Structure-Guided Engineering of CYP109B4: From Monooxygenase to Peroxygenase for Selective Steroid Hydroxylation
Xiaodong Zhang1, Yongchao Wang2, Xiaoqing Jiang1
1School of life science, State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei University, #368 Youyi Road, Wuhan 430062, P. R. China.
Abstract:
Cytochrome P450 CYP109B4 is a characterized 16β-steroid hydroxylase with significant potential in steroid drug biosynthesis, but its application is limited by reliance on costly coenzymes and complex electron transfer processes. To address this, we constructed a hydrogen peroxide (H2O2)-driven catalytic system through structure-guided engineering. Screening a combinatorial variant library identified the L240V/S387F double mutant, which exhibited emergent peroxygenase activity (baseline TTN = 11). Using focused rational iterative site-specific mutagenesis (FRISM), we obtained optimized variant B4-Pm9 exhibiting a 4-fold higher total turnover number (TTN = 44). Coupling with an alcohol dehydrogenase (Aldo)-based H2O2 regeneration system further enhanced catalytic efficiency, achieving a TTN of 348 (31-fold improvement over L240V/S387F mutant). Computational studies revealed the H2O2 activation mechanism in Pm7 and Pm9. Notably, variant B4-Pm7 demonstrated a novel mechanism of H2O2 cleavage mediated by the "alcohol-hydroxyl" moiety, showcasing a significant breakthrough in the realm of P450 peroxygenase.
More Related Videos
13:05Enzymatic Synthesis of Epoxidized Metabolites of Docosahexaenoic, Eicosapentaenoic, and Arachidonic Acids
Published on: June 28, 2019
09:33Formation of Covalent DNA Adducts by Enzymatically Activated Carcinogens and Drugs In Vitro and Their Determination by 32P-postlabeling
Published on: March 20, 2018
Related Concept Videos
01:17Drug Metabolism: Phase I Reactions
02:44Oxidation of Alkenes: Syn Dihydroxylation with Osmium Tetraoxide
02:17Reduction of Alkenes: Asymmetric Catalytic Hydrogenation
The metal catalyst used can be either heterogeneous or homogeneous. When hydrogenation of an alkene generates a chiral center, a pair of enantiomeric products is expected to form. However, an enantiomeric excess of one of the products can be facilitated using an enantioselective reaction or an...
02:36Regioselectivity and Stereochemistry of Hydroboration
Hydroboration proceeds in a concerted fashion with the attack of borane on the π bond, giving a cyclic four-centered transition state. The –BH2 group is bonded to the less substituted carbon and –H to the more substituted carbon. The concerted nature requires the simultaneous addition of –H and –BH2 across the same face of the alkene giving syn stereochemistry.
01:19Phase I Reactions: Oxidation of Aliphatic and Aromatic Carbon-Containing Systems
Oxidation reactions are fundamental in aromatic carbon-containing systems. An example is the hydroxylation of phenobarbital, a process that transforms it into...
03:08Hydroboration-Oxidation of Alkenes