Structural Insights into the Dual-Substrate Recognition and Catalytic Mechanisms of a Bifunctional Acetyl Ester–Xyloside Hydrolase from Caldicellulosiruptor lactoaceticus

Abstract

Enzymes are usually characterized by their evolutionarily conserved catalytic domains; however, this work presents the incidental gain-of-function of an enzyme in a loop region by natural evolution of its amino acids. A bifunctional acetyl ester−xyloside hydrolase (CLH10) was heterologously expressed, purified, and characterized. The primary sequence of CLH10 contains the fragments of the conserved sequence of esterase and glycosidase, which distribute in a mixed type. The crystal structure revealed that the primary sequence folded into two independent structural regions to undertake both acetyl esterase and β-1,4xylanase hydrolase functions. CLH10 is capable of cleaving both the β-1,4-xylosidic bond-linked main chain and the ester bond-linked acetylated side chain of xylan, which renders it valuable because it can degrade acetylated xylan within one enzyme. Significantly, the β-1,4-xylanase activity of CLH10 appears to have been fortuitously obtained because of the variable Asp10 and Glu139 located in its loop region, which suggested that the exposed loop region might act as a potential hot-spot for the design and generation of promising enzyme function in both directed evolution and rational protein design.

Rational Design for Broadened Substrate Specificity and Enhanced Activity of a Novel Acetyl Xylan Esterase from Bacteroides thetaiotaomicron

“…Among the three closest homologs, the activities of CLH10 and Cest-2923 were determined, and both enzymes had significantly higher activities toward p NPA than BT Axe1. As shown in Table S2, CLH10 had a catalytic efficiency ( k cat / K m ) of 8.46 × 10 3 toward p NPA, whereas activity against p NPB was not reported . Cest-2923 was active toward both p NPA and p NPB, with k cat / K m values of 1.12 × 10 4 and 162, respectively (Table S2).…”

“…As shown in Table S2, CLH10 had a catalytic efficiency (k cat / K m ) of 8.46 × 10 3 toward pNPA, whereas activity against pNPB was not reported. 39 Cest-2923 was active toward both pNPA and pNPB, with k cat /K m values of 1.12 × 10 4 and 162, respectively (Table S2). 41 Specifically, Cest-2923 displayed acyl-length selectivity against distinct p-nitrophenyl ester substrates, with C 2 > C 4 > C 8 > C 12 > C 14 , showing a preference for short acyl-length esters.…”

“…The resulting “oxyanion hole” is usually stabilized by the N atoms of the surrounding residues (e.g., Gly, Asn, or/and Arg) via hydrogen bonds . According to the results of molecular docking analysis and the comparison with the residues stabilizing the oxyanion hole of CLH10 from Caldicellulosiruptor lactoaceticus, it is inferred that Gly59 and Gly60 may be involved in the stabilization of the “oxyanion holes” of BT Axe1 (Figure A). To test the hypothesis, site-directed mutagenesis experiments were designed.…”

“…To perform rational design of BT Axe1, we constructed a structural similarity dendrogram via the DALI server using eleven structures with a Z-score higher than 24.7 . The top three closest homologs included acetyl ester–xyloside hydrolase from Caldicellulosiruptor lactoaceticus (CLH10), carboxylesterase from Lactobacillus plantarum WCFS1 (Cest-2923), and a putative sugar hydrolase from Lactobacillus plantarum (YeeB), which had sequence similarity to BT Axe1 of 28.9, 20.2, and 23.8%, respectively (Figure S5). All of these proteins exhibited the typical α/β hydrolase fold and the consensus G–X–S–X–G motif in the loop between β6 and α4.…”

Activity-Based Protein Profiling for the Identification of Novel Carbohydrate-Active Enzymes Involved in Xylan Degradation in the Hyperthermophilic Euryarchaeon Thermococcus sp. Strain 2319x1E

“…There are several examples for bifunctional enzymes with two different glycoside hydrolase functions ( Shi et al, 2010 ; Ferrara et al, 2014 ; Liang et al, 2018 ). Especially for xylanases, bi- or multifunctionality is common, including acetyl ester-xyloside hydrolases ( Khandeparker and Numan, 2008 ; Cao et al, 2019 ). However, as far as we know, a bifunctional enzyme with maltose-forming α-amylase and deacetylase activity has not been reported yet.…”