Dataset Information

Substrate-Assisted Hydroxylation and O-Demethylation in the Peroxidase-like Cytochrome P450 Enzyme CYP121.

ABSTRACT: CYP121 is a P450 enzyme from Mycobacterium tuberculosis that catalyzes a C-C coupling reaction between the two aromatic rings on its native substrate cyclo(l-Tyr-l-Tyr) (cYY) to form mycocyclosin, a necessary product for cell survival. Unlike the typical P450 enzymes for hydroxylation, CYP121 is believed to behave like a peroxidase and conduct radical-mediated C-C bond formation. Here, we probe whether the phenolic hydrogen of the substrate is the site of the postulated hydrogen atom abstraction for radical formation. We synthesized a singly O-methylated substrate analogue, cYF-4-OMe, and characterized its interaction with CYP121 by ultraviolet-visible and electron paramagnetic resonance spectroscopies and X-ray crystallography. We found that cYF-4-OMe can function as a substrate of CYP121 using the established assay via the peroxide shunt. Analysis of the enzymatic reaction revealed an O-demethylation of cYF-4-OMe instead of cyclization, yielding cYY and formaldehyde. A hydroxylated substrate, cYF-4-OMeOH, is expected to be the intermediate product, which was trapped and structurally characterized by X-ray crystallography. We further determined that the deformylation reaction of cYF-4-OMeOH proceeds via an alkyl-oxygen rather than aryl-oxygen bond cleavage by the ¹⁸O-labeling studies. Finally, the pH dependence catalytic study on the native substrate and the methoxy analogue further supports the mechanistic understanding that the hydrogen atom abstraction is the critical first oxidation step exerted by a heme-based oxidant during the cyclization reaction of cYY. The switch in catalytic activity reveals the power of CYP121 as a P450 enzyme and provides insight into the peroxidase-like catalytic mechanism.

SUBMITTER: Nguyen RC

PROVIDER: S-EPMC7207044 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Publications

Substrate-Assisted Hydroxylation and <i>O</i>-Demethylation in the Peroxidase-like Cytochrome P450 Enzyme CYP121.

Nguyen Romie C RC Yang Yu Y Wang Yifan Y Davis Ian I Liu Aimin A

ACS catalysis 20191220 2

CYP121 is a P450 enzyme from <i>Mycobacterium tuberculosis</i> that catalyzes a C-C coupling reaction between the two aromatic rings on its native substrate cyclo(l-Tyr-l-Tyr) (cYY) to form mycocyclosin, a necessary product for cell survival. Unlike the typical P450 enzymes for hydroxylation, CYP121 is believed to behave like a peroxidase and conduct radical-mediated C-C bond formation. Here, we probe whether the phenolic hydrogen of the substrate is the site of the postulated hydrogen atom abst ...[more]

PMID: 32391185

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Project description:Our previous study showed that CYP105D7, a substrate-promiscuous P450, catalyzes the hydroxylation of 1-deoxypentalenic acid, diclofenac, naringenin, and compactin. In this study, 14 steroid compounds were screened using recombinant Escherichia coli cells harboring genes encoding CYP105D7 and redox partners (Pdx/Pdr, RhFRED, and FdxH/FprD), and the screening identified steroid A-ring 2?- and D-ring 16?-hydroxylation activity. Wild-type CYP105D7 was able to catalyze the hydroxylation of five steroids (testosterone, progesterone, 4-androstene-3,17-dione, adrenosterone, and cortisone) with low (<10%) conversion rates. Structure-guided site-directed mutagenesis of arginine residues around the substrate entrance and active site showed that the R70A and R190A single mutants and an R70A/R190A double mutant exhibited greatly enhanced conversion rates for steroid hydroxylation. For the conversion of testosterone in particular, the R70A/R190A mutant's k cat/Km values increased 1.35-fold and the in vivo conversion rates increased significantly by almost 9-fold with high regio- and stereoselectivity. Molecular docking analysis revealed that when Arg70 and Arg190 were replaced with alanine, the volume of the substrate access and binding pocket increased 1.08-fold, which might facilitate improvement of the hydroxylation efficiency of steroids.IMPORTANCE Cytochrome P450 monooxygenases (P450s) are able to introduce oxygen atoms into nonreactive hydrocarbon compounds under mild conditions, thereby offering significant advantages compared to chemical catalysts. Promiscuous P450s with broad substrate specificity and reaction diversity have significant potential for applications in various fields, including synthetic biology. The study of the function, molecular mechanisms, and rational engineering of substrate-promiscuous P450s from microbial sources is important to fulfill this potential. Here, we present a microbial substrate-promiscuous P450, CYP105D7, which can catalyze hydroxylation of steroids. The loss of the bulky side chains of Arg70 and Arg190 in the active site and substrate entrance resulted in an up to 9-fold increase in the substrate conversion rate. These findings will support future rational and semirational engineering of P450s for applications as biocatalysts.

Dataset Information

Substrate-Assisted Hydroxylation and O-Demethylation in the Peroxidase-like Cytochrome P450 Enzyme CYP121.

Publications

Substrate-Assisted Hydroxylation and <i>O</i>-Demethylation in the Peroxidase-like Cytochrome P450 Enzyme CYP121.

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