Structure–function analyses reveal that a glucuronoyl esterase from Teredinibacter turnerae interacts with carbohydrates and aromatic compounds

Abstract

Glucuronoyl esterases (GEs) catalyze the cleavage of ester linkages found between lignin and glucuronic acid moieties on glucuronoxylan in plant biomass. As such, GEs represent promising biochemical tools in industrial processing of these recalcitrant resources. However, details on how GEs interact with their natural substrates are sparse, calling for thorough structure-function studies. Presented here is the structure and biochemical characterization of a GE, Tt CE15A, from the bacterium Teredinibacter turnerae , a symbiont of wood-boring shipworms. To gain deeper insight into enzyme–substrate interactions, inhibition studies were performed with both the WT Tt CE15A and variants in which we, by using site-directed mutagenesis, substituted residues suggested to have key roles in binding to or interacting with the aromatic and carbohydrate structures of its uronic acid ester substrates. Our results support the hypothesis that two aromatic residues (Phe-174 and Trp-376), conserved in bacterial GEs, interact with aromatic and carbohydrate structures of these substrates in the enzyme active site, respectively. The solved crystal structure of Tt CE15A revealed features previously not observed in either fungal or bacterial GEs, with a large inserted N-terminal region neighboring the active site and a differently positioned residue of the catalytic triad. The findings highlight key interactions between GEs and complex lignin-carbohydrate ester substrates and advance our understanding of the substrate specificities of these enzymes in biomass conversion.

Structural and biochemical studies of the glucuronoyl esterase OtCE15A illuminate its interaction with lignocellulosic components

“…Substitution of either the catalytic serine (Ser-267) or histidine (His-408) with alanine significantly compromised the Ot CE15A turnover rate ( k cat ), reducing the rate by 17,000- and 1,700-fold, respectively. Compared with the Tt CE15A S281A variant, where the k cat only decreased 370-fold (11), the S267A variant of Ot CE15A was dramatically crippled. Ot CE15A is an interesting member of CE15 and the α/β-hydrolase superfamily in that it features acidic residues at both the canonical and noncanonical positions (Glu-290 and Asp-356) (Fig.…”

“…In recent work, we have revealed that some bacterial CE15 members have more promiscuous substrate specificities, where certain enzymes are able to hydrolyze both glucuronoate and galacturonoate (GalA) esters (7). The first structurally and biochemically characterized CE15 members were of fungal origins, and only recently have bacterial CE15 members been investigated (7–11). The only GE protein structure currently deposited with a ligand is St GE2, a variant containing an alanine substitution of the catalytic serine, which is in complex with a methyl ester of the monosaccharide 4- O -methyl glucuronate (4- O -MeGlcA) (8).…”

“…As GEs have been shown to act on LCCs, to liberate glucuronoxylan from lignin for further metabolism, the physiological substrate for GEs is most likely glucuronoxylan or glucuronoxylooligosaccharides rather than small 4- O -MeGlcA moieties (4–6). Our recent characterization of the bacterial GE Tt CE15A from Teredinibacter turnerae showed that GEs are indeed able to interact with oligosaccharides (xylotriose appended with 4- O -MeGlcA) and that this interaction is mediated by key residues in the active site (11). However, direct molecular evidence of GE-xylan interactions are still lacking, and further structural information is needed.…”

“…However, recent characterizations of bacterial CE15 members have revealed how the location of acidic residue of the catalytic triad is not conserved among GEs. In fungal GEs, the catalytic acid is located in a noncanonical position relative to typical α/β-hydrolases, but in the structures of the bacterial CE15 members MZ0003, discovered in a marine arctic metagenome, and Tt CE15A the putative acidic residue was located on the canonical loop (10, 11). In Tt CE15A, substitution of its catalytic acid residue to alanine led to a >30-fold reduction in turnover rate, and, interestingly, full activity could not be rescued in the variant by introducing the acidic residue at the noncanonical position exhibited in fungal and many other bacterial GEs.…”

The acid-base-nucleophile catalytic triad in ABH-fold enzymes is coordinated by a set of structural elements

“…Residue X acid+2 , one of the conserved structural elements that is involved in the coordination of the imidazole ring of the catalytic histidine, is usually a hydrophobic or an aromatic residue located at the entrance of the active site cleft, mostly reviewed for its role in ligand binding [51][52][53][54][55][56] or the release of products of catalysis [51,57]. Consequently, the site-directed mutagenesis of residue X acid+2 in different ABH enzymes has led to various results, which are consistent with its role in ligand binding, including the compromising [52,54,55,58,59] or the enhancement [52] of catalytic activity, the alteration of transport tunnels [57,60], the modification of substrate specificity [53,55,56] and the inversion of enantioselectivity [52]. For the remaining residues that coordinate the imidazole ring of the catalytic histidine, including residue X acid+3 , we have only found a few mutational studies that have resulted in reduced activity [58,61,62], with a single study highlighting the residue's role in the stability of the enzyme and the catalytic activity rather than in ligand binding [58].…”