Enabling Peroxygenase Activity in Cytochrome P450 Monooxygenases by Engineering Hydrogen Peroxide Tunnels

Abstract

Given prominent physicochemical similarities between H2O2 and water, we report a new strategy for promoting the peroxygenase activity of P450 enzymes by engineering their water tunnels to facilitate H2O2 access to the heme center buried therein. Specifically, the H2O2-driven activities of two native NADH-dependent P450 enzymes (CYP199A4 and CYP153A M.aq ) increase significantly (by >183-fold and >15-fold, respectively). Additionally, the amount of H2O2 required for an artificial P450 peroxygenase facilitated by a dual-functional small molecule to obtain the desired product is reduced by 95%–97.5% (with ∼95% coupling efficiency). Structural analysis suggests that mutating the residue at the bottleneck of the water tunnel may open a second pathway for H2O2 to flow to the heme center (in addition to the natural substrate tunnel). This study highlights a promising, generalizable strategy whereby P450 monooxygenases can be modified to adopt peroxygenase activity through H2O2 tunnel engineering, thus broadening the application scope of P450s in synthetic chemistry and synthetic biology.

Biosynthesis of 3-Hydroxyphloretin Using Rational Design of 4-Hydroxyphenylacetate 3-Monooxygenase

“…After the analysis of molecular docking and sequence alignment of HpaB, residues F292, F297, and F301 and loop 209–212 were selected for SM to improve the yield of Pa HpaB to 3-hydroxyphloretin. In addition, ferulic acid and phloretin have similar chemical structures (Figure S1); in the previous study in our laboratory, mutants L213A and Q376N with a higher conversion efficiency to ferulic acid obtained through the substrate channel strategy would also be used in the hydroxylation of phloretin.…”

Natural alternative heme-environments allow efficient peroxygenase activity by cytochrome P450 monooxygenases

“…Recently it was demonstrated that engineering H 2 O tunnels to H 2 O 2 tunnels promote peroxygenase activity in CYPs. 48 As Ssca CYP was crystallized in the absence of substrate, we assume the second wider channel to be the substrate and H 2 O 2 channel.…”

Accurate Computation of Aqueous pKas of Biologically Relevant Organic Acids: Overcoming the Challenges Posed by Multiple Conformers, Tautomeric Equilibria, and Disparate Functional Groups with the Fully Black-Box pK-Yay Method

“…The reactions of acids with bases permeate throughout chemistry. , The resultant acid–base chemistry is very important in areas as diverse as enzyme driven biochemistry, − atmospheric chemistry, − materials science, − basic organic chemistry, − organometallic chemistry, − and nanochemistry. − Owing to both its ubiquity and significance, students learn about the concept of the acid dissociation constant ( K a ) and its logarithmic analogue p K a in various solvents, right from high school . Yet, both experimentally determining p K a values − and computing them via simulations continue to be actively pursued as key areas of contemporary research in chemistry. − …”