Some serine hydrolases also catalyze a promiscuous reaction -reversible perhydrolysis of carboxylic acids to make peroxycarboxylic acids. Five x-ray crystal structures of these carboxylic acid perhydrolases show a proline in the oxyanion loop. Here, we test whether this proline is essential for high perhydrolysis activity using Pseudomonas fluorescens esterase (PFE). The L29P variant of this esterase catalyzes perhydrolysis 43-fold faster (k cat comparison) than wild type. Surprisingly, saturation mutagenesis at the 29 position of PFE identified six other amino acid substitutions that increase perhydrolysis of acetic acid at least fourfold over wild type. The best variant, L29I PFE, catalyzed perhydrolysis 83 times faster (k cat comparison) than wild-type PFE and twice as fast as L29P PFE. Despite the different amino acid in the oxyanion loop, L29I PFE shows a similar selectivity for hydrogen peroxide over water as L29P PFE ( 0 = 170 M −1 vs. 160 M −1 ), and a similar fast formation of acetyl-enzyme (140 U/mg vs. 62 U/mg). X-ray crystal structures of L29I PFE with and without bound acetate show an unusual mixture of two different oxyanion loop conformations. The type II -turn conformation resembles the wild-type structure and is unlikely to increase perhydrolysis, but the type I -turn conformation creates a binding site for a second acetate. Modeling suggests that a previously proposed mechanism for L29P PFE can be extended to include L29I PFE where an acetate accepts a hydrogen bond to promote faster formation of the acetyl enzyme.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.