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Binding modes of CYP106A2 redox partners determine differences in progesterone hydroxylation product patterns.

ABSTRACT: Natural redox partners of bacterial cytochrome P450s (P450s) are mostly unknown. Therefore, substrate conversions are performed with heterologous redox partners; in the case of CYP106A2 from Bacillus megaterium ATCC 13368, bovine adrenodoxin (Adx) and adrenodoxin reductase (AdR). Our aim was to optimize the redox system for CYP106A2 for improved product formation by testing 11 different combinations of redox partners. We found that electron transfer protein 1(516-618) showed the highest yield of the main product, 15?-hydroxyprogesterone, and, furthermore, produced a reduced amount of unwanted polyhydroxylated side products. Molecular protein-protein docking indicated that this is caused by subtle structural changes leading to alternative binding modes of both redox enzymes. Stopped-flow measurements analyzing the CYP106A2 reduction and showing substantial differences in the apparent rate constants supported this conclusion. The study provides for the first time to our knowledge rational explanations for differences in product patterns of a cytochrome P450 caused by difference in the binding mode of the redox partners.

SUBMITTER: Sagadin T

PROVIDER: S-EPMC6123783 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Binding modes of CYP106A2 redox partners determine differences in progesterone hydroxylation product patterns.

Sagadin Tanja T Riehm Jan L JL Milhim Mohammed M Hutter Michael C MC Bernhardt Rita R

Communications biology 20180730

Natural redox partners of bacterial cytochrome P450s (P450s) are mostly unknown. Therefore, substrate conversions are performed with heterologous redox partners; in the case of CYP106A2 from <i>Bacillus megaterium</i> ATCC 13368, bovine adrenodoxin (Adx) and adrenodoxin reductase (AdR). Our aim was to optimize the redox system for CYP106A2 for improved product formation by testing 11 different combinations of redox partners. We found that electron transfer protein 1(516-618) showed the highest y ...[more]

PMID: 30271979

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Project description:BackgroundThe importance of understanding the detailed mechanism of cysteine biosynthesis in bacteria is underscored by the fact that cysteine is the only sulfur donor for all cellular components containing reduced sulfur. O-acetylserine sulfhydrylase (OASS) catalyzes this crucial last step in the cysteine biosynthesis and has been recognized as an important gene for the survival and virulence of pathogenic bacteria. Structural and kinetic studies have contributed to the understanding of mechanistic aspects of OASS, but details of ligand recognition features of OASS are not available. In the absence of any detailed study on the energetics of ligand binding, we have studied the thermodynamics of OASS from Salmonella typhimurium (StOASS), Haemophilus influenzae (HiOASS), and Mycobacterium tuberculosis (MtOASS) binding to their substrate O-acetylserine (OAS), substrate analogue (methionine), and product (cysteine).ResultsLigand binding properties of three OASS enzymes are studied under defined solution conditions. Both substrate and product binding is an exothermic reaction, but their thermodynamic signatures are very different. Cysteine binding to OASS shows that both enthalpy and entropy contribute significantly to the binding free energy at all temperatures (10-30°C) examined. The analyses of interaction between OASS with OAS (substrate) or methionine (substrate analogue) revealed a completely different mode of binding. Binding of both OAS and methionine to OASS is dominated by a favorable entropy change, with minor contribution from enthalpy change (?H(St-Met) = -1.5 ± 0.1 kJ/mol; T?S(St-Met) = 8.2 kJ/mol) at 20°C. Our salt dependent ligand binding studies indicate that methionine binding affinity is more sensitive to [NaCl] as compared to cysteine affinity.ConclusionsWe show that OASS from three different pathogenic bacteria bind substrate and product through two different mechanisms. Results indicate that predominantly entropy driven methionine binding is not mediated through classical hydrophobic binding, instead, may involve desolvation of the polar active site. We speculate that OASS in general, may exhibit two different binding mechanisms for recognizing substrates and products.

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Binding modes of CYP106A2 redox partners determine differences in progesterone hydroxylation product patterns.

Publications

Binding modes of CYP106A2 redox partners determine differences in progesterone hydroxylation product patterns.

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