Characterization of a novel family VIII esterase EstM2 from soil metagenome capable of hydrolyzing estrogenic phthalates

Abstract

Background: Microbes are rich sources of enzymes and esterases are one of the most important classes of enzymes because of their potential for application in the field of food, agriculture, pharmaceuticals and bioremediation. Due to limitations in their cultivation, only a small fraction of the complex microbial communities can be cultured from natural habitats. Thus to explore the catalytic potential of uncultured organisms, the metagenomic approach has turned out to be an effective alternative method for direct mining of enzymes of interest. Based on activity-based screening method, an esterase-positive clone was obtained from metagenomic libraries. Results: Functional screening of a soil metagenomic fosmid library, followed by transposon mutagenesis led to the identification of a 1179 bp esterase gene, estM2, that encodes a 392 amino acids long protein (EstM2) with a translated molecular weight of 43.12 kDa. Overproduction, purification and biochemical characterization of the recombinant protein demonstrated carboxylesterase activity towards short-chain fatty acyl esters with optimal activity for p-nitrophenyl butyrate at pH 8.0 and 37 °C. Amino acid sequence analysis and subsequent phylogenetic analysis suggested that EstM2 belongs to the family VIII esterases that bear modest similarities to class C β-lactamases. EstM2 possessed the conserved S-x-x-K motif of class C β-lactamases but did not exhibit β-lactamase activity. Guided by molecular docking analysis, EstM2 was shown to hydrolyze a wide range of di-and monoesters of alkyl-, aryl-and benzyl-substituted phthalates. Thus, EstM2 displays an atypical hydrolytic potential of biotechnological significance within family VIII esterases. Conclusions: This study has led to the discovery of a new member of family VIII esterases. To the best of our knowledge, this is the first phthalate hydrolase (EstM2), isolated from a soil metagenomic library that belongs to a family possessing β-lactamase like catalytic triad. Based on its catalytic potential towards hydrolysis of both phthalate diesters and phthalate monoesters, this enzyme may find use to counter the growing pollution caused by phthalatebased plasticizers in diverse geological environment and in other aspects of biotechnological applications.

Discovery and Design of Family VIII Carboxylesterases as Highly Efficient Acyltransferases

“…Based on sequence alignments, the division of family VIII carboxylesterases into three subclasses was previously suggested. [15] EstCE1, EstM2, 5ZWQ, 5GMX, and 4IVI belong either to subclass VIII.1 or VIII.2, both sharing a conserved WGG motif (WSG for 5ZWQ) in the region where the socalled KTG-box exists in class C b-lactamases ( Figure S6). [22] Due to its proximity to the catalytic triad, this motif appears to largely influence the physical properties of the ligandbinding site.…”

“…[6,8] EstCE1 catalyzes acetyl transfer to benzyl alcohol 66 times faster than hydrolysis of the acyl donor, thereby outperforming even one of the most efficient MsAcT variants (K97A; 50-fold faster) recently reported by our group. [6] Strikingly, EstM2 [15] catalyzes the acetylation of benzyl alcohol 149 times faster than donor hydrolysis, making it roughly 20 times more efficient than wild-type MsAcT. Only EstA (PDB 3ZYT) [16] did not show increased relative activity in the presence of benzyl alcohol, suggesting that it is not capable of catalyzing the acetylation of benzyl alcohol.…”

Phthalate hydrolase: distribution, diversity and molecular evolution

“…Involvement of phthalate hydrolase in the metabolic pathways of the degradation of PAEs have largely been postulated based on the characterization of metabolites and/or other biochemical analyses (Eaton, 2001; Nishioka et al ., 2006; Zhang et al ., 2014b; Lu et al ., 2020; Sarkar et al ., 2020; Du et al ., 2021). In contrast, characterization of phthalate hydrolase at enzymatic or genetic level is relatively limited.…”

Entdeckung und Design promiskuitiver Acyltransferase‐Aktivität in Carboxylesterasen der Familie VIII

“…Auf Grundlage von Sequenzvergleichen wurde kürzlich die Aufteilung von Carboxylesterasen der Familie VIII in drei Unterklassen vorgeschlagen. [15] Demnach gehçren EstCE1, EstM2, 5ZWQ, 5GMX und 4IVI entweder zur Unterklasse VIII.1 oder VIII.2, welche beide ein konserviertes WGG-Motiv (WSG für 5ZWQ) in der Region aufweisen, in der die so genannte KTG-Box in b-Lactamasen der Klasse C zu finden ist (Abbildung S6). [22] Aufgrund der Nähe zur katalytischen Triade scheint dieses Motiv die physikalischen Eigenschaften der Ligandenbindestelle tiefgreifend zu beeinflussen.…”

“…[6,8] EstCE1 katalysiert den Acetyltransfer zu Benzylalkohol 66-mal schneller als die Hydrolyse des Acyldonors und übertrifft damit sogar eine der effizientesten MsAcT-Varianten (K97A; 50-mal schneller), über die unsere Gruppe kürzlich berichtete. [6] Bemerkenswert ist, dass EstM2 [15] die Acetylierung von Benzylalkohol sogar 149-mal schneller katalysiert als die Donor-Hydrolyse und damit etwa 20-mal effizienter als MsAcT ist. Lediglich EstA (PDB 3ZYT) [16] zeigte keine erhçhte relative Aktivität in Gegenwart von Benzylalkohol, was darauf hindeutet, dass dieses Enzym nicht in der Lage ist, Benzylalkohol zu acetylieren.…”