Mazurkewich_2019_J.Biol.Chem_294_19978

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 chemically recalcitrant materials. However, details on how GEs interact and catalyze degradation of their natural substrates are sparse, calling for thorough enzyme structure-function studies. GEs belong to carbohydrate esterase family 15 (CE15), which is part of the larger alpha/beta hydrolase superfamily. We present here a structural and mechanistic investigation of the bacterial GE OtCE15A. GEs contain a Ser-His-Asp/Glu catalytic triad, but the location of the catalytic acid in GEs is known to be variable, and OtCE15A possesses two putative catalytic acidic residues in its active site. Through site-directed mutagenesis, we demonstrate here that these residues are functionally redundant, possibly indicating the evolutionary route toward new functionalities within the CE15 family. Structures determined with the bound products glucuronate and galacturonate, as well as a covalently bound intermediate, provided insights into the catalytic mechanism of CE15. A structure of OtCE15A with the glucuronoxylooligosaccharide 2(3)-(4-O-methyl-alpha-D-glucuronyl)-xylotriose (XUX) disclosed that the enzyme can indeed interact with polysaccharides from the plant cell wall, and an additional structure with the disaccharide xylobiose revealed an enzyme surface binding site that might indicate a mechanism by which the enzyme recognizes long glucuronoxylan chains. These results indicate that OtCE15A, and likely most CE15 family enzymes, can utilize glucuronoxylooligosaccharide esters and support the proposal that these enzymes are active on lignin-carbohydrate complexes in plant biomass.