the Monte Carlo approach, we prepared models of ToMO-substrate and ToMO-reaction intermediate complexes which allowed us to provide a molecular explanation for the regioselectivities of wild-type and mutant ToMO enzymes. Furthermore, using binding energy values calculated by energy analyses of the complexes and a simple mathematical model of the hydroxylation reaction, we were able to predict quantitatively the regioselectivities of the majority of the variant proteins with good accuracy. The results show not only that the fine-tuning of ToMO regioselectivity can be achieved through a careful alteration of the shape of the active site but also that the effects of the mutations on regioselectivity can be quantitatively predicted a priori. in the second (3, 5). Moreover, each TMO and PH shows its own characteristic regioselectivity. T4MO produces more than 96% p-cresol from toluene (25), whereas ToMO produces a mixture of the three isomers of cresol (5). PHs usually produce a large excess of o-cresol—70 and 90% in the cases of PH from Pseudomonas sp. strain OX1 (5) and T2MO from Burkholderia cepacia G4 (22), respectively. Thus, it appears that the BMM family constitutes an archive of powerful catalysts that could be used to construct new catalysts for the bioremediation of environmentally harmful substances and for industrial biosyntheses. Certainly, an understanding of the molecular determinants of BMM substrate specificity, regioselectivity and enantioselectivity properties is preliminary to the rational design of new, improved catalysts, as proved by the large number of studies on BMM catalytic mechanisms and on synthetic analogues capable of catalyzing reactions similar to those catalyzed by BMMs (14, 28, 39). The results of several structural and functional studies suggest that the catalytic mechanisms of BMMs are very similar (13, 19). The major subunit (A in TMOs and ␣ in MMOs) of the hydroxylase component (the H complex) contains a di-iron cluster bound to four glutamate and two histidine residues. These residues, and several other conserved hydrophilic residues, form an H bond network on one side of the iron ions (13, 19). On the other side, nonconserved hydrophobic residues form the substrate binding pocket (13, 19). The catalytically active diferrous form, interacting with dioxygen, produces a di-iron(III) intermediate (the peroxo intermediate) which, at least in the case of MMOs, turns into a di-iron(IV) form known as diamond core (19). The peroxo and diamond core intermediates each transfer one oxygen atom to the substrate (19). The possible intermediates involved in the transfer of

Bacterial multicomponent monooxygenases (BMMs) are a large and heterogeneous family of nonheme di-iron enzymes which share the very interesting ability to activate dioxygen and transfer a single oxygen atom to a wide variety of substrates (13, 23). Aliphatic and aromatic hydrocarbons are converted, respectively, into alcohols and phenols (3, 6, 15, 21, 22, 38), alkenes are converted into epoxides (8), and sulfur-containing compounds are oxidized into sulfoxides and sulfones (10). As BMMs allow bacteria to grow on hydrocarbons or xenobiotics as the sole source of carbon and energy, several members of this protein family, including the soluble methane monooxygenases (MMOs) (15, 21), alkene monooxygenases (8), phenol hydroxylases (PHs)/toluene 2-monooxygenases (T2MOs) (3, 22), and toluene monooxygenases (TMOs) such as toluene 4-monooxygenase (T4MO) from Pseudomonas mendocina KR1 (38) and toluene/o-xylene monooxygenase (ToMO) from Pseudomonas sp. strain OX1 (6), have been characterized thoroughly. All these enzymes possess defined substrate specificity, regioselectivity, and enantioselectivity properties. For example, TMOs and PHs perform two consecutive hydroxylation reactions with aromatic rings, but usually TMOs are more efficient in the first hydroxylation step, whereas PHs are more efficient

* Corresponding author: Dipartimento di Biologia Strutturale e Funzionale, Universita` di Napoli Federico II, Complesso Universitario di Monte S. Angelo, Via Cinthia 4, 80126 Naples, Italy. Phone: 39081-679208. Fax: 39-081-679313. E-mail: [email protected]. ‡ Valeria Cafaro and Eugenio Notomista contributed equally to the paper. † Supplemental material for this article may be found at http://aem .asm.org/. 䌤 Published ahead of print on 12 December 2008. 823

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Received 21 August 2008/Accepted 28 November 2008

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oxygen to the aromatic ring (16, 19, 20) are shown in Fig. 1. The reactive species, likely a di-iron(III) intermediate, attacks the pi-electron system of the aromatic ring, forming epoxide 1 or delocalized carbocation 2. The opening of the epoxide ring eventually provides the delocalized carbocation. The migration of a hydride from the sp3-hybridized carbon to the adjacent atom then converts the carbocation to the more stable ketone 3. Finally, the dissociation of the ketone and its tautomerization yield phenolic product 4. The different regioselectivities shown by BMMs have been attributed previously to differences in the shape of the active site pocket, an idea supported by the fact that several point mutations in the active sites of T4MO, toluene p-monooxygenase, ToMO, MMO, and B. cepacia G4 T2MO cause large variations in the regioselectivities of these BMMs (2, 7, 26, 30, 34, 35). In a previous paper (4), we have reported that mutations at position 103 of the A subunit change the regioselectivity of ToMO from Pseudomonas sp. strain OX1. However, a complete study of the active site pocket of ToMO A is still lacking. In this paper, we report the effects of the substitution of six residues in the ToMO A active site on substrate specificity and regioselectivity. Furthermore, we present a detailed analysis of the molecular determinants of regioselectivity based on the docking of substrates and hypothetical intermediates of the aromatic hydroxylation reaction into the active site of the crystallographic structure of ToMO (Protein Data Bank [PDB] code 1T0Q [32]), followed by a Monte Carlo optimization. The results show that (i) the fine-tuning of TMO regioselectivity can be achieved through a careful alteration of the shape of the active site pocket and that (ii) the effects of mutations on regioselectivity can be quantitatively predicted using the procedure described herein. MATERIALS AND METHODS Materials. Bacterial cultures, plasmid purifications, and transformations were performed according to the procedures of Sambrook et al. (31). Escherichia coli strains JM109 and CJ236 and vector pET22b(⫹) were from Novagen. Plasmid pBZ1260 (1) used for the expression of the ToMO cluster was kindly supplied by P. Barbieri (Dipartimento di Biologia Strutturale e Funzionale, Universita` dell’Insubria, Varese, Italy). E. coli strain JM101 was purchased from Boehringer. The pGEM-3Z expression vector, Wizard SV gel, and the PCR clean-up system for the elution of DNA fragments from agarose gel were obtained from Promega. Enzymes and other reagents for DNA manipulation were from New England Biolabs. The oligonucleotides were synthesized at MWG-Biotech

(Ebersberg, Germany). All other chemicals were from Sigma. The expression and purification of recombinant catechol 2,3-dioxygenase from Pseudomonas sp. strain OX1 are described elsewhere (36). ToMO A mutagenesis. Plasmids for the expression of ToMO complexes with mutated ToMO A subunits were prepared by site-directed mutagenesis of plasmid pTOU as described previously (4). Sequences of the mutagenic oligonucleotides are reported in Table S1 in the supplemental material. Determination of apparent kinetic parameters and identification of products. Assays were performed as described previously (4, 5) using E. coli JM109 cells transformed with plasmid pBZ1260 or plasmid pTOU, which expresses wild-type ToMO or ToMO mutant enzymes, respectively. All kinetic parameters were determined using whole cells (4, 5). Enzymatic activity on phenol was measured by monitoring the production of catechol in continuous coupled assays with recombinant catechol 2,3-dioxygenase from Pseudomonas sp. strain OX1 (5). The determination of apparent kinetic parameters for benzene, toluene, o-xylene, and naphthalene was carried out by a discontinuous assay (5). For the calculation of the kcat values, amounts of proteins were calculated as described previously (5). All the ToMO mutant enzymes showed expression levels similar to that of the wild-type enzyme. Reaction products were identified as described previously (5). All the regiospecificity studies were performed using substrate concentrations higher than the Km values. Under these conditions, absolute yields of products were proportional to kcat values. Modeling of substrates and intermediates into the active site of ToMO A. Substrates and reaction intermediates were docked into the active site of ToMO A by using the Monte Carlo energy minimization strategy. The ZMM-MVM molecular modeling package (ZMM Software Inc. [http://www.zmmsoft.com]) was used for all calculations. This software allows conformational searches using generalized coordinates such as torsion and bond angles instead of conventional Cartesian coordinates (40). Atom-atom interactions were evaluated using assisted model building with energy refinement force fields (37) with a cutoff distance of 8 Å. Conformational energy calculations included van der Waals, electrostatic, H bond, and torsion components. A hydration component was not included. Electrostatic interactions were assessed with a relative dielectric constant of 4. Substrate and intermediate structures were prepared using the PyMOL software (DeLano Scientific LLC). Geometry was optimized using the Zl module of ZMM. Partial charges were attributed using the complete neglect of differential overlap method in the HyperChem software (HyperCube Inc. [http://www.hyper .com]). The X-ray structure of the ToMO A-thioglycolate complex (PDB code 1T0Q) was used to build the models of the ToMO A-substrate and ToMO A-intermediate complexes. To reduce computational time, a double-shell model of the enzyme was built. The inner shell included 28 ToMO A residues surrounding the active site cavity. During energy calculation procedures, the side chain torsion angles—but not the backbone torsion angles—of these residues were allowed to vary. Due to the asymmetric shape of the cavity, which is flat with the di-iron cluster on one end and evolutionarily nonconserved hydrophobic residues on the opposite side, the residues of the inner shell were selected manually. They included the ligands of the iron ions, all residues with at least one side chain atom contributing to the hydrophobic part of the active site cavity, and all residues with at least one side chain atom less than 5 Å from the previous residues. The outer shell included 139 residues, which did not belong to the inner shell

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FIG. 1. Possible intermediates in the aromatic hydroxylation reaction catalyzed by ToMO. Intermediates 1, 2, and 3 are an epoxide, a carbocation, and a ketone, respectively. R1 and R2 are hydrogen atoms or methyl groups. The geometrical features of the di-iron(III)-(hydro)peroxide intermediate and the details of the O-O bond cleavage reaction are not known.

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and were located less than about 16 Å from the active site pocket. During energy calculation procedures, both the backbone and the side chain torsion angles of the outer shell residues were not allowed to vary. To further restrict the conformational freedom of the iron cluster and of the protein-ligand complexes, two flat-bottom parabolic penalty functions—the socalled constraints—available in ZMM were used. These functions increase the conformational energy of the system if it deviates from specified parameters. The atom-atom distance constraint applies a force to the system when the distance between two specified atoms deviates from a specified value or interval. This type of constraint was used to fix the distances between the two iron ions and between each iron ion and the surrounding atoms, including the bridging water molecule and the terminal water molecule. Atom-atom distance constraints were also used to fix the conformation of the ligands of the di-iron cluster. A force constant of 1,000 kcal/mol/Å was used. The atom-atomic coordinate constraint applies a force to the system when an atom moves farther than a specified distance from particular Cartesian coordinates. This constrain was used to prevent the movement of the Oε1 atom of the Glu103 side chain more than 0.5 Å from the original position observed in the crystallographic structure. A ⫹2 charge was arbitrarily assigned to each iron atom, both to account for electron density transfer from the ligands to iron ions and to avoid strong electrostatic attractive and repulsive interactions with negative and positive atoms, respectively, of substrates and intermediates. Similarly, both the bridging and terminal solvent molecules observed in the 1T0Q structure were modeled as neutral water molecules rather than OH⫺ ions. van der Waals radii of iron ions and oxygen atoms of water molecules were arbitrarily set to 0.8 and 1.5 Å, respectively, in order to reduce steric hindrance inside the di-iron cluster. Complexes with total energies of up to 8 kcal/mol higher than that of the lowest-energy complex were stored for the analysis of energy contributions. Total energy was partitioned into intrareceptor, intraligand, and receptor-ligand energies and energies of the constraints. Receptor-ligand energy was further parti-

tioned into van der Waals, electrostatic, and H bond components. Moreover, receptor-ligand energy was also partitioned (i) per active site residue in order to evaluate the contribution of each residue to the binding of ligands and (ii) per ligand atom in order to evaluate the contributions of the ring and methyl substituents. Intrareceptor energies gave an estimation of the energy costs for receptor (ToMO A) conformational changes upon ligand binding. The docking procedure is described in detail in the supplemental material. The PDB files for the initial manually generated complexes and the ZMM instruction files containing the lists of mobile residues, constraints, and parameters used during calculations are available upon request.

RESULTS AND DISCUSSION Kinetic model for ToMO regioselectivity. The results of several studies of BMMs suggest that the regioselectivities of these enzymes depend on the shape of the active site cavity, which is believed to influence the orientations of substrates and reaction intermediates during catalysis (4, 7, 9, 19, 26, 34). The ToMO active site pocket is buried deeply inside the ToMO A subunit and is in contact with the surface through a long tunnel whose diameter is large enough to allow the entrance of substrates and the exit of products (32). The active site cavity (Fig. 2A and B) shows a lens-like shape. The distance between two atoms on opposite sides of the edge is about 10.5 to 12.5 Å. Assuming a van der Waals radius of about 2 Å, these distances correspond to an inner diameter of 6.5 to 8.5 Å.

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FIG. 2. Active site pocket of ToMO A. Panels A and B show the active site of the crystal structure of ToMO A (PDB code 1T0Q). Only residues contributing to define the edge of the cavity are shown. In panel B, the grid cuts the cavity in such a way as to provide the largest section. Carbon atoms are shown in red (Glu134 and Glu231), green (Ala107), yellow (Met180), blue (Glu103), magenta (Phe176), cyan (Ile100), and orange (Phe205). (C) Superimposition of the PDB code 1T0Q crystal structure (colored as in panels A and B) onto the structure of the complex ToMO-CCI 2 for the reaction leading to phenol production from benzene (carbon atoms are shown in white). (D) Superimposition of the complex ToMO-CCI 2 (carbon atoms are shown in white) onto the complex ToMO-ketone 3 (carbon atoms are shown in green) for the reaction leading to phenol production from benzene. THG, thioglycolate. CCI indicates CCI 2 and KTI indicates ketone 3.

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K‡o-m ⫽ 关ET‡m兴/关ET‡o兴 ⫽ Ko-mK‡m/K‡o ⫽ exp共 ⫺ ⌬G‡o-m/RT兲 (1) FIG. 3. Kinetic models for the hydroxylation reaction of toluene and o-xylene. CCIs 5 to 12 are the CCIs deriving from toluene and o-xylene shown in Fig. 4. oC, mC, and pC, o-, m-, and p-cresols.

K‡m-p ⫽ 关ET‡p兴/关ET‡m兴 ⫽ Km-pK‡p/K‡m ⫽ exp共 ⫺ ⌬G‡m-p/RT兲 (2) K‡o-p ⫽ 关ET‡p兴/关ET‡o兴 ⫽ Ko-mKm-pK‡p/K‡o ⫽ exp共 ⫺ ⌬G‡o-p/RT兲

The distance between two atoms placed above and below the plane of the cavity is about 8.5 Å; hence, the thickness of the cavity is about 4.5 Å. Therefore, the cavity is slightly larger than a benzene molecule, which has a thickness of about 4 Å and a diameter of about 6.4 Å. The grid in Fig. 2B cuts the cavity into two halves in such a way as to highlight the section with the larger surface. The edge of the pocket (Fig. 2A and B) is formed by residues Ala107, Met180, Glu103, Phe176, Ile100, and Phe205 and two iron ligands, Glu134, adjacent to Ala107, and Glu231, adjacent to Phe205. It should be noted that in the case of Glu103, only the methylene groups contribute to the pocket (4, 32). Thr201 and Phe196 form one of the faces of the cavity, whereas Glu104 forms the opposite one. In a previous paper (4), we explained the regiospecificity of ToMO A for toluene by hypothesizing that there are at least three different positions in the active site pocket which can accommodate the methyl group of toluene. These three subsites can orient the methyl group of the substrate such that its ortho, meta, or para carbon is presented to the di-iron center. Thus, it is the difference in the affinities of the subsites for the binding of the methyl group which determines the relative abundances of three different enzyme-substrate complexes, which can account for the observed distribution of cresols produced by ToMO. Using a manual docking procedure (4), we mapped an ortho subsite located among Ala107, Met180, and Glu103, a meta subsite among Glu103, Phe176, and Ile100, and a para subsite located between Ile100 and Phe205.

(3) where ⌬G‡o-m, ⌬G‡m-p, and ⌬G‡o-p are the free-energy differences for the three conversions, R is the gas constant, and T is the absolute temperature. This new model also includes an undefined number of unproductive ET (ETu) complexes. It should be noted that the number and the stability of these unproductive complexes do not influence regioselectivity but only the apparent kcat value, as they decrease the relative abundances of the ETo, ETp, and ETm complexes and of the corresponding ET‡ complexes at equilibrium. Each ET‡ provides the corresponding EI, which in turn releases a cresol isomer. Each ET‡-EI transformation should proceed with the same rate, ␯, which is given by the following well-known relation: ␯ ⫽ kT/h, where k and h are the Boltzmann and Planck constants, respectively, and T is the absolute temperature. According to model i (Fig. 3), the relative abundances of o-, m-, and p-cresol isomers produced by the enzyme are determined by the relative abundances of the three transition state ET‡ complexes at equilibrium. Therefore, calculating the energy differences, ⌬G‡o-m, ⌬G‡m-p, and ⌬G‡o-p, should allow the prediction of the percentages of cresols formed. Two components should contribute to these energy differences: (i) the covalent bond energy (bE‡), which includes the energy of the bonds among ligand atoms and between ligand and protein atoms, for example, those between the oxygen atom transferred to the substrate and each iron of the cluster

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Figure 3 shows a new, more complex kinetic model of ToMO regioselectivity. According to model i, toluene would bind to the active site in three different catalytically productive orientations, thus giving rise to three different enzyme-toluene (ET) complexes that lead to the production of o-, m-, and p-cresol isomers (complexes ETo, ETm, and ETp, respectively) through at least one enzyme-intermediate (EI) complex (EIo, EIm, or EIp, corresponding to o-, m-, or p-cresol, respectively). According to this model, the ET-EI conversion is the ratelimiting step. As the interactions between toluene and the active site cavity are limited to van der Waals interactions and as the active site cavity is larger than the toluene molecule, it is likely that the interconversion of the ET complexes is fast with respect to their transformation to cresols. The new model can be described by six equilibrium constants: Ko-m, Ko-p, and Km-p for the conversions ETo ^ ETm, ETo ^ ETp, and ETm ^ ETp, respectively, and K‡o, K‡m, and K‡p for the conversion of each productive ET complex to the corresponding transition state (ET‡) complex. Each ET‡ complex can turn into the other two activated complexes through the ET complexes. Therefore, we can define three equilibrium constants for the conversions ET‡o ^ ET‡m, ET‡o ^ ET‡p, and ET‡m ^ ET‡p, which will be the products of the constants defined above:

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(Fig. 1), and (ii) the ligand-protein noncovalent bond energy (nbE‡). Assuming that bE‡ is scarcely influenced by the position of the substituent, the ⌬G‡ values should depend essentially on the nbE‡ contributions: ⌬G‡o-m ⫽ 共nbE‡m ⫺ nbE‡o兲 ⫹ 共bE‡m ⫺ bE‡o兲 ⫽ 共nbE‡m ⫺ nbE‡o兲 ⫽ ⌬nbE

‡

o-m

(4)

⌬G‡m-p ⫽ 共nbE‡p ⫺ nbE‡m兲 ⫹ 共bE‡p ⫺ bE‡m兲 ⫽ 共nbE‡p ⫺ nbE‡m兲

TABLE 1. Interaction energy values for ToMO A-benzene reaction intermediates Interaction energy (kcal/mol) Ligand

Epoxide Carbocation Ketonea Ketoneb a b

m-p

(5)

van der Waals

Electrostatic

Total

⫺12.27 ⫺15.06 ⫺13.41 ⫺13.35

⫺6.42 ⫺19.44 ⫺9.08 ⫺9.43

⫺18.69 ⫺34.50 ⫺22.49 ⫺22.78

sp3 carbon near Thr201. sp3 carbon near Ala107.

⌬G‡o-p ⫽ 共nbE‡p ⫺ nbE‡o兲 ⫹ 共bE‡p ⫺ bE‡o兲 ⫽ 共nbE‡p ⫺ nbE‡o兲 ⫽ ⌬nbE‡o-p

(6)

The kinetic model ii shown in Fig. 3 illustrates the case of o-xylene. Two productive enzyme-substrate complexes (referred to hereinafter as EX complexes, where X represents the substrate)—EX2,3 and EX3,4—and the corresponding EX‡ complexes yield 2,3-dimethylphenol (2,3-DMP) and 3,4-DMP, respectively. In the following sections, we discuss several pieces of evidence which support these kinetic models. Modeling toluene and o-xylene into the active site of ToMO A. We have tried to dock substrates into the active site cavity of ToMO A by the Monte Carlo method, as it allows effective exploration of the conformational space with less central processing unit time than other, more time-consuming methods such as molecular dynamics. In all docking experiments, the backbone of ToMO A and the structure of the di-iron cluster were held rigid whereas at least two layers of side chains around the active site cavity were allowed to move to improve the fit of ligands inside the cavity. The Monte Carlo energy minimization of ToMO A without ligands in the active site cavity showed that only three residues contributing to the surface of the cavity, i.e., Ile100, Thr201, and Phe205, were particularly mobile, being able to assume several conformations. However, the mobility of Phe205 was limited to the ␹2 torsion angle. In the second layer of residues, Leu208, Leu272, Gln204 and, to a lesser extent, His96 were able to adopt different conformations. When toluene and o-xylene were docked into the active site, more than 10 low-energy orientations for each substrate were found, giving rise to several different binding complexes (data not shown). These results suggest that aromatic substrates can assume several binding orientations inside the active site, in agreement with the models in Fig. 3. Modeling the intermediates of benzene into the active site of ToMO A. As it is well-known that active sites are complementary to activated transition states and to unstable intermediates rather than to substrates and products (some examples can be found in references 11, 12, 24, 27, and 33 and references therein), we tried to identify catalytically productive binding modes through the docking of the intermediates of the hydroxylation reaction. As shown in Fig. 1, two or three intermediates are supposed to be involved in the conversions of aromatic hydrocarbons to phenols (16): (i) an aromatic carbocation, (ii) an unsaturated ketone, and possibly (iii) an epoxide. The carbocationic intermediate (CCI) has a critical role in the regioselectivity of the reaction because, after its formation, the

nature of the product is irreversibly defined. In contrast, the epoxide intermediate formed from toluene or from oxylene can yield two different isomers, depending on which C-O bond of the epoxide ring undergoes cleavage. We initially tested this procedure by docking the three possible intermediates of benzene hydroxylation into the active site of wild-type ToMO. Benzene was chosen instead of toluene or o-xylene because in this case a single molecular species exists for each intermediate. Docking was carried out by fixing the oxygen atom transferred to the aromatic ring at the same coordinates found for the bridging oxygen of the thioglycolate anion in the crystal structure of ToMO A (PDB code 1T0Q). The assumption that the oxygen atom is bound to the di-iron cluster even after its transfer to the substrate (Fig. 1) limits the degrees of freedom of the intermediates and provides a fixed point which can be used as a rotation center for the ligand. Binding energy values for the ToMO-benzene intermediate complexes reported in Table 1 indicate that the CCI fits the active site better than the other intermediates. The main contribution to the tight binding of the CCI depends on electrostatic interactions (Table 1), but van der Waals contacts also play an important role. In the CCI, the oxygen atom is bound to a single carbon atom of the ring and the C-O bond forms an angle of ⬃130° with the ring, which can thus be placed almost exactly in the plane of the cavity as shown in Fig. 2C, thus maximizing the steric interaction with the cavity. On the other hand, in the molecule of the epoxide intermediate, the sixatom ring and the epoxide ring form an angle of 105° and the oxygen atom lies above the central point of the C-C bond of the epoxide ring. As a consequence, when the oxygen atom is located at the bridging position of the di-iron cluster, too-close contacts between the six-atom ring and the active site cavity and between the three-atom ring and the di-iron cluster are generated (data not shown). As for the ketonic intermediate, the oxygen atom is in the same plane as the carbon atom ring. This geometry prevents the positioning of the ring in the plane of the cavity but, interestingly, pushes it toward the tunnel (Fig. 2D) which connects the active site to the exterior of the molecule. An interesting observation which stems from these docking experiments is that the orientation of the carbocation (Fig. 2C) is very similar to that of the thioglycolate found in the crystal structure of ToMO (this feature is even more evident in the case of the carbocations deriving from o-xylene, as discussed below). Modeling the intermediates of toluene and o-xylene into the active site of ToMO A. Given the increased stability of the

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⫽ ⌬nbE

‡

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ToMO-CCI complex relative to the ToMO-epoxide and ToMO-ketone complexes, in the case of benzene we decided to pay particular attention to the modeling of the ToMO-CCI complexes corresponding to toluene and o-xylene. Docking the CCIs of toluene and o-xylene is a complex procedure because several isomers exist. As shown in Fig. 4, toluene may generate up to five CCIs and four possible intermediates may be produced from reactions starting with o-xylene. Intermediate couples 5 and 6, 7 and 8, 10 and 11, and 12 and 13 are enantiomers. CCIs 5 and 6 yield o-cresol, CCIs 7 and 8 yield m-cresol, CCIs 10 and 11 yield 2,3-DMP, and CCIs 12 and 13 yield 3,4-DMP. CCI 9 yields p-cresol. Our docking experiments indicate that only CCIs 5, 7, and 9 from toluene and CCIs 10 and 12 from o-xylene can bind to the active site of wild-type ToMO with the same orientation as the CCI derived from benzene (data not shown), i.e., with the ring in the plane of the grids in Fig. 2B and C. As described in the following sections, using only the binding energy values relative

to these five intermediates, we obtained very good agreement between predicted and experimentally determined percentages; therefore, we will discuss only the docking analyses of these intermediates. Figure 5A shows a model of the positioning of CCIs 10 and 12, which lead to 2,3-DMP and 3,4-DMP, respectively, into the active site. The models of CCIs 5 and 7, leading to o- and m-cresol, respectively, are completely superimposable onto the model of CCI 10 (data not shown), whereas CCI 9, which yields p-cresol, has an orientation similar to that of CCI 12 (data not shown). Figure 5B shows that the ring of CCI 12 is placed exactly in the plane of the grid in Fig. 2 and that it mimics the orientation of thioglycolate even better than the CCI of benzene shown in Fig. 2C. Thus, our data indicate that CCIs 5, 7, 9, 10, and 12 dock into the active site of ToMO A and place their methyl groups into subsites located on the border of the pocket, as hypothesized previously (4). However, the ToMOCCI complexes suggest rather different positioning of the subsites for methyl groups from that in the original model. The model in Fig. 5A shows that only the ortho and para subsites are unambiguously defined. In this new model, the ortho subsite, located among residues Glu134, Leu192, and Ala107, is closer to the di-iron cluster than that in our previous model whereas the new para subsite is defined by residues Glu103, Phe176, and Ile100 (the meta subsite in the original model). Moreover, two alternative meta subsites (designated m1a and m1b) can be mapped. The existence of two alternative meta subsites may depend on the close proximity of the ortho and para subsites. This geometry is incompatible with the simultaneous docking of three adjacent methyl groups. Therefore, when CCI 10 is docked into the cavity, the ortho methyl groups block the ortho subsite whereas the meta methyl group partially fills the para subsite, thus defining the m1b subsite (residues Glu103, Phe176, and Met180). On the other hand, when CCI 12 is docked into the cavity, the para methyl group fills the para subsite, whereas the meta group partly occupies the ortho subsite, thus defining the m1a subsite (residues Ala107, Glu103, and Met180). The van der Waals contributions of the methyl groups of CCI 10 (subsites o and m1b) are ⫺1.12 and ⫺2.02 kcal/mol, respectively, whereas their contributions in the case of CCI 12 (subsites m1a and p) are ⫺1.64 and ⫺1.65 kcal/mol, respectively. Hence, it seems that a methyl group positioned into the m1b subsite gives a greater contribution to the binding energy than one positioned into the m1a subsite. An indirect confirmation of this observation comes from the docking of CCI 7, which leads to m-cresol. In this case, the single methyl group is predicted to occupy subsite m1b. In order to further analyze the subsites of the active site, we docked the intermediate of the hydroxylation of m-xylene, which yields 2,4-DMP, into the cavity (Fig. 5C). In this case, both the ortho and para methyl groups were forced slightly out from the ortho and para subsites. Because of the steric hindrance between the ortho methyl and residue Glu134 and between the para methyl and residue Ile100, the contributions of the ortho and para methyl groups to the binding energy decreased to ⫺0.05 and ⫺0.63 kcal/mol, respectively. These two values are significantly lower than those found for the intermediates deriving from o-xylene (see above). Interestingly, the total van der Waals contributions of CCIs 10 and 12 and of the intermediate produced from m-xylene to the binding energy

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FIG. 4. Chemical structures of the possible CCIs deriving from toluene and o-xylene. CCIs 5 and 6, CCIs 7 and 8, and CCI 9 are the possible intermediates for the transformation of toluene into o-, m-, and p-cresols, respectively. CCIs 10 and 11 and CCIs 12 and 13 are the possible intermediates for the transformation of o-xylene into 2,3- and 3,4-DMPs, respectively.

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were ⫺15.54, ⫺16.01, and ⫺13.37 kcal/mol, respectively. This finding would indicate that the cavity is better tailored to accommodate the intermediates from the physiological substrate o-xylene than those deriving from a nonphysiological substrate such as m-xylene. In conclusion, our docking data provide a very detailed map of the active site residues potentially involved in regioselectivity. From binding energies to percentages of products. In the hypothesis that the CCI is the first intermediate formed during the hydroxylation reaction, as the toluene3CCI reaction is endergonic, the ET‡ transition states should be similar to enzyme-CCI complexes. Therefore, the ⌬nbE‡o-m, ⌬nbE‡m-p, and ⌬nbE‡o-p values defined by equations 4 to 6 can be estimated through the Monte Carlo docking of the CCIs. Using equations 1 to 6 and the binding energy values provided by the ZMM software for toluene CCIs 5, 7, and 9, we predicted the percentages of cresols. Predicted percentages of o-, m-, and p-cresols (39.7, 19.9, and 40.4%, respectively) were very similar to experimentally determined percentages (36, 19, and 45%, respectively). Similarly, using the binding energy values for o-xylene CCIs 10 and 12, we found that predicted percentages of 2,3-, and 3,4-DMPs (17.9 and 82.1%, respectively) were very similar to experimentally determined percentages (19 and 81%, respectively). Thus, it may be concluded that the hypotheses of completely steric control of regioselectivity and the use of the ToMO A-CCI noncovalent bond energies for calculating the relative stabilities of the ET‡ or EX‡ complexes are essentially correct. As a control, the docking procedure was repeated using the two possible epoxides deriving from toluene, i.e., toluene 2,3epoxide and toluene 3,4-epoxide, which provide o-cresol/mcresol and m-cresol/p-cresol, respectively. The binding energy of toluene 3,4-epoxide was found to be about 2 kcal/mol higher than that of toluene 2,3-epoxide. In a system at equilibrium, this energy difference would imply the formation of less than 5% 2,3-epoxide. Even assuming that the 2,3-epoxide intermediate converts entirely to o-cresol, this finding is not in agreement with the experimentally determined percentage of o-cresol (36%). Similarly, in the case of the ketonic intermediates, the isomer leading to m-cresol showed the higher binding energy (data not shown), in disagreement with the experimental data. Modeling the CCIs of toluene and o-xylene in the active sites of ToMO A mutant forms. To further test our hypothesis, all the residues located on the edge of the cavity (Ala107, Met180, Glu103, Phe176, Ile100, and Phe205) were selected for mutational studies to experimentally verify whether changes at these sites would affect the regioselectivity of the enzyme in a predictable way. These residues were all changed to hydrophobic residues to preserve the hydrophobic nature of the pocket. Ala107 was changed to larger residues, such as Val and Ile, in order to hinder the ortho site. Met180 was changed to Ile in an enzyme already carrying the mutation E103G in order to obtain a double-mutant enzyme designated (E103G, M180I)ToMO A, in which all the residues facing the active site pocket are identical to the corresponding residues present in homologous T4MO. Glu103, previously mutated to Gly, Leu, and Met (4), was changed to the ␤-branched residues Val and Ile in order to

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FIG. 5. Structures of the ToMO-CCI 10 and ToMO-CCI 12 complexes. (A) Superimposition of the ToMO-CCI 10 complex (carbon atoms are in white) onto the ToMO-CCI 12 complex (carbon atoms are in green). (B) Superimposition of the PDB structure 1T0Q (carbon atoms are in magenta) onto the structure of the ToMO-CCI 12 complex (carbon atoms are in green). (C) Superimposition of the complexes ToMO-CCI 10 and ToMO-CCI 12 (colored as in panel A) onto the complex of ToMO with the CCI of the reaction m-xylene 3 2,4-DMP (carbon atoms are in magenta). The surface of the cavity of the complex ToMO-CCI 10 is shown as a mesh. The mesh is colored to show the contributions of residues Glu134, Ala107, Met180, Glu103, Phe176, and Ile100.

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TABLE 2. Apparent kcat values of ToMO and ToMO mutant proteins on benzene, toluene, o-xylene, and naphthalene kcat (s⫺1)a for substrate: Phenol

Benzene

Wild type I100A I100V I100M I100L I100F I100W F176I F176L A107V A107I F205L E103G E103L E103 M E103V E103I E103G, I100L E103V, I100V E103G, M180I

1.0 0.019 0.4 0.35 0.76 0.15 0 0.25 0.32 0.07 0.027 0.01 1.0 0.89 0.29 0.24 0.21 0.53 0.30 0.50

0.37 0.05 0.22 0.14 0.3 0.13 0.018 0.43 0.36 0.064 0.0017 0.0015 0.43 0.29 0.26 0.22 0.03 0.58 0.11 0.22

Toluene

o-Xylene

Naphthalene

0.43 0.26 0.03 0.025 0.27 0.19 0.18 0.078 0.56 0.3 0.3 0.16 Very low Very low 0.28 0.21 0.32 0.23 0.024 0.032 0.0009 0.0005 0.00094 0.0006 0.42 0.42 0.32 0.36 0.3 0.2 0.22 0.11 0.03 0.01 0.67 0.75 0.097 0.029 0.34 0.2

0.033 0.0015 0.011

0.016 0.016

a Standard errors for kcat values were ⬍20% for phenol and ⬍25% for benzene, toluene, o-xylene, and naphthalene.

increase hindrance in the region between the meta and para sites. Phe176 was changed to Leu and Ile in order to enlarge this region. Ile100 contributes to defining the hypothetical para site, but it is also at the boundary between the active site pocket and the tunnel which connects the pocket to the surface of the protein. Moreover, it is less closely packed than the other residues of the active site and bulges from the surface of the pocket. The corresponding residue in MMOs, Leu110, has been defined previously as the gate that controls the access to the active site (2, 29). Therefore, to explore the entire range of side chain dimensions, Ile100 was changed to Ala, Val, Leu, Met, Phe, and Trp.

TABLE 3. Comparison between experimentally determined and calculated percentages of cresol isomers produced by wild-type ToMO and ToMO mutant proteins ToMO variant or mutation(s)

Wild type E103G E103G, M180I A107V A107I F176I F176L E103V E103I E103M E103L a

Binding energya (kcal/mol) for: ET‡o

ET‡m1b

⫺34.69 ⫺34.00 ⫺32.74 ⫺27.50 ⫺27.30 ⫺33.28 ⫺33.27 ⫺33.70 ⫺33.73 ⫺33.86 ⫺33.46

⫺34.28 ⫺33.91 ⫺33.85 NAd NA ⫺33.31 ⫺33.40 ⫺33.03 ⫺33.29 ⫺31.61

ET‡m1a

NA NA ⫺33.35

Experimentally determined %c of:

Calculated % ofb: ET‡p

o-C

m-C

p-C

o-C

m-C

p-C

⫺34.70 ⫺35.44 ⫺35.28 ⫺34.04 ⫺33.90 ⫺34.91 ⫺34.82 ⫺33.45 ⫺32.89 ⫺33.21 ⫺32.26

39.7 7.5 1.2 0.0 0.0 5.6 6.3 45.2 64.6 58.5 85.1

19.9 6.5 8.1 2.9 3.3 5.9 7.8 25.1 19.8 22.3 3.7

40.4 86.0 90.7 97.1 96.7 88.4 85.9 29.7 15.6 19.2 11.2

36 9 6 0 0 7 6 49 59 47 20

19 6 10 6 4 6.5 7 34 24 19 11

45 85 84 94 96 87.5 87 17 17 34 69

Total binding energy for the complex ToMO variant-CCI 5 (ET‡o), ToMO variant-CCI 7 (ET‡m1b and ET‡m1a), or ToMO variant-CCI 9 (ET‡p). o-, m-, and p-C, o-, m-, and p-cresols. c Error, ⬍1%. d NA, not applicable. The hindrance of the branched side chains of Val and Ile forces the meta methyl group to occupy a position intermediate between the m1b and p subsites. The binding energies for the (A107V)-ToMO–CCI 7 and (A107I)-ToMO–CCI 7 complexes are ⫺31.95 and ⫺31.90 kcal/mol, respectively. b

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ToMO variant or mutation(s)

Phe205, which is located at the boundary between the active site pocket and the tunnel, like Ile100, was changed to Leu. All mutant enzymes were assayed with phenol, benzene, toluene, o-xylene, and naphthalene. The majority of the mutations did not change the catalytic efficiency with respect to that of wild-type ToMO or caused only minor changes. Only mutations I100A, I100W, F205L, A107V, and A107I were found to reduce significantly the kcat values for all the substrates (Table 2). Possible explanations are discussed briefly in the supplemental material. The docking procedure for the prediction of the regioselectivity was repeated using the Monte Carlo-optimized models of ToMO A mutant enzymes. As in wild-type ToMO A, the backbone and the structure of the di-iron cluster were held rigid; i.e., we assumed that the mutations did not significantly change the structure of the protein. Recently, Murray and coworkers have published the crystallographic structure of ToMO carrying the mutation I100W [the (I100W)-ToMO enzyme] (18). In spite of the large increase in the hindrance of the side chain at position 100, the structure of the mutated ToMO A is essentially unchanged. This effect happens because the bulky tryptophan side chain is accommodated inside the active site cavity partially hindering it. As all the residues we have mutated are at the border of the active site cavity, it is likely that variations in the steric hindrance of these side chains can be easily accommodated without relevant changes to the backbone structure, as observed in the case of the I100W mutation. We want also to underline that the Monte Carlooptimized model of (I100W)-ToMO correctly predicted the orientation of the mutated side chain (data not shown). Tables 3 and 4 report the results obtained after the docking of toluene- and o-xylene-derived CCIs, respectively, to ToMO A mutant enzymes at positions 107, 103, 180, and 176. The regioselectivities of all mutant enzymes, with the exception of those of (E103L)-ToMO for toluene and (F176L)-ToMO for o-xylene, were predicted with fairly good accuracy. Minor differences between experimental and predicted percentages may depend on the small differences in binding energy values (⌬G‡)

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TABLE 4. Comparison between experimentally determined and calculated percentages of DMP isomers produced by wild-type ToMO and ToMO mutant proteins ToMO variant or mutation(s)

Binding energya (kcal/mol) for:

Experimentally determined %b of:

Calculated % of:

EX‡3,4

2,3-DMP

3,4-DMP

2,3-DMP

3,4-DMP

Wild type E103G E103G, M180I A107V A107I F176I F176L E103V E103I E103M E103L

⫺35.27 ⫺35.18 ⫺34.1 ⫺26.20 ⫺25.60 ⫺33.60 ⫺34.8 ⫺33.81 ⫺31.67 ⫺34.36 ⫺32.75

⫺36.17 ⫺36.39 ⫺36.23 ⫺32.65 ⫺31.80 ⫺35.25 ⫺35.47 ⫺34.15 ⫺33.20 ⫺35.02 ⫺33.76

17.9 11.5 2.7 0.0 0.0 5.8 24.4 36.0 7.0 24.7 15.4

82.1 88.5 97.3 100 100 94.2 75.6 64.0 93.0 75.3 84.6

19 1 2 0 0 2.5 2.5 39 13 18 6

81 99 98 100 100 97.5 97.5 61 87 82 94

I100A I100V I100L I100M I100F I100L, E103G I100V, E103V

⫺34.61 ⫺35.33 ⫺35.43 ⫺35.48 ⫺35.79 ⫺35.09 ⫺33.62

⫺35.85 ⫺36.01 ⫺36.16 ⫺36.51 ⫺36.84 ⫺36.29 ⫺34.17

11.0 24.1 22.6 15.0 14.5 11.6 28.3

89.0 75.9 77.4 85.0 85.5 88.4 71.7

18 12 12 28 19 4 34.5

82 88 82 72 81 96 65.5

a b

Total binding energy for the complex ToMO variant-CCI 10 (EX‡2,3) or ToMO variant-CCI 12 (EX‡3,4). Error, ⬍1%.

among ET‡o, ET‡m, ET‡p, EX‡3,4, and EX‡2,3 complexes. In fact, our approach predicts ⌬G‡ values of 0.1 to 0.3 kcal/mol, which are 1% or less of the calculated binding energy values (30 to 36 kcal/mol). Moreover, as the docking procedure provides the binding energy values of each EI, it is now possible to understand also how each mutation influences the stability of the interaction of toluene and o-xylene methyl groups at each subsite. For example, mutation E103G significantly reduces the production of o-cresol, increasing the difference in stability between the ortho and para orientations, which changes from 0.01 kcal/mol in the case of the wild-type protein to 1.44 kcal/mol in the case of the E103G mutant enzyme. As shown in Table 3, this increase is due both to the destabilization of the ortho orientation (complex ET‡o) and to the stabilization of the para orientation (complex ET‡p). Moreover, the ZMM software yields individual group contributions to the total binding energy (see Materials and Methods). Our data (not shown) indicate that the contribution of the methyl group in the ortho site to the binding energy, due essentially to van der Waals interactions, is barely influenced by the E3G mutation, changing from ⫺1.34 kcal/ mol for the wild-type protein to ⫺1.47 kcal/mol for the E103G mutant form. Rather, the predicted destabilization of the ET‡o complex (0.69 kcal/mol) depends on the loss of van der Waals contacts between the C-H groups at positions 3 and 4 of the ring (Fig. 4 numbering) and the larger active site pocket. On the other hand, the enlargement of the para subsite removes clashes between the para methyl group and the pocket and increases the contribution of the para methyl group to the binding energy (from ⫺0.76 to ⫺2.1 kcal/mol). In the case of o-xylene, the mutation E103G increases the relative stability of the EX‡3,4 complex, which provides 3,4-DMP, both increasing the stability of the EX‡3,4 complex and decreasing the stability of the EX‡2,3 complex (Table 4). The values for the individual

components of the calculated binding energy indicate that the increase in the stability of the EX‡3,4 complex is due mainly to improved binding of the methyl groups and that the decrease in the stability of the EX‡2,3 complex depends on the worse accommodation of the substrate ring. Mutations F176I and F176L have effects similar to that of mutation E103G on the regioselectivity of ToMO A, but these effects depend on different factors. Indeed, these two mutations leave the stability of the ET‡p complex (Table 3) almost unchanged with respect to that of the wild-type enzyme, whereas they decrease the stability of ET‡o and ET‡m complexes by 1.42 and 0.95 kcal/mol, respectively. Moreover, the values for the individual components of the binding energy indicate that the decrease in the stability of the ET‡o complex is due mainly to a loss of van der Waals contacts between the active site pocket and the ring of the substrate (about 0.9 kcal/mol) and that the contribution of the methyl group to the binding energy is not affected by the mutation (the change is less than 0.1 kcal/mol). In contrast, in the case of the ET‡p complex, the loss of van der Waals contacts between the active site pocket and the ring of the substrate (about 0.8 kcal/mol) is counterbalanced by an increased contribution by the methyl group of the substrate (about 1 kcal/mol). As for mutations E103I and E103V, it should be noted that they insert ␤-branched residues at position 103. This insertion generates clashes between the side chain and the ring of the substrates, thus lowering the stability of all the ToMO-CCI complexes with respect to that of the wild-type enzyme (Tables 3 and 4). The greater destabilization of the ET‡p complex than of ET‡o and ET‡m (Table 3) leads to increased production of o- and m-cresols, whereas slightly different conformations of the valine and isoleucine side chains (data not shown) make the (E103V)-ToMO–ET‡m1a complex more stable than the (E103I)-ToMO–ET‡m1b complex. These results give a molec-

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EX‡2,3

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TABLE 5. Comparison between experimentally determined and calculated percentages of cresol isomers produced by wild-type ToMO and ToMO variants mutated at position 100 ToMO variant or mutation(s)

ET‡o

ET‡m1b

⫺34.69 ⫺33.90 ⫺33.80 ⫺34.66 ⫺34.48 ⫺34.71 ⫺34.55 ⫺33.98 ⫺35.50

⫺34.28 ⫺33.88 ⫺33.86 ⫺33.98

⫺32.85

ET‡m1a

⫺34.15 ⫺34.52 ⫺34.40 ⫺33.81

Calculated % ofb:

e⫺⌬bE /RT ‡

ET‡p

ET‡m2

⫺34.70 ⫺35.07 ⫺34.30 ⫺34.35 ⫺35.04 ⫺35.85 ⫺36.52 ⫺35.30 ⫺33.46

⫺34.50 ⫺35.20 ⫺34.75 ⫺35.23 ⫺37.76 ⫺36.02 ⫺36.29 ⫺34.08 ⫺35.51

0.00 0.58 0.38 0.32 0.00 0.90 0.55 1.00 2.70

Experimentally determined %c of:

o-C

m-C

p-C

o-C

m-C

p-C

40.0 7.0 16.0 35.0 22.0 5.0 3.0 8.0 20.0

20.0 43.0 47.0 44.0 10.0 52.0 28.0 16.0 61.0

40.0 50.0 37.0 21.0 68.0 42.0 70.0 76.0 19.0

36 9 13 31 29 37 25 6 19.5

19 42 47 38 5 35 5 17 62

45 49 40 31 66 28 70 77 18.5

a

Total binding energy for the complex ToMO variant-CCI 5 (ET‡o), ToMO variant-CCI 7 (ET‡m1b and ET‡m1a), ToMO variant-CCI 9 (ET‡p), or ToMO variant-CCI 8 (ET‡m2). b o-, m-, and p-C, o-, m-, and p-cresols. c Error, ⬍1%.

ular reason for the enhanced production of m-cresol by mutant (E103V)-ToMO with respect to that by mutant (E103I)ToMO. Moreover, due to the slightly different orientations of the two side chains, mutation E103V decreases the stability of the EX‡3,4 complex much more than that of the EX‡2,3 complex and mutation E103I has the opposite effect (Table 4). As a consequence, mutation E103V increases the percentage of 2,3-DMP whereas mutation E103I decreases it with respect to that produced by wild-type ToMO. In both cases, the decrease in the stability of the EX‡2,3 and EX‡3,4 complexes is due to worse accommodation of both the substrate ring and the methyl groups. The Ala107 side chain, according to our model, contributes to the surface of the ortho site. In agreement with the model, the mutation of Ala107 to Val or Ile, whose side chains fill the cavity of the ortho site, completely abolishes the production of o-cresol and 2,3-DMP. Modeling intermediates in the case of Ile100 mutant enzymes. Several mutations at position 100, which defines the para subsite, surprisingly increase the production of m-cresol (Table 5). Replacement with alanine, leucine, phenylalanine, and valine increases the production of m-cresol, whereas replacement with methionine and tryptophan decreases it. It is also interesting that the effects of mutations at positions 100 and 103 are additive, as shown by the behavior of the doublemutant enzymes (I100L, E103G)-ToMO A and (I100V, E103V)-ToMO A. The individual mutations I100V and E103V increase the percentage of m-cresol from 19 to 47 and 34%, respectively, whereas the corresponding double mutant produces 62% m-cresol. Furthermore, mutation I100L increases the percentage of m-cresol from 19 to 38%, whereas mutation E103G decreases it to 6%. In this case, the double-mutant enzyme (I100L, E103G)-ToMO A produces 17% m-cresol. As shown in Fig. 5A, a second hypothetical meta site (the m2 site) may exist between Ile100 and Phe205 (the para subsite in our previous model [4]) which is occupied by the methyl group when CCI 8 (Fig. 4) is docked into the active site. However, the binding of CCI 8 is different from the binding of the other intermediates because the hindrance of the side chains of Ile100 and Phe205 forces the ring of the intermediate into a plane which forms an angle of about 25° with the plane that

contains intermediates 5, 7, 9, 10, and 12 (Fig. 6A). Thus, it may be that this different geometry of the wild-type ToMO A-CCI 8 complex—with the aromatic ring out of the plane of the grid in Fig. 2B—makes the complex catalytically unproductive. In fact, this geometrical constraint is the reason that, in the case of the wild-type protein, we have limited our docking analysis to intermediates 5, 7, 9, 10, and 12 only (see above). Several mutations of residue 100 remove part or all of the hindrance and allow intermediate 8 to dock to the active site in a conformation more similar to those of intermediates 5, 7, 9, 10, and 12 (Fig. 6A). This finding makes kinetic model i inadequate to predict the effects of mutations at position 100. To take into account the extra methyl subsite m2, we modified model i by introducing a fourth ET complex (ETm2) and the corresponding transition state ET‡m2 (model iii in Fig. 3). Model iii includes three extra ⌬G‡ values, ⌬G‡o-m2, ⌬G‡m1-m2, and ⌬G‡p-m2, which determine the relative abundance of the ET‡m2 complex with respect to total ET‡ complexes and, hence, the amount of m-cresol produced by the pathway ETm2 ^ ET‡m2 3 enzyme-CCI 8. As the orientation of CCI 8 inside the active site is different for each mutant protein and different from those of intermediates 5, 7, 9, 10, and 12, the hypothesis that the ⌬G‡ values depend essentially on the noncovalent bond energy contributions is no longer valid. On the contrary, because of nonoptimal binding of the activated substrate to the cluster, the covalent bond energy component of the ET‡m2 complex, bE‡m2, should be generally higher than that of the other three ET‡ complexes, bE‡: ⌬G‡o-m2 ⫽ 关共nbE‡m2 ⫺ nbE‡o兲 ⫹ 共bE‡m2 ⫺ bE‡兲兴 ⫽ 共⌬nbE‡m2-o ⫹ ⌬ bE ‡兲

(7)

⌬G‡m1-m2 ⫽ 关共nbE‡m2 ⫺ nbE‡m1兲 ⫹ 共bE‡m2 ⫺ bE‡兲兴 ⫽ 共⌬nbE‡m2-m1 ⫹ ⌬ bE ‡兲

(8)

⌬G‡p-m2 ⫽ 关共nbE‡m2 ⫺ nbE‡p兲 ⫹ 共bE‡m2 ⫺ bE‡兲兴 ⫽ 共⌬nbE‡m2-p ⫹ ⌬bE‡兲 where ⌬bE‡ ⫽ (bE‡m2 ⫺ bE‡) ⱖ 0.

(9)

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Wild type I100A I100V I100L I100 M I100F I100W I100L, E103G I100V, E103V

Binding energy (kcal/mol)a for:

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Therefore, the equilibrium constants which determine the relative abundance of the ET‡m2 complex can be expressed as function of the ⌬bE‡ values: K‡o-m2 ⫽ 关ET‡m2兴/关ET‡o兴 ⫽ exp共 ⫺ ⌬nbE‡m2-o/RT兲 ⫻ exp共 ⫺ ⌬bE‡/RT兲

(10)

K‡m1-m2 ⫽ 关ET‡m2兴/关ET‡m1兴 ⫽ exp共 ⫺ ⌬nbE‡m2-m1/RT兲 ⫻ exp共 ⫺ ⌬bE‡/RT兲

(11)

K‡p-m2 ⫽ 关ET‡m2兴/关ET‡p兴 ⫽ exp共 ⫺ ⌬nbE‡m2-p/RT兲 ⫻ exp共 ⫺ ⌬bE‡/RT兲

(12)

Using the Monte Carlo In equations 10 through 12, correct to add symbol “⫻” between “exp . . .” expressions [e.g., exp(⫺⌬nbE‡m2-p/RT) ⫻ exp(⫺⌬bE‡/RT)]? If not, please clarify relationship between these expressions; should space be-

tween them be deleted, or should a chem point or some other symbol be added in place of the multiplication sign?៮ strategy, we predicted nbE‡o, nbE‡m1, nbE‡p, and nbE‡m2 for each mutation at position 100. Then, using equations 1 to 12, we determined the exp(⫺⌬bE‡/RT) values, which provided predicted cresol percentages similar to the experimentally determined ones. Table 5 shows that, except for mutations I100F and I100W, for each mutation at position 100, a single exp(⫺⌬bE‡/ RT) value exists which yields good agreement between experimental and predicted percentages of products. Moreover, Fig. 6B shows that there is good correlation between these exp(⫺⌬bE‡/RT) values and the angle ␦ formed between the plane in which intermediates 5, 7, 9, 10, and 12 lie (the plane of the grid in Fig. 2) and the plane where intermediate 8 is located (Fig. 6A). This interesting finding suggests that the energy of the transition state increases with the angle ␦ and further supports the hypothesis that the predicted Monte Carlo-minimized complexes ToMO A-benzene-CCI and ToMO A-CCI 5, 7, 9, 10 or 12 illustrate the geometry of the catalytically productive orientation of aromatic substrates inside the ToMO A active site pocket. Moreover, we want also to underline that our model provides a simple explanation for the additive effects of mutations at positions 100 and 103. In fact, according to our model, the observed percentage of m-cresol is the sum of the percentages of m-cresol produced from toluene docked with its methyl group in subsite m1 and from toluene docked with its methyl group in subsite m2. Moreover, the residue at position 103 prevalently contributes to the m1 site, whereas the residue at position 100 contributes to the m2 site. Thus, the combined effect of a mutation at position 103, which improves interactions at subsite m1, and a mutation at position 100, which partially opens subsite m2, is the production of a percentage of m-cresol close to the sum of the percentages produced by the single mutations. This is the case for the double mutant (I100V, E103V)-ToMO. In the case of the double mutant (I100L, E103G)-ToMO, the effect of mutation E103G, which decreases the ability of the m1 site to anchor a methyl group, is counterbalanced by the effect of mutation I100L on the catalytic efficiency of the m2 site. Consequently, no apparent change in m-cresol production is observed. As for mutations I100F and I100W, the lower level of agreement between predicted and experimental data may be due to the fact that mutations I100F and I100W increase significantly the volume of the side chain at position 100. As described above, the crystallographic structure of the (I100W)-ToMO mutant enzyme (18) shows that the tryptophan side chain points toward the active site cavity, partially hindering it. Most likely, the simultaneous accommodation inside the active site cavity of the bulky side chain of tryptophan—or phenylalanine—and of the substrate may require changes in the conformation of the ToMO A backbone which cannot be predicted by our strategy, as all the docking experiments were carried out by holding the backbone of ToMO A in a rigid position and allowing the movement of only the side chains closer to the cavity. As for o-xylene, our docking data indicate that there is no mutation at position 100 which allows the positioning of intermediates 11 and 13 in the active site with a conformation similar to those of intermediates 10 and 12 (data not shown). Thus, in the case of o-xylene, there is no need for hypothesizing

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FIG. 6. Comparison between the orientation of CCI 10 docked into wild-type ToMO (WT) and the orientations of CCI 8 docked into wild-type and mutant ToMOs. (A) The surface of the cavity of the ToMO-CCI 10 complex is shown as a mesh. ␦ is the angle between the ring of CCI 10 docked into wild-type ToMO and CCI 8 docked into wild-type and mutant ToMOs. (B) Correlation between angle ␦ shown in panel A and exp(⫺⌬bE‡/RT) values. Data for the mutation I100V were not used in the linear fit.

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TABLE 6. Comparison between experimentally determined and calculated percentages of naphthol isomers produced by wild-type ToMO and ToMO mutant proteins ToMO variant or mutation

Wild type F176I F176L I100A I100V

Binding energya (kcal/mol) for:

Experimentally determined %c of:

Calculated %b of:

EN‡␣

EN‡␤1

EN‡␤2

␣-N

␤-N produced by EN‡␤1

␤-N produced by EN‡␤2

␣-N

␤-N

⫺38.66 ⫺36.32 ⫺37.29 ⫺38.58 ⫺38.02

⫺31.08 ⫺32.88 ⫺28.65 ⫺33.37 ⫺30.30

⫺35.49 ⫺34.10 ⫺34.17 ⫺36.10 ⫺35.91

99.5 97.4 99.5 98.5 97.3

0.0 0.3 0.0 0.02 0.0

0.5 2.3 0.5 1.5 2.7

87 57 57 53 81

13 43 43 47 19

a kinetic model which takes into account the involvement of an m2 subsite. Consequently, we used kinetic model ii and calculated the data reported in the lower part of Table 4. Also in this case, the predicted regioselectivities of the mutant enzymes were in good agreement with the experimental values. Regioselectivity on naphthalene. From a steric point of view, naphthalene can be described as an ortho disubstituted benzene derivative bearing groups larger than methyl groups. Four CCIs can be produced from naphthalene, similar to those shown in Fig. 4 for o-xylene. Our docking data indicate that only intermediates equivalent to CCIs 10, 12, and 13 in Fig. 4 can fit the active site (in Table 6, these complexes are named EN‡␣, EN‡␤1, and EN‡␤2, respectively, and their models are shown in Fig. S5 in the supplemental material). However, none of these three intermediates can assume exactly the same orientation as the intermediates derived from benzene, toluene, and o-xylene. This finding is in agreement with the observation that the kcat value of wild-type ToMO for naphthalene is considerably lower than those measured for the more physiological substrates. Data in Table 6 show that in the case of naphthalene, our predictions are only qualitatively correct. In fact, for all the mutant enzymes we have studied, the calculated ␣-naphthol/␤-naphthol ratio was higher than that observed experimentally. However, the model correctly predicts that mutations which reduce the volume of the residues at positions 100 and 176 (Ile and Phe, respectively) increase the percentage of ␤-naphthol. Ile and Phe side chains likely impair the correct positioning of the naphthalene reaction intermediates corresponding to CCIs 12 and 13 (i.e., the intermediates which form the complexes EN‡␤1 and EN‡␤2, respectively). The poor quantitative agreement of our predictions with experimental data may depend on the rigidity of the backbone and of the major part of the side chains in the docking procedure. It may be possible that greater flexibility of the backbone is needed to accommodate this large substrate. Moreover, it should also be remembered that the models shown in Fig. 3 are based on the hypothesis that EX complexes are generated through fast equilibrium events. From a molecular point of view, this means that substrates can easily change their orientation inside the active site. Most likely, the hypothesis holds true for monocyclic, small substrates, but it could not hold in the case of a bulky molecule like naphthalene (see Fig. S5 in the supplemental material). If naphthalene, entering the active site, generates an EN␤ complex (as the docking of naphthalene

CCIs suggests [see Fig. S5C in the supplemental material]) and the rate of conversion of this initial complex to EN␣ is not higher than the rate of the hydroxylation reaction, more ␤-naphthol than the amount predicted by a model based on fast equilibrium events will be formed. Regioselectivity on polar substrates: the case of phenol. An intriguing feature of several multicomponent monooxygenases is their specificity in the second hydroxylation step, which produces exclusively catechol from phenol and (di)methylcatechols from cresols and DMPs. It should also be remembered that Tao et al. and Vardar and Wood (34, 35), using random mutagenesis, were able to obtain several ToMO and T4MO mutant forms which produce hydroquinone in different amounts. Mutant (I100Q)-ToMO is particularly interesting, as it produces 80% hydroquinone and only 20% catechol, the physiological product of ToMO. Even if a detailed analysis is beyond the scope of this paper, we have tested our approach with this mutant enzyme and with wild-type ToMO. We have docked the two possible enantiomeric CCIs of the phenol-to-catechol hydroxylation reaction (CCIs 14 and 15 in Fig. 7) into the ToMO active site on the hypothesis that, like those for methyl groups, subsites for the hydroxyl group should exist. Our results indicate that the hydroxyl groups can be posi-

FIG. 7. Chemical structures of the CCIs deriving from phenol. CCIs 14 and 15 are the possible intermediates of the phenol-catechol reaction. CCI 16 is the intermediate of the phenol-hydroquinone reaction. The positive charge is delocalized on both the ring and the OH group.

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a Total binding energy for the complex ToMO variant-naphthalene analogue of CCI 10 (EN‡␣), ToMO variant-naphthalene analogue of CCI 12 (EN‡␤1), or ToMO variant-naphthalene analogue of CCI 13 (EN‡␤2). b ␣- and ␤-N, ␣- and ␤-naphthol. c Error, ⬍1%.

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REGIOSELECTIVITY OF PSEUDOMONAS SP. STRAIN OX1 ToMO ACKNOWLEDGMENTS

We are indebted to Giuseppe D’Alessio, Matthew H. Sazinsky, and Anna Tramontano for critically reading the manuscript. This work was supported by grants from the Ministry of University and Research (PRIN/2002 and PRIN/2004). REFERENCES 1. Bertoni, G., F. Bolognesi, E. Galli, and P. Barbieri. 1996. Cloning of the genes for and characterization of the early stages of toluene catabolism in Pseudomonas stutzeri OX1. Appl. Environ. Microbiol. 62:3704–3711. 2. Borodina, E., T. Nichol, M. G. Dumont, T. J. Smith, and J. C. Murrell. 2007. Mutagenesis of the “leucine gate” to explore the basis of catalytic versatility in soluble methane monooxygenase. Appl. Environ. Microbiol. 73:6460– 6467. 3. Cafaro, V., V. Izzo, R. Scognamiglio, E. Notomista, P. Capasso, A. Casbarra, P. Pucci, and A. Di Donato. 2004. Phenol hydroxylase and toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1: interplay between two enzymes. Appl. Environ. Microbiol. 70:2211–2219. 4. Cafaro, V., E. Notomista, P. Capasso, and A. Di Donato. 2005. Mutation of glutamic acid 103 of toluene o-xylene monooxygenase as a means to control the catabolic efficiency of a recombinant upper pathway for degradation of methylated aromatic compounds. Appl. Environ. Microbiol. 71:4744–4750. 5. Cafaro, V., E. Notomista, P. Capasso, and A. Di Donato. 2005. Regiospecificity of two multicomponent monooxygenases from Pseudomonas stutzeri OX1: molecular basis for catabolic adaptation of this microorganism to methylated aromatic compounds. Appl. Environ. Microbiol. 71:4736–4743. 6. Cafaro, V., R. Scognamiglio, A. Viggiani, V. Izzo, I. Passaro, E. Notomista, F. Dal Piaz, A. Amoresano, A. Casbarra, P. Pucci, and A. Di Donato. 2002. Expression and purification of the recombinant subunits of toluene/o-xylene monooxygenase and reconstitution of the active complex. Eur. J. Biochem. 269:5689–5699. 7. Fishman, A., Y. Tao, L. Rui, and T. K. Wood. 21 October 2004. Controlling the regiospecific oxidation of aromatics via active site engineering of toluene para-monooxygenase of Ralstonia pickettii PKO1. J. Biol. Chem. 280:506– 514. [Epub ahead of print.] 8. Gallagher, S. C., R. Cammark, and H. Dalton. 1997. Alkene monooxygenase from Nocardia corallina B-276 is a member of the class of dinuclear iron proteins capable of stereospecific epoxygenation reactions. Eur. J. Biochem. 247:635–641. 9. Hemmi, H., J. M. Studts, Y. K. Chae, J. Song, J. L. Markley, and B. G. Fox. 2001. Solution structure of the toluene 4-monooxygenase effector protein (T4moD). Biochemistry 40:3512–3524. 10. Horinouchi, M., K. Kasuga, H. Nojiri, H. Yamane, and T. Omori. 1997. Cloning and characterization of genes encoding an enzyme which oxidizes dimethyl sulfide in Acinetobacter sp. strain 20B. FEMS Microbiol. Lett. 155:99–105. 11. Kraut, D. A., P. A. Sigala, B. Pybus, C. W. Liu, D. Ringe, G. A. Petsko, and D. Herschlag. 28 March 2006. Testing electrostatic complementarity in enzyme catalysis: hydrogen bonding in the ketosteroid isomerase oxyanion hole. PLoS Biol. 4:e99. [Epub ahead of print.] 12. Lahiri, S. D., G. Zhang, D. Dunaway-Mariano, and K. N. Allen. 13 March 2003. The pentacovalent phosphorus intermediate of a phosphoryl transfer reaction. Science 299:2067–2071. [Epub ahead of print.] 13. Leahy, J. G., P. J. Batchelor, and S. M. Morcomb. 2003. Evolution of the soluble diiron monooxygenases. FEMS Microbiol. Rev. 27:449–479. 14. Lippard, S. J. 2005. Hydroxylation of C-H bonds at carboxylate-bridged diiron centres. Philos. Trans. A 363:861–877, 1035–1040. 15. Lipscomb, J. D. 1994. Biochemistry of the soluble methane monooxygenase. Annu. Rev. Microbiol. 48:371–399. 16. Mitchell, K. H., C. E. Rogge, T. Gierahn, and B. G. Fox. 14 March 2003. Insight into the mechanism of aromatic hydroxylation by toluene 4-monooxygenase by use of specifically deuterated toluene and p-xylene. Proc. Natl. Acad. Sci. USA 100:3784–3789. [Epub ahead of print.] 17. Mitchell, K. H., J. M. Studts, and B. G. Fox. 2002. Combined participation of hydroxylase active site residues and effector protein binding in a para to ortho modulation of toluene 4-monooxygenase regiospecificity. Biochemistry 41:3176–3188. 18. Murray, L. J., R. Garcia-Serres, M. S. McCormick, R. Davydov, S. G. Naik, S. H. Kim, B. M. Hoffman, B. H. Huynh, and S. J. Lippard. 29 November 2007. Dioxygen activation at non-heme diiron centers: oxidation of a proximal residue in the I100W variant of toluene/o-xylene monooxygenase hydroxylase. Biochemistry 46:14795–14809. [Epub ahead of print.] 19. Murray, L. J., and S. J. Lippard. 23 May 2007. Substrate trafficking and dioxygen activation in bacterial multicomponent monooxygenases. Acc. Chem. Res. 40:466–474. [Epub ahead of print.] 20. Murray, L. J., S. G. Naik, D. O. Ortillo, R. Garcia-Serres, J. K. Lee, B. H. Huynh, and S. J. Lippard. 30 October 2007. Characterization of the areneoxidizing intermediate in ToMOH as a diiron(III) species. J. Am. Chem. Soc. 129:14500–14510. [Epub ahead of print.] 21. Murrell, J. C., B. Gilbert, and I. R. McDonald. 2000. Molecular biology and regulation of methane monooxygenase. Arch. Microbiol. 173:325–332.

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tioned in two hydrophilic sites defined by residues Glu134 and Glu197 on one side of the di-iron cluster (see Fig. S6A in the supplemental material) and by Glu231 on the other side (see Fig. S6B in the supplemental material). The contributions of the hydrogen bonds to the binding energy are about 0.5 kcal/ mol in both cases. When the intermediate leading to hydroquinone (CCI 16 in Fig. 7) was docked into the active site of ToMO, we noticed that no hydrogen bond partner was present in the catalytic pocket, thus giving a molecular basis for the inability of the wild-type enzyme to produce hydroquinone. On the contrary, in the (I100Q)-ToMO mutant enzyme, the carbonyl group of the Gln100 side chain could form a hydrogen bond with the hydroxyl group on the intermediate, leading to hydroquinone (see Fig. S6C in the supplemental material), with an estimated contribution to the binding energy of 0.7 kcal/mol. This finding led to a prediction which is in agreement with the experimental observation that the (I100Q)-ToMO mutant enzyme produces more hydroquinone than catechol. Conclusions. BMMs have broad substrate specificities, coupled with specific regioselectivity properties, in the hydroxylation reaction of aromatic substrates. These features are metabolically relevant, because they are the basis for the capabilities of several microorganisms to grow on selected molecules. Moreover, given the catalytic potentials of BMMs, they may constitute a powerful tool for the bioremediation of harmful substances and may serve as specific biocatalysts in (regio)selective syntheses. Results from several structural and functional studies suggest that the different regioselectivities of BMMs depend on differences in the shape of the active site pocket (2, 4, 7, 17, 26, 30, 34, 35). However, a detailed description of the molecular basis for the regioselectivities of these enzymes is still lacking. This situation is particularly inconvenient because it impairs the possibility to attempt rational modifications to produce new catalysts and/or new microorganisms endowed with specific, advantageous properties. In this study, we have developed a procedure based on the docking of the intermediates of the hydroxylation reaction into the active site pocket of a specific monooxygenase, ToMO A. This approach allows for (i) a detailed analysis of the molecular determinants of the enzyme’s regioselectivity, (ii) the prediction of the regioselectivity properties of mutant forms of the enzyme, in the absence of any experimental data, and (iii) the prediction of the catalytically productive orientation of a substrate inside the active site pocket. Thus, this procedure is a valuable tool for the design of mutant monooxygenases for use in biosynthesis and bioremediation procedures, and its applicability may also be extended to other kinds of substrates and other multicomponent monooxygenases. Finally, the results of the docking experiments reported in this paper have very interesting implications for the catalytic mechanism of TMOs. The optimal fit between the ToMO active site pocket and the delocalized carbocation and the good agreement between experimentally determined regioselectivity and the regioselectivity predicted using the delocalized carbocations as ligands strongly suggest that the delocalized carbocation is a crucial intermediate in aromatic hydroxylation reactions.

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