New Structural Motif for Carboxylic Acid Perhydrolases

Abstract

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.

Thermostability enhancement of the Pseudomonas fluorescens esterase I by in vivo folding selection in Thermus thermophilus

“…This effect was accompanied by the apparition of lactamase activity and the reduction in the activity with the model substrate pNPA (Table ), as previously reported (Ding & Kazlauskas, ). In fact, L30P seems to be a promiscuous generalist (Khersonsky & Tawfik, ) showing esterase, lactonase (Ding & Kazlauskas, ; D. L. Yin et al, ), perhydrolase (D. T. Yin et al, ), and lactamase activities (Torres et al, ). In addition, T m of Q11L/L30P/A191S was 3.5°C and 8.1°C higher than those of Q11L/A191S and the wild‐type enzyme, respectively.…”

“…In fact, L30P seems to be a promiscuous generalist (Khersonsky & Tawfik, 2010) showing esterase, lactonase (Ding & Kazlauskas, 2017;D. L. Yin et al, 2010), perhydrolase (D. T. Yin et al, 2013), and lactamase activities (Torres et al, 2012). In addition, T m of Q11L/L30P/A191S was 3.5°C and 8.1°C higher than those of Q11L/ A191S and the wild-type enzyme, respectively.…”

“…3.1.1.2) is a monomeric enzyme of 31 kDa that can be overexpressed in Escherichia coli (Choi, Jeohn, Rhee, & Yoo, ). Site‐directed mutagenesis studies have been previously carried out on PFEI aiming to increase its activity in the hydrolysis of epoxides (Jochens et al, ) and lactones (Ding & Kazlauskas, ; D. L. Yin et al, ), in the perhydrolysis of carboxylic acids (D. T. Yin, Purpero, Fujii, Jing, & Kazlauskas, ), and to improve the enantioselectivity in the kinetic resolution of diverse chiral substrates (Schließmann, Hidalgo, Berenguer, & Bornscheuer, ; Torres et al, ). In addition, simultaneous site‐saturation mutagenesis was performed on three surface positions of PFEI to enhance its thermal stability (Jochens, Aerts, & Bornscheuer, ).…”

Lactonization reactions through hydrolase-catalyzed peracid formation. Use of lipases for chemoenzymatic Baeyer–Villiger oxidations of cyclobutanones

“…Within this class of enzymes, perhydrolases are able to efficiently catalyze perhydrolysis reactions for the formation of peracids [5,6], but the unusual participation of lipases for global oxidative process has attracted the attention of different research groups in the last decade [5,7,8]. Thus, examples of lipase-mediated epoxidation [9][10][11][12][13][14], Baeyer-Villiger reactions [15][16][17][18][19], perhydrolysis of carboxylic acid and esters [20], sequential Baeyer-Villiger reaction and ring-opening polymerization [21], and also consecutive esterification and Baeyer-Villiger cascade reactions [22] have appeared in the literature, giving access to synthetically useful oxygenated heterocycles through clean and selective transformations under mild reaction conditions.…”

Uncovering divergent evolution of α/β-hydrolases: a surprising residue substitution needed to convert Hevea brasiliensis hydroxynitrile lyase into an esterase

“…3, 4 Other subtleties in the mechanism of serine hydrolases include questions of whether there is a tunnel to allow water access to the active site, 5, 6 how acyl transferases avoid hydrolysis to favor acyl transfer 7 and how perhydrolases favor hydrogen peroxide over water. 8 Nevertheless, all these reactions are similar and involve similar transitions states.…”