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Supporting information For the manuscript Hajighasemi M. et al.
Biochemical and Structural Insights into Enzymatic Depolymerization of Polylactic Acid and Other Polyesters by Microbial Carboxylesterases Mahbod Hajighasemi,† Boguslaw P. Nocek,‡ Anatoli Tchigvintsev,† Greg Brown,† Robert Flick,† Xiaohui Xu,† Hong Cui,† Tran Hai,§ Andrzej Joachimiak,‡ Peter N. Golyshin,§ Alexei Savchenko,† Elizabeth A. Edwards,*,† and Alexander F. Yakunin*,†
†
Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5, Canada
‡
The Bioscience Division, Argonne National Laboratory, Argonne, Illinois 60439, United States §
School of Biological Sciences, University of Bangor, Gwynedd LL57 2UW, U.K.
*E-mail: [email protected], *E-mail: [email protected]
S1
Supplementary Figures Figure S1. Turbidimetric assay of PLA depolymerization activity of ABO2449. Figure S2. Sequence alignment of RPA1511, ABO2449, and two representative family V esterases. Figure S3. Phylogenetic analysis of esterase-type PLA depolymerases and other polyester degrading enzymes. Figure S4. Effect of detergents on esterase activity of RPA1511, ABO2449 and PlaM4. Figure S5. GPC analysis of the reaction products of enzymatic hydrolysis of solid PLA18. Figure S6. Effect of pH and NaCl on esterase activity of RPA1511 and ABO2449. Figure S7. Structural model of ABO2449 and its catalytic triad.
Supplementary Tables
Table S1. 86 ester substrates used for enzyme screening. Table S2. Molecular weight characteristics of the reaction products of enzymatic hydrolysis of solid PLA18 determined by GPC. Table S3. LC-MS analysis of oligomeric composition of different lactic acid species released by (A) RPA1511 and (B) ABO2449 from PLA10 substrate. Table S4. Kinetic parameters of the wild-type (WT) and mutant ABO2449 with various substrates Table S5. Crystallographic data collection and model refinement statics.
S2
Figure S1. Turbidimetric assay of PLA depolymerization activity of ABO2449. (A) Changes in optical density (OD580) of emulsified PLA10 during incubation with purified ABO2449 (1 mg). (B) Test tubes with emulsified PLA10 as substrate and purified ABO2449 representing different time points from panel A.
S3
Figure S2. Sequence alignment of RPA1511, ABO2449, and two representative family V esterases. Residues conserved in all aligned proteins are boxed with the serine hydrolase catalytic triad shown in grey background. The residues of RPA1511 and ABO2449 mutated in the present study are marked with triangles, whereas the secondary structure elements of RPA1511 are shown above the alignment. The compared proteins are RPA1511 (Q6N9M9), ABO2449 (Q0VLQ1), the PHA depolymerase PhaB from Pseudomonas oleovorans (P26495) and the Psychrobacter immobilis lipase 1 (X67712).
S4
Figure S3. Phylogenetic analysis of esterase-type PLA depolymerases and other polyester degrading enzymes. Phylogenetic relatedness of ABO2449 and RPA1511 to representative esterases from the families I – VIII (based on Arpigny and Jaeger, 199951). The numbers on the nodes correspond to the percent recovery in 1,000 bootstrap resamplings. The reference enzymes for esterase families include (UniProt IDs) P95419 (family I), p10480 (II), Q56008 (III), H9UPC6 (IV), P26495 (V), P73192 (VI), Q01470 (VII) and Q44050 (VIII).
S5
Figure S4. Effect of detergents on esterase activity of RPA1511, ABO2449 and PlaM4. Esterase activity of purified proteins (5 µg of RPA1511, 0.5 µg of ABO2449, 4 µg of PlaM4) was measured at 30 °C using 1.5 mM α-naphthyl propionate (for RPA1511 and ABO2449) or 1.5 mM p-NP-butyrate as the substrate. Triton X-100, Plysurf or Tween-20 were added at indicated concentrations to reaction mixtures. Results are means ± S.D. from at least two independent determinations.
S6
Figure S5. GPC analysis of the reaction products of enzymatic hydrolysis of solid PLA18. Reaction mixtures (1.0 ml) contained 0.4 M Tris-HCl (pH 8.0), solid PLA18 (20 mg), and enzyme (50 µg). After 2 hours of incubation at 35 °C, residual solid PLA was collected by centrifugation, dissolved in THF, cleared by centrifugation through syringe filters and analyzed by GPC as described in the Experimental Section. The numbers on the peaks indicate the molecular weight in daltons.
S7
Figure S6. Effect of pH and NaCl on esterase activity of RPA1511 and ABO2449. Esterase activity of purified proteins was measured at 30 °C using α-naphthyl propionate (1.5 mM) as substrate. For pH range, the following buffers were used (50 mM): MES (pH 6.0), HEPES (pH 7.0, 7.5, 8.0), Tris-HCl (pH 8.0, 8.5), CHES (pH 9.0, 9.5, 10.0), CAPS (pH 10.0, 10.5, 11.0).
S8
Figure S7. Structural model of ABO2449 and its catalytic triad. The ABO2449 model was generated using the protein structure prediction server Phyre2 and the structure of the aclacinomycin methylesterase RdmC (PDB code 1Q0R, 28% sequence identity to ABO2449). The catalytic triad residues (Ser120, His275, and Asp247) are shown as sticks along a ABO2449 ribbon (gray).
S9
Table S1. 86 ester substrates used for enzyme screening 1. (+)-Methyl (R)-2-chloropropionate 2. (−)-Methyl (S)-2-chloropropionate
30. Ethyl hexanoate 31. Ethyl iodoacetate
59. Methyl chloroacetate 60. Methyl cinnamate
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43.
61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71.
(+)-Methyl D-lactate (−)-Methyl L-lactate (1S)-(+)-Menthyl acetate (1R)-(−)-Menthyl acetate (1S)-(+)-Neomenthyl acetate (1R)-(−)-Neomenthyl acetate (S)-(+)-Glycidyl butyrate (R)-(−)-Glycidyl butyrate 2-Bromoethyl acetate Acetylcholine chloride Bromoacetic acid
14. Butyl acetate 15. Ethyl (R)-(−)-3-hydroxybutyrate 16. Ethyl (S)-(−)-4-chloro-3hydroxybutyrate 17. Ethyl 2-bromopropionate 18. Ethyl 2-chloropropionate 19. Ethyl 2-ethylacetoacetate 20. Ethyl 2-methylacetoacetate 21. Ethyl 3-bromopropionate 22. Ethyl 4-bromobutyrate 23. Ethyl 4-chloroacetoacetate 24. Ethyl 4-hydroxy-3methoxycinnamate 25. Ethyl acetate 26. Ethyl bromoacetate 27. Ethyl chloroacetate 28. Ethyl cinnamate 29. Ethyl decanoate
44. 45. 46. 47. 48. 49. 50. 51. 52.
Ethyl octanoate Ethyl propionate Ethyl propionylacetate Ethyl tribromoacetate Ethyl trifluoroacetate Ethyl α-bromoisobutyrate Glycine ethyl ester hydrochloride Isoamyl cinnamate Isobutyl acetate Isobutyl cinnamate Isopropenyl acetate Methyl (R)-(+)-3-bromo-2methylpropionate Methyl (S)-(+)-3-hydroxy-2methylpropionate Methyl (R)-(−)-3-hydroxy-2methylpropionate Methyl (S)-(+)-mandelate Methyl (R)-(−)-mandelate Methyl (R)-3-hydroxybutyrate Methyl (S)-(+)-3-hydroxybutyrate Methyl 2-chloro-3hydroxypropionate Methyl 2-hydroxyisobutyrate Methyl 2,2-dimethyl-3hydroxypropionate
Methyl glycolate Methyl propionate Methyl pyruvate Methyl trans-cinnamate Phenethyl cinnamate Phenyl acetate Propyl acetate tert-Butyl 3-hydroxypropionate tert-Butyl cinnamate Triacetin (C 2:0) Tripropionin (C 3:0)
72. Tributyrin (C 4:0) 73. Tricaproin (C 6:0) 74. Tricaprylin (C 8:0) 75. 76. 77. 78.
Tricaprin (C 10:0) Trilinolein (C 18:2) Vinyl acetate Vinyl benzoate
79. Vinyl butyrate 80. Vinyl cinnamate 81. Vinyl crotonate
53. Methyl 3-bromopropionate
82. Vinyl decanoate
54. 55. 56. 57. 58.
83. 84. 85. 86.
Methyl 4-(hydroxymethyl)benzoate Methyl acetoacetate Methyl benzoate Methyl bromoacetate Methyl butyrate
Vinyl laurate Vinyl methacrylate Vinyl pivalate Vinyl propionate
S10
Table S2. Molecular weight characteristics of the reaction products of enzymatic hydrolysis of solid PLA18 determined by GPC. PLA substrate peak
PLA product peaks
Treatment
Mpa
Mnb
PDIc
Mp
Mn
PDI
Mp
Mn
PDI
RPA1511
29,448
17,458
2.34
250
186
1.95
-
-
-
Blank
29,076
17,381
2.35
-
-
-
-
-
-
ABO2449
29,690
17,799
2.29
528
374
1.30
66
72
1.13
Blank+detergent
29,318
15,396
2.58
480
268
1.65
60
58
1.03
a
Mp: molecular weight of the highest peak Mn: number average molecular weight c PDI: polydispersity index, Mw.Mn-1 b
S11
Table S3. LC-MS analysis of oligomeric composition of different lactic acid species released by (A) RPA1511 and (B) ABO2449 from PLA10 substrate.
A (RPA1511) Oligomeric State 2 3 4 5 6 7 8 9 10
Chemical Formula C6H10O5 C9H14O7 C12H18O9 C15H22O11 C18H26O13 C21H30O15 C24H34O17 C27H38O19 C30H42O21
m/z Theoretical Observed 161.0455 161.0429 233.0667 233.0642 305.0878 305.0851 377.1089 377.1057 449.1301 449.1259 521.1512 521.1463 593.1723 593.1674 665.1935 665.1882 737.2146 737.2087
Δ ppm 16.1 10.7 8.8 8.5 9.4 9.4 8.3 8.0 8.0
Signal Intensity 2.0E+08 1.1E+07 4.2E+07 5.1E+07 5.4E+07 3.0E+07 2.7E+07 1.2E+07 8.0E+06
Chemical Formula C6H10O5 C9H14O7 C12H18O9 C15H22O11 C18H26O13 C21H30O15 C24H34O17 C27H38O19 C30H42O21
m/z Theoretical Observed 161.0455 161.0429 233.0667 ND* 305.0878 ND 377.1089 ND 449.1301 ND 521.1512 ND 593.1723 ND 665.1935 ND 737.2146 ND
Δ ppm 16.1 ND ND ND ND ND ND ND ND
Signal Intensity 1.3E+08 ND ND ND ND ND ND ND ND
B (ABO2449) Oligomeric State 2 3 4 5 6 7 8 9 10
*ND = not detectable
S12
Table S4. Kinetic parameters of the wild-type (WT) and mutant ABO2449 with various substrates
Protein
kcat/Km
Km (mM)
kcat (s )
α-Naphthyl acetate
0.24 ± 0.02
195.10 ± 4.36
0.81
α-Naphthyl propionate
0.63 ± 0.09
354.90 ± 19.65
0.56
pNP-acetate
0.96 ± 0.13
351.30 ± 25.43
0.37
pNP-butyrate
0.57 ± 0.03
365.40 ± 8.69
0.64
pNP-valerate
0.44 ± 0.03
304.50 ± 8.38
0.69
Ethyl iodoacetate
13.03 ± 0.45
16.06 ± 0.33
0.0012
Tributyrin
0.25 ± 0.04
20.05 ± 1.08
0.080
Phenyl acetate
8.50 ± 0.45
8.40 ± 0.24
0.0010
Vinyl laurate
0.12 ± 0.01
3.81 ± 0.06
0.032
L32A
α-Naphthyl acetate
0.25 ± 0.02
27.21 ± 0.80
0.11
S33A
α-Naphthyl acetate
0.48 ± 0.15
37.60 ± 3.83
0.078
F38A
α-Naphthyl acetate
0.50 ± 0.05
90.29 ± 3.52
0.18
M144A
α-Naphthyl acetate
1.35 ± 0.24
127.70 ± 12.31
0.095
L152A
α-Naphthyl acetate
0.22 ± 0.02
40.58 ± 1.09
0.18
L163A
α-Naphthyl acetate
0.40 ± 0.03
94.95 ± 2.09
0.24
M183A
α-Naphthyl acetate
0.51 ± 0.07
153.40 ± 7.80
0.30
F219A
α-Naphthyl acetate
0.10 ± 0.03
31.42 ± 1.82
0.31
L249A
α-Naphthyl acetate
0.41 ± 0.09
12.30 ± 0.97
0.030
P302R
α-Naphthyl acetate
0.23 ± 0.02
117.20 ± 2.51
0.51
WT
Variable substrate
-1
(s-1.µM-1)
S13
Table S5. Crystallographic data collection and model refinement statics. Values for the highestresolution shell are shown in parentheses.
Parameters
RPA1511
Beamline Data collection
SBC-19ID
Space group
I222
Cell dimensions a, b, c (Å)
80 87.4 93.7
Wavelength (Å)
0.9794
Resolution range (Å)
32 - 2.20 (2.28 - 2.20)
Unique reflections
16885
Rmerge
0.077
I/σI
15.2 (1.7)
Completeness (%)
99.3 (98.1)
Refinement Total reflections
63870
Rwork
0.181 (0.231)
Rfree
0.221 (0.329)
Number of non-hydrogen atoms
2237
protein
2146
ligands
22
water
69
Rmsd Bond lengths (Å)
0.005
Bond angles (°)
0.85
Average B-factors protein
43.5
ligands
72.5
water
42.3
Ramachandran favored/outliers (%)
98/0
Clashscore Protein residues
0.46 287
PDB accession code
4PSU
S14
Biochemical and Structural Insights into Enzymatic Depolymerization of Polylactic Acid and Other Polyesters by Microbial Carboxylesterases Mahbod Hajighasemi,† Boguslaw P. Nocek,‡ Anatoli Tchigvintsev,† Greg Brown,† Robert Flick,† Xiaohui Xu,† Hong Cui,† Tran Hai,§ Andrzej Joachimiak,‡ Peter N. Golyshin,§ Alexei Savchenko,† Elizabeth A. Edwards,*,† and Alexander F. Yakunin*,†
†
Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5, Canada
‡
The Bioscience Division, Argonne National Laboratory, Argonne, Illinois 60439, United States §
School of Biological Sciences, University of Bangor, Gwynedd LL57 2UW, U.K.
*E-mail: [email protected], *E-mail: [email protected]
S1
Supplementary Figures Figure S1. Turbidimetric assay of PLA depolymerization activity of ABO2449. Figure S2. Sequence alignment of RPA1511, ABO2449, and two representative family V esterases. Figure S3. Phylogenetic analysis of esterase-type PLA depolymerases and other polyester degrading enzymes. Figure S4. Effect of detergents on esterase activity of RPA1511, ABO2449 and PlaM4. Figure S5. GPC analysis of the reaction products of enzymatic hydrolysis of solid PLA18. Figure S6. Effect of pH and NaCl on esterase activity of RPA1511 and ABO2449. Figure S7. Structural model of ABO2449 and its catalytic triad.
Supplementary Tables
Table S1. 86 ester substrates used for enzyme screening. Table S2. Molecular weight characteristics of the reaction products of enzymatic hydrolysis of solid PLA18 determined by GPC. Table S3. LC-MS analysis of oligomeric composition of different lactic acid species released by (A) RPA1511 and (B) ABO2449 from PLA10 substrate. Table S4. Kinetic parameters of the wild-type (WT) and mutant ABO2449 with various substrates Table S5. Crystallographic data collection and model refinement statics.
S2
Figure S1. Turbidimetric assay of PLA depolymerization activity of ABO2449. (A) Changes in optical density (OD580) of emulsified PLA10 during incubation with purified ABO2449 (1 mg). (B) Test tubes with emulsified PLA10 as substrate and purified ABO2449 representing different time points from panel A.
S3
Figure S2. Sequence alignment of RPA1511, ABO2449, and two representative family V esterases. Residues conserved in all aligned proteins are boxed with the serine hydrolase catalytic triad shown in grey background. The residues of RPA1511 and ABO2449 mutated in the present study are marked with triangles, whereas the secondary structure elements of RPA1511 are shown above the alignment. The compared proteins are RPA1511 (Q6N9M9), ABO2449 (Q0VLQ1), the PHA depolymerase PhaB from Pseudomonas oleovorans (P26495) and the Psychrobacter immobilis lipase 1 (X67712).
S4
Figure S3. Phylogenetic analysis of esterase-type PLA depolymerases and other polyester degrading enzymes. Phylogenetic relatedness of ABO2449 and RPA1511 to representative esterases from the families I – VIII (based on Arpigny and Jaeger, 199951). The numbers on the nodes correspond to the percent recovery in 1,000 bootstrap resamplings. The reference enzymes for esterase families include (UniProt IDs) P95419 (family I), p10480 (II), Q56008 (III), H9UPC6 (IV), P26495 (V), P73192 (VI), Q01470 (VII) and Q44050 (VIII).
S5
Figure S4. Effect of detergents on esterase activity of RPA1511, ABO2449 and PlaM4. Esterase activity of purified proteins (5 µg of RPA1511, 0.5 µg of ABO2449, 4 µg of PlaM4) was measured at 30 °C using 1.5 mM α-naphthyl propionate (for RPA1511 and ABO2449) or 1.5 mM p-NP-butyrate as the substrate. Triton X-100, Plysurf or Tween-20 were added at indicated concentrations to reaction mixtures. Results are means ± S.D. from at least two independent determinations.
S6
Figure S5. GPC analysis of the reaction products of enzymatic hydrolysis of solid PLA18. Reaction mixtures (1.0 ml) contained 0.4 M Tris-HCl (pH 8.0), solid PLA18 (20 mg), and enzyme (50 µg). After 2 hours of incubation at 35 °C, residual solid PLA was collected by centrifugation, dissolved in THF, cleared by centrifugation through syringe filters and analyzed by GPC as described in the Experimental Section. The numbers on the peaks indicate the molecular weight in daltons.
S7
Figure S6. Effect of pH and NaCl on esterase activity of RPA1511 and ABO2449. Esterase activity of purified proteins was measured at 30 °C using α-naphthyl propionate (1.5 mM) as substrate. For pH range, the following buffers were used (50 mM): MES (pH 6.0), HEPES (pH 7.0, 7.5, 8.0), Tris-HCl (pH 8.0, 8.5), CHES (pH 9.0, 9.5, 10.0), CAPS (pH 10.0, 10.5, 11.0).
S8
Figure S7. Structural model of ABO2449 and its catalytic triad. The ABO2449 model was generated using the protein structure prediction server Phyre2 and the structure of the aclacinomycin methylesterase RdmC (PDB code 1Q0R, 28% sequence identity to ABO2449). The catalytic triad residues (Ser120, His275, and Asp247) are shown as sticks along a ABO2449 ribbon (gray).
S9
Table S1. 86 ester substrates used for enzyme screening 1. (+)-Methyl (R)-2-chloropropionate 2. (−)-Methyl (S)-2-chloropropionate
30. Ethyl hexanoate 31. Ethyl iodoacetate
59. Methyl chloroacetate 60. Methyl cinnamate
3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13.
32. 33. 34. 35. 36. 37. 38. 39. 40. 41. 42. 43.
61. 62. 63. 64. 65. 66. 67. 68. 69. 70. 71.
(+)-Methyl D-lactate (−)-Methyl L-lactate (1S)-(+)-Menthyl acetate (1R)-(−)-Menthyl acetate (1S)-(+)-Neomenthyl acetate (1R)-(−)-Neomenthyl acetate (S)-(+)-Glycidyl butyrate (R)-(−)-Glycidyl butyrate 2-Bromoethyl acetate Acetylcholine chloride Bromoacetic acid
14. Butyl acetate 15. Ethyl (R)-(−)-3-hydroxybutyrate 16. Ethyl (S)-(−)-4-chloro-3hydroxybutyrate 17. Ethyl 2-bromopropionate 18. Ethyl 2-chloropropionate 19. Ethyl 2-ethylacetoacetate 20. Ethyl 2-methylacetoacetate 21. Ethyl 3-bromopropionate 22. Ethyl 4-bromobutyrate 23. Ethyl 4-chloroacetoacetate 24. Ethyl 4-hydroxy-3methoxycinnamate 25. Ethyl acetate 26. Ethyl bromoacetate 27. Ethyl chloroacetate 28. Ethyl cinnamate 29. Ethyl decanoate
44. 45. 46. 47. 48. 49. 50. 51. 52.
Ethyl octanoate Ethyl propionate Ethyl propionylacetate Ethyl tribromoacetate Ethyl trifluoroacetate Ethyl α-bromoisobutyrate Glycine ethyl ester hydrochloride Isoamyl cinnamate Isobutyl acetate Isobutyl cinnamate Isopropenyl acetate Methyl (R)-(+)-3-bromo-2methylpropionate Methyl (S)-(+)-3-hydroxy-2methylpropionate Methyl (R)-(−)-3-hydroxy-2methylpropionate Methyl (S)-(+)-mandelate Methyl (R)-(−)-mandelate Methyl (R)-3-hydroxybutyrate Methyl (S)-(+)-3-hydroxybutyrate Methyl 2-chloro-3hydroxypropionate Methyl 2-hydroxyisobutyrate Methyl 2,2-dimethyl-3hydroxypropionate
Methyl glycolate Methyl propionate Methyl pyruvate Methyl trans-cinnamate Phenethyl cinnamate Phenyl acetate Propyl acetate tert-Butyl 3-hydroxypropionate tert-Butyl cinnamate Triacetin (C 2:0) Tripropionin (C 3:0)
72. Tributyrin (C 4:0) 73. Tricaproin (C 6:0) 74. Tricaprylin (C 8:0) 75. 76. 77. 78.
Tricaprin (C 10:0) Trilinolein (C 18:2) Vinyl acetate Vinyl benzoate
79. Vinyl butyrate 80. Vinyl cinnamate 81. Vinyl crotonate
53. Methyl 3-bromopropionate
82. Vinyl decanoate
54. 55. 56. 57. 58.
83. 84. 85. 86.
Methyl 4-(hydroxymethyl)benzoate Methyl acetoacetate Methyl benzoate Methyl bromoacetate Methyl butyrate
Vinyl laurate Vinyl methacrylate Vinyl pivalate Vinyl propionate
S10
Table S2. Molecular weight characteristics of the reaction products of enzymatic hydrolysis of solid PLA18 determined by GPC. PLA substrate peak
PLA product peaks
Treatment
Mpa
Mnb
PDIc
Mp
Mn
PDI
Mp
Mn
PDI
RPA1511
29,448
17,458
2.34
250
186
1.95
-
-
-
Blank
29,076
17,381
2.35
-
-
-
-
-
-
ABO2449
29,690
17,799
2.29
528
374
1.30
66
72
1.13
Blank+detergent
29,318
15,396
2.58
480
268
1.65
60
58
1.03
a
Mp: molecular weight of the highest peak Mn: number average molecular weight c PDI: polydispersity index, Mw.Mn-1 b
S11
Table S3. LC-MS analysis of oligomeric composition of different lactic acid species released by (A) RPA1511 and (B) ABO2449 from PLA10 substrate.
A (RPA1511) Oligomeric State 2 3 4 5 6 7 8 9 10
Chemical Formula C6H10O5 C9H14O7 C12H18O9 C15H22O11 C18H26O13 C21H30O15 C24H34O17 C27H38O19 C30H42O21
m/z Theoretical Observed 161.0455 161.0429 233.0667 233.0642 305.0878 305.0851 377.1089 377.1057 449.1301 449.1259 521.1512 521.1463 593.1723 593.1674 665.1935 665.1882 737.2146 737.2087
Δ ppm 16.1 10.7 8.8 8.5 9.4 9.4 8.3 8.0 8.0
Signal Intensity 2.0E+08 1.1E+07 4.2E+07 5.1E+07 5.4E+07 3.0E+07 2.7E+07 1.2E+07 8.0E+06
Chemical Formula C6H10O5 C9H14O7 C12H18O9 C15H22O11 C18H26O13 C21H30O15 C24H34O17 C27H38O19 C30H42O21
m/z Theoretical Observed 161.0455 161.0429 233.0667 ND* 305.0878 ND 377.1089 ND 449.1301 ND 521.1512 ND 593.1723 ND 665.1935 ND 737.2146 ND
Δ ppm 16.1 ND ND ND ND ND ND ND ND
Signal Intensity 1.3E+08 ND ND ND ND ND ND ND ND
B (ABO2449) Oligomeric State 2 3 4 5 6 7 8 9 10
*ND = not detectable
S12
Table S4. Kinetic parameters of the wild-type (WT) and mutant ABO2449 with various substrates
Protein
kcat/Km
Km (mM)
kcat (s )
α-Naphthyl acetate
0.24 ± 0.02
195.10 ± 4.36
0.81
α-Naphthyl propionate
0.63 ± 0.09
354.90 ± 19.65
0.56
pNP-acetate
0.96 ± 0.13
351.30 ± 25.43
0.37
pNP-butyrate
0.57 ± 0.03
365.40 ± 8.69
0.64
pNP-valerate
0.44 ± 0.03
304.50 ± 8.38
0.69
Ethyl iodoacetate
13.03 ± 0.45
16.06 ± 0.33
0.0012
Tributyrin
0.25 ± 0.04
20.05 ± 1.08
0.080
Phenyl acetate
8.50 ± 0.45
8.40 ± 0.24
0.0010
Vinyl laurate
0.12 ± 0.01
3.81 ± 0.06
0.032
L32A
α-Naphthyl acetate
0.25 ± 0.02
27.21 ± 0.80
0.11
S33A
α-Naphthyl acetate
0.48 ± 0.15
37.60 ± 3.83
0.078
F38A
α-Naphthyl acetate
0.50 ± 0.05
90.29 ± 3.52
0.18
M144A
α-Naphthyl acetate
1.35 ± 0.24
127.70 ± 12.31
0.095
L152A
α-Naphthyl acetate
0.22 ± 0.02
40.58 ± 1.09
0.18
L163A
α-Naphthyl acetate
0.40 ± 0.03
94.95 ± 2.09
0.24
M183A
α-Naphthyl acetate
0.51 ± 0.07
153.40 ± 7.80
0.30
F219A
α-Naphthyl acetate
0.10 ± 0.03
31.42 ± 1.82
0.31
L249A
α-Naphthyl acetate
0.41 ± 0.09
12.30 ± 0.97
0.030
P302R
α-Naphthyl acetate
0.23 ± 0.02
117.20 ± 2.51
0.51
WT
Variable substrate
-1
(s-1.µM-1)
S13
Table S5. Crystallographic data collection and model refinement statics. Values for the highestresolution shell are shown in parentheses.
Parameters
RPA1511
Beamline Data collection
SBC-19ID
Space group
I222
Cell dimensions a, b, c (Å)
80 87.4 93.7
Wavelength (Å)
0.9794
Resolution range (Å)
32 - 2.20 (2.28 - 2.20)
Unique reflections
16885
Rmerge
0.077
I/σI
15.2 (1.7)
Completeness (%)
99.3 (98.1)
Refinement Total reflections
63870
Rwork
0.181 (0.231)
Rfree
0.221 (0.329)
Number of non-hydrogen atoms
2237
protein
2146
ligands
22
water
69
Rmsd Bond lengths (Å)
0.005
Bond angles (°)
0.85
Average B-factors protein
43.5
ligands
72.5
water
42.3
Ramachandran favored/outliers (%)
98/0
Clashscore Protein residues
0.46 287
PDB accession code
4PSU
S14
