Abstract
The microbial degradation of the plant cell wall is an important biological process that is highly relevant to environmentally significant industries such as the bioenergy and biorefining sectors. A major component of the wall is glucuronoxylan, a β1,4-linked xylose polysaccharide that is decorated with α-linked glucuronic and/or methylglucuronic acid (GlcA/MeGlcA). Recently three members of a glycoside hydrolase family, GH115, were shown to hydrolyze MeGlcA side chains from the internal regions of xylan, an activity that has not previously been described. Here we show that a dominant member of the human microbiota, Bacteroides ovatus, contains a GH115 enzyme, BoAgu115A, which displays glucuronoxylan α-(4-O-methyl)-glucuronidase activity. The enzyme is significantly more active against substrates in which the xylose decorated with GlcA/MeGlcA is flanked by one or more xylose residues. The crystal structure of BoAgu115A revealed a four-domain protein in which the active site, comprising a pocket that abuts a cleft-like structure, is housed in the second domain that adopts a TIM barrel-fold. The third domain, a five-helical bundle, and the C-terminal β-sandwich domain make inter-chain contacts leading to protein dimerization. Informed by the structure of the enzyme in complex with GlcA in its open ring form, in conjunction with mutagenesis studies, the potential substrate binding and catalytically significant amino acids were identified. Based on the catalytic importance of residues located on a highly flexible loop, the enzyme is required to undergo a substantial conformational change to form a productive Michaelis complex with glucuronoxylan.
Highlights
The structure of GH115 glucuronidases that remove glucuronic acid from xylan chains is unknown
BoAgu115A Is a Xylan-specific ␣-Glucuronidase—The genome of B. ovatus encodes seven proteins that are members of GH115. To explore their potential enzymatic activities, six of the seven B. ovatus GH115 proteins were expressed in E. coli in soluble form (BACOVA_00249 could not be expressed in E. coli), purified by immobilized metal affinity chromatography (IMAC) to electrophoretic homogeneity, and their activity against glucuronoxylans was explored using Polysaccharide Analysis using Carbohydrate gel Electrophoresis (PACE)
The data showed that BoAgu115A released glucuronic acid (GlcA) and 4-O-methyl-GlcA (MeGlcA) from wild type Arabidopsis glucuronoxylan, but not from an Arabidopsis mutant completely lacking MeGlcA and GlcA decorations on xylan [23] (Fig. 2A)
Summary
The structure of GH115 glucuronidases that remove glucuronic acid from xylan chains is unknown. The major matrix polysaccharide of plant cell walls, consists of 1,4-linked D-xylose residues decorated at O2 and/or O3 with L-arabinofuranose and acetyl groups, and exclusively at O2 with ␣-D-glucuronic acid (GlcA) or 4-Omethyl-GlcA (MeGlcA) (Fig. 1) [8]. These decorated regions make the polymer backbone recalcitrant to attack by most endo-xylanases and block the action of xylosidases. The majority of ␣-glucuronidases that hydrolyze the GlcA and MeGlcA decorations are located in GH67 [9, 10] These enzymes, do not attack glucuronoxylan, they only remove uronic acid from the non-reducing end of glucuronoxylooligosaccharides [9, 10]. Mutagenesis and structural data indicate that a significant conformational change is required to assemble the catalytic apparatus in BoAgu115A
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