Structure–function analysis for the hydroxylation of Δ4 C21‐steroids by the myxobacterial CYP260B1

Salamanca-Pinzón, S. Giovanna; Khatri, Yogan; Carius, Yvonne; Keller, Lena; Müller, Rolf; Lancaster, C. Roy D.; Bernhardt, Rita

doi:10.1002/1873-3468.12217

Cited by 13 publications

(23 citation statements)

References 43 publications

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“…The crystal structure of CYP260A1 in the space group P3 2 21 was solved at 2.67 Å resolution. As a model, the crystal structure of CYP260B1 from S. cellulosum So ce56 (PDB entry http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5HIW) with a sequence identity of 47% was used for the molecular replacement. The crystal structure of CYP260A1 consisted of four molecules in the asymmetric unit (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…All datasets were indexed, integrated, and scaled using Mosflm and Scala from the CCP4 program suite . The CYP260A1 structure was solved by molecular replacement using the chain A of the CYP260B1 (PDB http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5HIW) with the program Molrep . Model building was performed with the program COOT and refinement using REFMAC5 .…”

Section: Methodsmentioning

confidence: 99%

“…In this regard, bacterial P450s could be the best alternatives. However, there are only few bacterial P450s which can convert steroidal molecules including CYP106A1 , CYP106A2 , CYP125 , CYP142 , CYP154C5 , and CYP260 .…”

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confidence: 99%

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Structural characterization of CYP260A1 from Sorangium cellulosum to investigate the 1α‐hydroxylation of a mineralocorticoid

et al. 2016

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In this study, we report the crystal structure of the cytochrome P450 CYP260A1 (PDB 5LIV) from the myxobacterium Sorangium cellulosum So ce56. In addition, we investigated the hydroxylation of 11-deoxycorticosterone by CYP260A1 by reconstituting the enzyme with the surrogate redox partners adrenodoxin and adrenodoxin reductase. The major product of this steroid conversion was identified as 1a-hydroxy-11-deoxycorticosterone, a novel D4 C-21 steroidal derivative. Furthermore, we docked the substrate into the crystal structure and replaced Ser326, the residue responsible for substrate orientation, with asparagine and observed that the mutant S326N displayed higher activity and selectivity for the formation of 1a-hydroxy-11-deoxycorticosterone compared to the wild-type CYP260A1. Thus, our findings highlight the usefulness of the obtained crystal structure of CYP260A1 in identifying biotechnologically more efficient reactions.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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Structural characterization of CYP260A1 from Sorangium cellulosum to investigate the 1α‐hydroxylation of a mineralocorticoid

et al. 2016

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“…CYPs such as CYP106A1, CYP106A2, CYP109B1, CYP109E1, CYP154C3, CYP154C5, CYP260A1, and CYP260B1, originating from bacterial sources, are known to hydroxylate steroids . CYP154C8 shows high similarity with CYP154C3 (74 %) and CYP154C5 (66 %), both of which are reported to hydroxylate steroids at C16α.…”

Section: Introductionmentioning

confidence: 99%

Effects of Alternative Redox Partners and Oxidizing Agents on CYP154C8 Catalytic Activity and Product Distribution

Dangi

Park

2018

ChemBioChem

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CYP154C8 catalyzes the hydroxylation of diverse steroids, as has previously been demonstrated, by using an NADH-dependent system including putidaredoxin and putidaredoxin reductase as redox partner proteins carrying electrons from NADH. In other reactions, CYP154C8 reconstituted with spinach ferredoxin and NADPH-dependent ferredoxin reductase displayed catalytic activity different from that of the NADH-dependent system. The NADPH-dependent system showed multistep oxidation of progesterone and other substrates including androstenedione, testosterone, and nandrolone. (Diacetoxyiodo)benzene was employed to generate compound I (FeO ), actively supporting the redox reactions catalyzed by CYP154C8. In addition to 16α-hydroxylation, progesterone and 11-oxoprogesterone also underwent hydroxylation at the 6β-position in reactions supported by (diacetoxyiodo)benzene. CYP154C8 was active in the presence of high concentrations (>10 mm) of H O , with optimum conversion surprisingly being achieved at ≈75 mm H O . More importantly, H O tolerance by CYP154C8 was evident in the very low heme oxidation rate constant (K) even at high concentrations of H O . Our results demonstrate that alternative redox partners and oxidizing agents influence the catalytic efficiency and product distribution of a cytochrome P450 enzyme. More importantly, these choices affected the type and selectivity of reaction catalyzed by the P450 enzyme.

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“…The automated docking program autodock (version 4.20) was applied for docking of cinnamaldehyde into the substrate‐free crystal structure of CYP260B1 (PDB http://www.rcsb.org/pdb/search/structidSearch.do?structureId=5HIW) . The Windows version 1.5.6 of autodock Tools was used to compute Kollman charges for the enzyme CYP260B1 and Gasteiger–Marsili charges for the ligands .…”

Section: Methodsmentioning

confidence: 99%

Investigating the roles of T224 and T232 in the oxidation of cinnamaldehyde catalyzed by myxobacterial CYP260B1

et al. 2016

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Although the oxidation of aldehydes to carboxylic acids is mainly catalyzed by aldehyde dehydrogenases in nature, cytochromes P450 are also able to perform such reactions. In this study, we demonstrate the oxidation of cinnamaldehyde to cinnamic acid by the myxobacterial CYP260B1. Following our docking studies of the aldehyde, we generated T224A and T234A mutants of CYP260B1 by site-directed mutagenesis to disrupt the substrate positioning and proton delivery, respectively. Furthermore, we used the kinetic solvent isotope effect on the steady-state turnover of the substrate to investigate the reactive intermediate capable of performing the catalysis. Our results suggest that the aldehyde oxidation occurs via a nucleophilic attack of the ferric peroxoanion.

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Structure–function analysis for the hydroxylation of Δ4 C21‐steroids by the myxobacterial CYP260B1

Cited by 13 publications

References 43 publications

Structural characterization of CYP260A1 from Sorangium cellulosum to investigate the 1α‐hydroxylation of a mineralocorticoid

Structural characterization of CYP260A1 from Sorangium cellulosum to investigate the 1α‐hydroxylation of a mineralocorticoid

Effects of Alternative Redox Partners and Oxidizing Agents on CYP154C8 Catalytic Activity and Product Distribution

Investigating the roles of T224 and T232 in the oxidation of cinnamaldehyde catalyzed by myxobacterial CYP260B1

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