Engineering CYP153A_M.aq to Oxyfunctionalize its Inhibitor Dodecylamine Using a LC/MS Based Rapid Flow Analysis Screening

Abstract

The catalytic space of the P450 monooxygenase CYP153AM.aq was opened from a terminal (ω‐) fatty acid hydroxylase to a catalyst capable of performing ω‐hydroxylation of dodecylamine, which is a potent inhibitor for the wild‐type enzyme. A simple screening method named Rapid‐flow Analysis of Product Peaks (RAPP) was established and applied to measure saturation libraries directly from a 96‐deepwell plate in 36 seconds per sample. The obtained variants are less inhibited by the amine, although concurrently show less affinity towards the acid. Molecular modelling and molecular dynamics simulations showed significant effects of the mutations on the substrate tunnel architectures.

A Career in Catalysis: Bernhard Hauer

“…By engineering access channels of the same enzyme (CYP153AM.aq), Bernhard's group successfully enabled terminal hydroxylation of dodecylamine, an inhibitor of the wild-type enzyme. 198 In that study, it was shown how mutations in channels increased the accessibility of the substrate while at the same time affecting hydrogen-bonding interactions. The new hydrogen-bonding network resulted in the fixation of dodecylamine in the active site in an orientation compatible with terminal hydroxylation (Figure 13).…”

“…M.aqRLT showed a 151-fold improved catalytic efficiency and led to increased protein stiffness concomitant with enhanced binding affinity manifested in a 25-fold reduced K M (Figure ). By engineering access channels of the same enzyme (CYP153AM.aq), Bernhard’s group successfully enabled terminal hydroxylation of dodecylamine, an inhibitor of the wild-type enzyme . In that study, it was shown how mutations in channels increased the accessibility of the substrate while at the same time affecting hydrogen-bonding interactions.…”

“…This work was performed in collaboration with the Damborsky group at Loschmidt Laboratories, Masarky University Brno (Czech Republic). Reproduced from ref . Copyright 2022 American Chemical Society.…”