Investigation of the requirements for efficient and selective cytochrome P450 monooxygenase catalysis across different reactions

Podgorski, Matthew N.; Coleman, Tom; Chao, Rebecca R.; Voss, James J. De; Bruning, John B.; Bell, Stephen

doi:10.1016/j.jinorgbio.2019.110913

Cited by 25 publications

(69 citation statements)

References 39 publications

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“… c The slow rate of the CYP199A4 D251N mutant means that the hydrogen peroxide production could not be determined accurately . The data for the wild-type (WT) enzyme, from Table , are included for comparison. …”

Section: Resultsmentioning

confidence: 98%

“…We have previously shown that the T252A mutant of CYP199A4 (CYP199A4 T252A ) decreased the coupling efficiency of the enzyme turnovers with a concomitant increase in the hydrogen peroxide uncoupling pathway . Although the switch in activity is not as dramatic as that observed with P450cam, it does imply an increase in the levels of the ferric hydroperoxy, Cpd 0, or Fe(III):(H 2 O 2 ) intermediates (Scheme ).…”

Section: Resultsmentioning

confidence: 99%

“… b The leak rate of the mutant enzymes in the absence of the substrate was comparable to the wild-type leak rate (CYP199A4 T252A , 8.7 and CYP199A4 D251N , 7.5 nmol nmol-CYP –1 min –1 ). The data are reported as measured and have not been corrected for the leak rate. c The slow rate of the CYP199A4 D251N mutant means that the hydrogen peroxide production could not be determined accurately . The data for the wild-type (WT) enzyme, from Table , are included for comparison. …”

Section: Resultsmentioning

confidence: 99%

“…Both the crystal structure of CYP199A4 with VBA and the frozen-solution EPR measurements suggest that a coordinated water molecule is retained to a significant extent when the substrate binds, despite the observation of an 80% spin-state shift. While quite unusual, examples of six-coordinate heme centers in the crystal structures of P450s which have a significant high-spin component as measured by UV–vis spectroscopy, have been reported previously, including in, for example, adamantine-bound P450cam . Hydrogen bonding to the distal water of P450 enzymes has been hypothesized to contribute to the ferric low-spin state of the resting form.…”

Section: Discussionmentioning

confidence: 99%

“…c The slow rate of the CYP199A4 D251N mutant means that the hydrogen peroxide production could not be determined accurately. 73 The data for the wild-type (WT) enzyme, from of the CYP199A4 T252A reaction. As with the oxidation of 4methoxybenzoic acid, there was an increase in the peroxide uncoupling pathway in this turnover (T252A, 59%; WT, 2.5%).…”

Section: Acs Catalysismentioning

confidence: 99%

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Understanding the Mechanistic Requirements for Efficient and Stereoselective Alkene Epoxidation by a Cytochrome P450 Enzyme

et al. 2021

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The cytochrome P450 (CYP) family of heme monooxygenase enzymes commonly catalyzes enantioselective hydroxylation and epoxidation reactions. Epoxidation reactions have been hypothesized to proceed via multiple mechanisms involving different reactive intermediates. Here, we use activity, spectroscopic, structural, and molecular dynamics data to investigate the activity and stereoselectivity of 4-vinylbenzoic acid epoxidation by the bacterial enzyme CYP199A4 from Rhodopseudomonas palustris HaA2. The epoxidation of 4-vinylbenzoic acid by CYP199A4 proceeded with high enantioselectivity, giving the (S)-epoxide in 99% ee at an activity of 220 nmol nmol-CYP–1 min–1. Optical and EPR spectroscopy, redox potential measurements, and the crystal structure of 4-vinylbenzoic acid-bound CYP199A4 indicated the partial retention of an aqua ligand at the heme center in the presence of the substrate, providing a justification of the lower activity (∼20%) compared to the oxidative demethylation of 4-methoxybenzoic acid. Mutagenesis at the conserved acid–alcohol pair (D251/T252), which perturbs the generation of the reactive oxygen intermediates, was employed to investigate their role in epoxidation reactions. The T252A mutant increased the rate of turnover of the catalytic cycle, but an elevation in hydrogen peroxide generation via uncoupling resulted in a similar rate of epoxide formation. The activity of epoxidation significantly reduced with the D251N mutant. The chemoselectivity and stereoselectivity of the epoxidation reaction were maintained in the turnovers by these mutants. Overall, there was little evidence that other intermediates, aside from the archetypal reactive ferryl porphyrin cation radical, Compound I, contributed significantly to the epoxidation reaction. The observation of the high selectivity for the (S)-enantiomer was rationalized by molecular dynamics simulations. When the arrangement of the alkene and the active intermediate approached an ideal transition state structure for epoxidation, one face of the alkene was more often exposed to the iron oxo unit.

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Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%