Engineering C–C Bond Cleavage Activity into a P450 Monooxygenase Enzyme

Abstract

The cytochrome P450 (CYP) superfamily of heme monooxygenases has demonstrated ability to facilitate hydroxylation, desaturation, sulfoxidation, epoxidation, heteroatom dealkylation, and carbon−carbon bond formation and cleavage (lyase) reactions. Seeking to study the carbon− carbon cleavage reaction of α-hydroxy ketones in mechanistic detail using a microbial P450, we synthesized α-hydroxy ketone probes based on the physiological substrate for a well-characterized benzoic acid metabolizing P450, CYP199A4. After observing low activity with wild-type CYP199A4, subsequent assays with an F182L mutant demonstrated enzyme-dependent C−C bond cleavage toward one of the α-hydroxy ketones. This C−C cleavage reaction was subject to an inverse kinetic solvent isotope effect analogous to that observed in the lyase activity of the human P450 CYP17A1, suggesting the involvement of a species earlier than Compound I in the catalytic cycle. Co-crystallization of F182L-CYP199A4 with this α-hydroxy ketone showed that the substrate bound in the active site with a preference for the (S)-enantiomer in a position which could mimic the topology of the lyase reaction in CYP17A1. Molecular dynamics simulations with an oxy-ferrous model of CYP199A4 revealed a displacement of the substrate to allow for oxygen binding and the formation of the lyase transition state proposed for CYP17A1. This demonstration that a correctly positioned α-hydroxy ketone substrate can realize lyase activity with an unusual inverse solvent isotope effect in an engineered microbial system opens the door for further detailed biophysical and structural characterization of CYP catalytic intermediates.

Simplification of Corticosteroids Biosynthetic Pathway by Engineering P450BM3

“…VD-BM3, featuring an engineered substrate access channel, has demonstrated a high efficiency in the hydroxylation conversion rate of PG but with poor regioselectivity. An effective strategy to enhance regioselectivity is to rationally engineer residues near the substrate to bring the catalytic group of the substrate closer to the enzyme’s active site. ,, The docking results of PG and VD-BM3 complexes revealed that S72 and L437 were located within a 4-Å range of the AB ring of PG and hence were selected for saturation mutagenesis to recalibrate the distances of the D ring of PG toward the heme center (Figure A, B). S72Q -VD-BM3 was capable of producing 97% a at an 80% conversion rate (Table ), while S72H-VD-BM3 produced 97% d at a 30% conversion rate.…”

Selective α-Hydroxyketone Formation and Subsequent C–C Bond Cleavage by Cytochrome P450 Monooxygenase Enzymes

“…Both substrates bound with significantly lower affinity than 4- n -propylbenzoic acid ( ≥ 95% high-spin, 0.54 μM) but with a higher affinity than 4-acetylbenzoic acid (140 μM, Table ). , …”

“…This also has an absorbance maximum at 379 nm but this was not used to determine the binding affinity (Figure ). f Despite the red shift in the Soret band, a type I difference spectrum was observed …”

Fungal P450 Deconstructs the 2,5-Diazabicyclo[2.2.2]octane Ring En Route to the Complete Biosynthesis of 21R-Citrinadin A

“…They usually mediate a typical monooxygenation reaction via the canonical “oxygen rebound” mechanism to insert a single oxygen atom into the C–H, CC, N–H, or S–H bonds . With an increasing number of characterized P450s, the substrate radical generated by the high active oxoiron­(IV) species can initiate more complex oxidation such as C–C bond formation and cleavage. , Notably, the nucleophilic attack of the heme-Fe III hydroperoxide complex contributes to the realization of some uncommon reactions, such as epoxidation catalyzed by Fma-P450 , and C–C bond cleavage mediated by Baeyer–Villiger rearrangement catalyzed by CYP 17, JCM 1, and SdnB . Accordingly, we propose that the CtdY catalysis may proceed through a mechanism in which the carbonyl of the amide bond in 20 might be attacked by heme-Fe III hydroperoxide species resulting in the cleavage of the C–N bond.…”