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.
Highlights
Glucuronoyl esterases (GEs) catalyze the cleavage of ester linkages between lignin and glucuronic acid moieties on glucuronoxylan in plant biomass
This is most evident in the OtCE15A-H408A complex structure with the GlcA covalent intermediate, where the arginine still contributes to the positioning of the carbonyl group
Similar to other ␣/-hydrolases, the carbohydrate esterase family 15 (CE15) members share some similarity around the stabilization of the oxyanion, aided by the main-chain amide nitrogen at the end of the helical dipole that is proximal to the catalytic serine
Summary
The activity of OtCE15A and nine other bacterial CE15 members has previously been characterized on model substrates, laying the foundation for these mechanistic studies [7] (Table S1). Relative to the apostructure previously determined, binding of GlcA resulted in minimal changes in the enzyme’s structure (all atom root mean square deviation of 0.639 Å), and the binding of the uronic acid to OtCE15A is very similar to that seen in the structure of the fungal StGE2 (catalytic S213A variant) in complex with the methyl ester of 4-O-MeGlcA (Fig. 2B) [8]. An S267A-GlcA complex structure was determined to high resolution (1.12 Å) and showed that in the absence of the catalytic serine, the glucuronate binds identically as in the WT protein (Fig. 2C) This high-resolution structure facilitated the observation of both glucuronate anomers being present. Following a 5-s substrate soak, we were able to determine the structure of the product GlcA covalently bound to the catalytic serine (Ser-267) through the C5 carboxylate (Fig. 4B).
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