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

Abstract

Glucuronoyl esterases (GEs) catalyze the cleavage of ester linkages 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 and catalyze degradation of their natural substrates are sparse, calling for thorough enzyme structure-function studies. Presented here is a structural and mechanistic investigation of the bacterial GE OtCE15A. GEs belong to the carbohydrate esterase family 15 (CE15), which is in turn part of the larger α/β-hydrolase superfamily. GEs contain a Ser-His-Asp/Glu catalytic triad, but the location of the catalytic acid in GEs has been shown to be variable, and OtCE15A possesses two putative catalytic acidic residues in the active site. Through site-directed mutagenesis, we demonstrate that these residues are functionally redundant, possibly indicating the evolutionary route toward new functionalities within the family. Structures determined with glucuronate, in both native and covalently bound intermediate states, and galacturonate provide insights into the catalytic mechanism of CE15. A structure of OtCE15A with the glucuronoxylooligosaccharide 23-(4-O-methyl-α-d-glucuronyl)-xylotriose (commonly referred to as XUX) shows that the enzyme can indeed interact with polysaccharides from the plant cell wall, and an additional structure with the disaccharide xylobiose revealed a surface binding site that could possibly indicate a recognition mechanism for long glucuronoxylan chains. Collectively, the results indicate that OtCE15A, and likely most of the CE15 family, can utilize esters of glucuronoxylooligosaccharides and support the proposal that these enzymes work on lignin-carbohydrate complexes in plant biomass.

The coordinated action of glucuronoyl esterase and α‐glucuronidase promotes the disassembly of lignin–carbohydrate complexes

“…These results support the prediction that xylans do not hinder glucuronoyl esterase access to target linkages [42,43], and indicate that glucuronoyl esterases likely act before α‐glucuronidases and do not merely release single MeGlc p A residues linked to lignin that remain after α‐glucuronidase action. This observation is also supported by crystal structures of GEs with bound MeGlc p A‐appended xylo‐oligosaccharide ligands [42,43].…”

“…Similarly, the glucuronoyl esterase from Cerrena unicolor ( Cu GE) releases uronic acid‐containing xylooligosaccharides from extracted birchwood [40,41]. Furthermore, structural characterization of Tt CE15A, Ot CE15A, and Cu GE showed enzyme interactions with lignin and carbohydrate components of hardwood xylan [38,42–43]. Whereas glucuronoyl esterases were already shown to increase the hydrolytic activity of a commercial enzyme cocktail on milled corn cob [39] and endo‐xylanase activity on LCCs from birchwood [40], the impact of glucuronoyl esterases on other accessory enzymes targeting xylan substitutions has not been reported.…”

Investigation of a thermostable multi-domain xylanase-glucuronoyl esterase enzyme from Caldicellulosiruptor kristjanssonii incorporating multiple carbohydrate-binding modules

“…Calkro_0402 is not found fused to a CE15 domain, and it might be possible that the xylanase domain in the C. kristjanssonii enzyme over time has adapted to better complement the GE domain in order to achieve maximum synergistic effects [54], in keeping with the observation that almost all GEs characterized to date have exhibited neutral-to-basic pH optima [31,32,57]. The temperature required for optimal activity of the CkXyn10C domain by itself was 65 °C (Fig.…”

“…This is also reflected in the fact that the active sites of GE enzymes are surface-exposed and in addition to xylanor xylooligosaccharide binding sites have large surfaces where lignin fragments could be accommodated [32,55,56]. In fact, a recent study showed how GE enzymes may bind longer xylooligosaccharides, where previously only monosaccharides had been successfully modeled as ligands in protein 3-D structures [57]. The reaction of the CkGE15A domain with the BnzGlcA substrate at 40 °C showed an approximately twofold reduction in K m and a threefold increase in k cat .…”

“…A standard curve with varying concentrations of glucose was used to convert absorbance values to concentration of reducing sugars released. Attempts to repeat the assay with commercially purchased corn xylan were unsuccessful, as the material proved to be short oligomers rather than xylan chains [57].…”