Structure‐guided design combined with evolutionary diversity led to the discovery of the xylose‐releasing exo‐xylanase activity in the glycoside hydrolase family 43

Abstract

Rational design is an important tool for sculpting functional and stability properties of proteins and its potential can be much magnified when combined with in vitro and natural evolutionary diversity. Herein, we report the structure‐guided design of a xylose‐releasing exo‐β‐1,4‐xylanase from an inactive member of glycoside hydrolase family 43 (GH43). Structural analysis revealed a nonconserved substitution (Lys247) that results in the disruption of the hydrogen bond network that supports catalysis. The mutation of this residue to a conserved serine restored the catalytic activity and crystal structure elucidation of the mutant confirmed the recovery of the proper orientation of the catalytically relevant histidine. Interestingly, the tailored enzyme can cleave both xylooligosaccharides and xylan, releasing xylose as the main product, being the first xylose‐releasing exo‐β‐1,4‐xylanase reported in the GH43 family. This enzyme presents a unique active‐site topology when compared with closely related β‐xylosidases, which is the absence of a hydrophobic barrier at the positive‐subsite region, allowing the accommodation of long substrates. Therefore, the combination of rational design for catalytic activation along with naturally occurring differences in the substrate binding interface led to the discovery of a novel activity within the GH43 family. In addition, these results demonstrate the importance of solvation of the β‐propeller hollow for GH43 catalytic function and expand our mechanistic understanding about the diverse modes of action of GH43 members, a key and polyspecific carbohydrate‐active enzyme family abundant in most plant cell‐wall‐degrading microorganisms.

Gut Microbiome of the Amazon Master of the Grasses Harbors Unprecedented Enzymatic Strategies for Plant Glycans Breakdown

“…Structural elucidation by SeMet phasing (Table S8) revealed a two-domain architecture with a β-sandwich accessory domain tightly bound to the catalytic domain ( Figure S8D). Distinct to all other GH43_12 members structurally characterized so far, in which the β-sandwich domain is composed only by C-terminal β-strands, the GH43_12 structure herein elucidated shows an N-terminal β-strand that integrates with C-terminal β-strands to form the β-sandwich domain (43)(44)(45) (Figure S8 D). It indicates a further level of structural complexity within the GH43 family that might be carefully considered when designing constructs and chimeras involving these instrumental enzymes for plant polysaccharides depolymerization.…”

Characterization of a GH Family 43 β-Xylosidase Having a Novel Carbohydrate-binding Module from <i>Paenibacillus xylaniclasticus</i> Strain TW1

“…According to the database, some of the known GH43 enzymes have a catalytic module at the N-terminus and an additional unknown-function module, similar to Px Xyl43A-UM, at the C-terminus. 13) 14) 15) 16) 17) However, the amino acid sequences of these unknown-function modules did not show similarity to that of the function-characterized modules, classified as CBM. Therefore, we decided to characterize the catalytic activity of the full-length Px Xyl43A as well as the carbohydrate-binding activity of the unknown-function module (UM) derived from Px Xyl43A.…”

Complete genome sequencing and investigation on the fiber-degrading potential of Bacillus amyloliquefaciens strain TL106 from the tibetan pig

“…GH32 family enzymes, can hydrolyze or synthesize of fructosan glycoside bonds, including fructanase and sucrase [52]. GH43 was an important member of xylan degradation [53]. GH51 can dissociate arabinogalactan and arabinomannan in cell walls, promoting the degradation of pectin [54].…”