Supporting Information A Heme Peroxidase with a Functional Role as ...
Supporting Information A Heme Peroxidase with a Functional Role as an L-Tyrosine Hydroxylase in the Biosynthesis of Anthramycin Katherine L. Connor†, Keri L. Colabroy§ and Barbara Gerratana†* †
Department of Chemistry and Biochemistry, University of Maryland, MD 20742, USA § Department of Chemistry, Muhlenberg College, Allentown, PA 18014, USA
*Address Correspondence to: Barbara Gerratana, Department of Chemistry and Biochemistry, Bldg 091, University of Maryland, College Park, MD 20742, USA. Telephone: 1 (301) 405-3949; Fax: 1 (301) 314-9121; E-mail: [email protected]
The following information includes 13 additional figures as supplementary data. The methods used in the acquisition of these data are described in the Experimental Procedures section of the manuscript. The content of the supplementary data is related to the manuscript as follows: 1) Supplemental Figures Figure S1 Figure S2 Figure S3-S6 Figure S7-S9 Figure S10- S13
Multiple sequence alignment of Orf13 with homologs Purification of Orf13 Spectra for cofactor identification and occupancy assessment Catalytic assessment and steady state kinetics Evaluation of substrate order and substrate specificity
2) Reference
1
1. SUPPLEMENTAL FIGURES
Figure S1. Multiple sequence alignment of the putative tyrosine hydroxylases Orf13 (S. refuineus; Accession No. ABW71844), TomI (S. achromogenes; Accession No. ACN39022), SibU (S. sibiricum; Accession No. ACN39744), LmbB2 (S. lincolnesis; Accession No. CAA55748), HrmE (S. griseoflavus; Accession No. AEH41783) and AMED_5527 (Amycolatopsis mediterranei; Accession No. YP_003767687) using ClustalW2. Highly conserved residues with potential catalytic relevance are marked by a red diamond (▼).
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Figure S2. SDS-PAGE of Orf13 purification with oxidative protection during gel filtration using (A) 10 mM Imidazole (B) 50 mM DTT or (C) without oxidative protection. Lane 1: un-induced pET24a/orf13 in BL21(DE3) E. coli, lane 2: induced Orf13 (MW 33.6 kDa), lane 3: cell free extract, lane 4: Orf13 pooled fractions after Ni-Sepharose chromatography (Q-Sepharose with 50 mM DTT; Fig. S2B), lane 5: Orf13 pooled fractions after S-200 HR gel filtration chromatography.
Figure S3. UV-visible absorption spectrum of Orf13 containing 75% heme b and 25% protoporphyrin IX (PPIX) before (solid line) and after (dashed line) buffer exchange to remove DTT (50 mM) from the storage buffer. Auto-oxidation of ferrous heme-iron to ferric heme-iron was observed by a shift in the Soret band position from 420 nm to 404 nm, respectively.
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Figure S4. A. ESI-MS of peak (TR 42 min, Fig. 3) collected from HPLC of the supernatant from denatured Orf13. The precursor ion of 616.3 m/z is the same m/z observed in the heme b standard sample collected by HPLC at 42 minutes. Heme b is 616 amu and the precursor ion m/z at exactly its molecular weight is due to a radical porphyrin cation (1). B. ESI-MS/MS fragmentation of the precursor ion at 616.3 m/z generated a product ion peak at 557 m/z. This mass is 59 amu less than the precursor ion and corresponds to the lost of –CH2COOH from one of the propanoic acid groups of heme b (2). The same fragmentation pattern was observed with the heme b standard sample.
Figure S5. UV-visible absorption spectrum of Orf13 containing 50% heme b and 50% protoporphyrin IX (PPIX) before (solid line) and after (dashed line) reduction with sodium dithionite. A single Soret band at 404 nm observed with ferric-heme iron splits into two distinct 4
Soret bands at 406 nm and 431 nm after reduction with dithionite. The Soret band species at 406 nm is PPIX and was confirmed by HPLC-MS and MS/MS analysis.
Figure S6. A. ESI-MS of peak (TR 44 min, Figure 3) collected from HPLC of the denatured Orf13 sample. The singly charged precursor ion m/z 563.5 is the same precursor ion m/z observed with PPIX standard sample collected from HPLC at 42 minutes and is in agreement with the MW of PPIX. B. The ESI-MS/MS fragmentation of m/z 563.5 precursor ion generated a predominant product ion peak at 504.3 m/z. This mass is 59 amu less than the precursor ion and corresponds to the lost of –CH2COOH from one of the propanoic acid groups of the porphyrin ring. The same fragmentation pattern was observed with the PPIX standard sample.
Figure S7. HPLC-FLD chromatograms of Orf13 activity assay in the presence of hydrogen peroxide. Final assay conditions: 2.5 µM Orf13 (76% heme b occupancy), 1 mM L-tyrosine and 0.1 mM H2O2 in 50 mM sodium phosphate (pH 8.0). The reaction was carried out at 37 °C for 15 minutes. L-DOPA production was observed by an increase of the peak at 4.65 minutes. Average retention times of L-DOPA and L-tyrosine standards are 4.5 and 5.5 minutes, respectively. 5
Figure S8. Michaelis-Menten curves of L-tyrosine hydroxylation by Orf13 for the hydrogen peroxide dependent reaction; (A) fixed variable concentrations of L-tyrosine at a fixed hydrogen peroxide (500 µM) concentration, (B) fixed variable concentrations of hydrogen peroxide at a fixed L-tyrosine (2 mM) concentration. Concentrations greater than 1 mM hydrogen peroxide affected the stability of the colorimetric reagents and degraded L-DOPA, the product detected in the assay. Double reciprocal analysis of hydrogen peroxide at fixed concentrations of L-tyrosine (5 mM, 2 mM and 0.45 mM) produced similar steady state rate constants within error of the original steady state analysis at 2 mM L-tyrosine. Assay conditions: Orf13 was pre-incubated for 5 minutes at 37 °C with L-tyrosine followed by addition of hydrogen peroxide to initiate the reaction. The reaction was carried out for 4 minutes at 37 °C. L-DOPA formation was measured using the L-DOPA colorimetric assay.
Figure S9. HPLC-FLD chromatograms of L-tyrosine hydroxylation by Orf13 in the presence Lascorbate (A) or dihydroxyfumaric acid (B). Average retention times of L-DOPA and L-tyrosine standards are 4.5 and 5.5 minutes, respectively. 6
Figure S10. Progress curves of the L-tyrosine hydroxylation reaction by Orf13 under different substrate pre-incubation conditions. Assay conditions: 0.5 µM Orf13, 1 mM L-tyrosine, and 400 µM hydrogen peroxide in 100 mM sodium phosphate (pH 8.0) at 37 °C. Orf13 (75% heme b) was pre-incubated 5 minutes at 37 °C with L-tyrosine (●), hydrogen peroxide (♦) or alone (▼). Reactions were initiated by addition of the substrate(s) not included during pre-incubation. LDOPA formation was measured by the L-DOPA colorimetric assay. Data interpolation is shown beyond the time range for initial rate measurements.
Figure S11. Chemical structures of L-tyrosine and substrate analogues used to evaluate the substrate specificity of the aromatic hydroxylation reaction by Orf13.
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Figure S12. Visible absorption spectra of catechol-nitrite complexes for catechol product formation by the aromatic hydroxylation of L-tyrosine and phenol substrate analogues by Orf13 in the presence of hydrogen peroxide. Final assay conditions: 1.5 µM Orf13 (50% heme b occupancy), 5 mM L-tyrosine or substrate analogue, 500 µM H2O2 in 100 mM sodium phosphate (pH 8.0). All reactions were performed at 37 °C, quenched at 2 minutes and underwent the colorimetric work described in Experimental Procedures. The substrate analogue 3-(4hydroxyphenyl) propanoic acid is abbreviated as 4-HPPPA.
Figure S13 – Progress curves of the hydroxylation of L-tyrosine and aromatic substrate analogues of DL-m-tyrosine, tyramine, 3-(4-hydroxyphenyl) propanoic acid and p-cresol by Orf13. Assay conditions: 1.5 µM Orf13 (50% heme b occupancy), 5 mM L-tyrosine or substrate 8
analog, 250 µM hydrogen peroxide in 100 mM sodium phosphate (pH 8.0) at room temperature. Orf13 was pre-incubated for 5 minutes with L-tyrosine or the substrate analog at room temperature. The reaction was initiated by addition of hydrogen peroxide and catechol product formation was measured using the L-DOPA colorimetric assay with the respective catechol product standard curve.
2. REFERENCE 1.
2.
Whiteaker, J. R., Fenselau, C. C., Fetterolf, D., Steele, D., and Wilson, D. (2004) Quantitative Determination of Heme for Forensic Characterization of Bacillus Spores Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry, Anal. Chem. 76, 2836-2841. Wang, Y., Gatti, P., Sadilek, M., Scott, C. R., Turecek, F., and Gelb, M. H. (2008) Direct Assay of Enzymes in Heme Biosynthesis for the Detection of Porphyrias by Tandem Mass Spectrometry. Uroporphyrinogen Decarboxylase and Coproporphyrinogen III Oxidase, Anal. Chem. 80, 2599-2605.
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Department of Chemistry and Biochemistry, University of Maryland, MD 20742, USA § Department of Chemistry, Muhlenberg College, Allentown, PA 18014, USA
*Address Correspondence to: Barbara Gerratana, Department of Chemistry and Biochemistry, Bldg 091, University of Maryland, College Park, MD 20742, USA. Telephone: 1 (301) 405-3949; Fax: 1 (301) 314-9121; E-mail: [email protected]
The following information includes 13 additional figures as supplementary data. The methods used in the acquisition of these data are described in the Experimental Procedures section of the manuscript. The content of the supplementary data is related to the manuscript as follows: 1) Supplemental Figures Figure S1 Figure S2 Figure S3-S6 Figure S7-S9 Figure S10- S13
Multiple sequence alignment of Orf13 with homologs Purification of Orf13 Spectra for cofactor identification and occupancy assessment Catalytic assessment and steady state kinetics Evaluation of substrate order and substrate specificity
2) Reference
1
1. SUPPLEMENTAL FIGURES
Figure S1. Multiple sequence alignment of the putative tyrosine hydroxylases Orf13 (S. refuineus; Accession No. ABW71844), TomI (S. achromogenes; Accession No. ACN39022), SibU (S. sibiricum; Accession No. ACN39744), LmbB2 (S. lincolnesis; Accession No. CAA55748), HrmE (S. griseoflavus; Accession No. AEH41783) and AMED_5527 (Amycolatopsis mediterranei; Accession No. YP_003767687) using ClustalW2. Highly conserved residues with potential catalytic relevance are marked by a red diamond (▼).
2
Figure S2. SDS-PAGE of Orf13 purification with oxidative protection during gel filtration using (A) 10 mM Imidazole (B) 50 mM DTT or (C) without oxidative protection. Lane 1: un-induced pET24a/orf13 in BL21(DE3) E. coli, lane 2: induced Orf13 (MW 33.6 kDa), lane 3: cell free extract, lane 4: Orf13 pooled fractions after Ni-Sepharose chromatography (Q-Sepharose with 50 mM DTT; Fig. S2B), lane 5: Orf13 pooled fractions after S-200 HR gel filtration chromatography.
Figure S3. UV-visible absorption spectrum of Orf13 containing 75% heme b and 25% protoporphyrin IX (PPIX) before (solid line) and after (dashed line) buffer exchange to remove DTT (50 mM) from the storage buffer. Auto-oxidation of ferrous heme-iron to ferric heme-iron was observed by a shift in the Soret band position from 420 nm to 404 nm, respectively.
3
Figure S4. A. ESI-MS of peak (TR 42 min, Fig. 3) collected from HPLC of the supernatant from denatured Orf13. The precursor ion of 616.3 m/z is the same m/z observed in the heme b standard sample collected by HPLC at 42 minutes. Heme b is 616 amu and the precursor ion m/z at exactly its molecular weight is due to a radical porphyrin cation (1). B. ESI-MS/MS fragmentation of the precursor ion at 616.3 m/z generated a product ion peak at 557 m/z. This mass is 59 amu less than the precursor ion and corresponds to the lost of –CH2COOH from one of the propanoic acid groups of heme b (2). The same fragmentation pattern was observed with the heme b standard sample.
Figure S5. UV-visible absorption spectrum of Orf13 containing 50% heme b and 50% protoporphyrin IX (PPIX) before (solid line) and after (dashed line) reduction with sodium dithionite. A single Soret band at 404 nm observed with ferric-heme iron splits into two distinct 4
Soret bands at 406 nm and 431 nm after reduction with dithionite. The Soret band species at 406 nm is PPIX and was confirmed by HPLC-MS and MS/MS analysis.
Figure S6. A. ESI-MS of peak (TR 44 min, Figure 3) collected from HPLC of the denatured Orf13 sample. The singly charged precursor ion m/z 563.5 is the same precursor ion m/z observed with PPIX standard sample collected from HPLC at 42 minutes and is in agreement with the MW of PPIX. B. The ESI-MS/MS fragmentation of m/z 563.5 precursor ion generated a predominant product ion peak at 504.3 m/z. This mass is 59 amu less than the precursor ion and corresponds to the lost of –CH2COOH from one of the propanoic acid groups of the porphyrin ring. The same fragmentation pattern was observed with the PPIX standard sample.
Figure S7. HPLC-FLD chromatograms of Orf13 activity assay in the presence of hydrogen peroxide. Final assay conditions: 2.5 µM Orf13 (76% heme b occupancy), 1 mM L-tyrosine and 0.1 mM H2O2 in 50 mM sodium phosphate (pH 8.0). The reaction was carried out at 37 °C for 15 minutes. L-DOPA production was observed by an increase of the peak at 4.65 minutes. Average retention times of L-DOPA and L-tyrosine standards are 4.5 and 5.5 minutes, respectively. 5
Figure S8. Michaelis-Menten curves of L-tyrosine hydroxylation by Orf13 for the hydrogen peroxide dependent reaction; (A) fixed variable concentrations of L-tyrosine at a fixed hydrogen peroxide (500 µM) concentration, (B) fixed variable concentrations of hydrogen peroxide at a fixed L-tyrosine (2 mM) concentration. Concentrations greater than 1 mM hydrogen peroxide affected the stability of the colorimetric reagents and degraded L-DOPA, the product detected in the assay. Double reciprocal analysis of hydrogen peroxide at fixed concentrations of L-tyrosine (5 mM, 2 mM and 0.45 mM) produced similar steady state rate constants within error of the original steady state analysis at 2 mM L-tyrosine. Assay conditions: Orf13 was pre-incubated for 5 minutes at 37 °C with L-tyrosine followed by addition of hydrogen peroxide to initiate the reaction. The reaction was carried out for 4 minutes at 37 °C. L-DOPA formation was measured using the L-DOPA colorimetric assay.
Figure S9. HPLC-FLD chromatograms of L-tyrosine hydroxylation by Orf13 in the presence Lascorbate (A) or dihydroxyfumaric acid (B). Average retention times of L-DOPA and L-tyrosine standards are 4.5 and 5.5 minutes, respectively. 6
Figure S10. Progress curves of the L-tyrosine hydroxylation reaction by Orf13 under different substrate pre-incubation conditions. Assay conditions: 0.5 µM Orf13, 1 mM L-tyrosine, and 400 µM hydrogen peroxide in 100 mM sodium phosphate (pH 8.0) at 37 °C. Orf13 (75% heme b) was pre-incubated 5 minutes at 37 °C with L-tyrosine (●), hydrogen peroxide (♦) or alone (▼). Reactions were initiated by addition of the substrate(s) not included during pre-incubation. LDOPA formation was measured by the L-DOPA colorimetric assay. Data interpolation is shown beyond the time range for initial rate measurements.
Figure S11. Chemical structures of L-tyrosine and substrate analogues used to evaluate the substrate specificity of the aromatic hydroxylation reaction by Orf13.
7
Figure S12. Visible absorption spectra of catechol-nitrite complexes for catechol product formation by the aromatic hydroxylation of L-tyrosine and phenol substrate analogues by Orf13 in the presence of hydrogen peroxide. Final assay conditions: 1.5 µM Orf13 (50% heme b occupancy), 5 mM L-tyrosine or substrate analogue, 500 µM H2O2 in 100 mM sodium phosphate (pH 8.0). All reactions were performed at 37 °C, quenched at 2 minutes and underwent the colorimetric work described in Experimental Procedures. The substrate analogue 3-(4hydroxyphenyl) propanoic acid is abbreviated as 4-HPPPA.
Figure S13 – Progress curves of the hydroxylation of L-tyrosine and aromatic substrate analogues of DL-m-tyrosine, tyramine, 3-(4-hydroxyphenyl) propanoic acid and p-cresol by Orf13. Assay conditions: 1.5 µM Orf13 (50% heme b occupancy), 5 mM L-tyrosine or substrate 8
analog, 250 µM hydrogen peroxide in 100 mM sodium phosphate (pH 8.0) at room temperature. Orf13 was pre-incubated for 5 minutes with L-tyrosine or the substrate analog at room temperature. The reaction was initiated by addition of hydrogen peroxide and catechol product formation was measured using the L-DOPA colorimetric assay with the respective catechol product standard curve.
2. REFERENCE 1.
2.
Whiteaker, J. R., Fenselau, C. C., Fetterolf, D., Steele, D., and Wilson, D. (2004) Quantitative Determination of Heme for Forensic Characterization of Bacillus Spores Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Spectrometry, Anal. Chem. 76, 2836-2841. Wang, Y., Gatti, P., Sadilek, M., Scott, C. R., Turecek, F., and Gelb, M. H. (2008) Direct Assay of Enzymes in Heme Biosynthesis for the Detection of Porphyrias by Tandem Mass Spectrometry. Uroporphyrinogen Decarboxylase and Coproporphyrinogen III Oxidase, Anal. Chem. 80, 2599-2605.
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