HjCel3A, GH3 β-1,3-glucosidase from Hypocrea jecorina Cte_2137, CBM35 of C. Fundamental pancreatic -cell function is to produce and secrete insulin in response to blood glucose levels.
chrysosporium SacteLam55A, GH55 exo-β-1,3-glucanase from Streptomycs sp. chrysosporium SeMet, selenomethionine Gal3, β-1,3-galactotriose WT, wild-type WT_Gal, WT bound with Gal E208Q_Gal3, E208Q bound with Gal3 E208A_Gal3, E208A bound with Gal3 PcCBM35, CBM35 domain of Pc1,3Gal43A Gal -1, Gal residue occupied subsite -1 Gal +1, Gal residue occupied subsite +1 Gal +2, Gal residue occupied subsite +2 Gal2, β-1,3-galactobiose Gal_site 1, the non-reducing terminal Gal residue of Gal3 bound to PcCBM35 Gal_site 2, the middle Gal residue of Gal3 bound to PcCBM35 Gal_site 3, the reducing terminal Gal residue of Gal3 bound to PcCBM35 HPLC, high-performance liquid chromatography PcLam55A, exo-β-1,3-glucanase from P. Our findings should also be helpful in engineering designer enzymes for efficient utilization of various types of biomass.Ībbreviations (in order of appearance): AGPs, arabinogalactan proteins Gal, D-galactose Pc1,3Gal43A, exo-β-1,3-galactanase from Phanerochaete chrysosporium GH, glycoside hydrolase GH43_sub24, GH family 43 subfamily 24 CBM, carbohydrate binding module Ct1,3Gal43A, exo-β-1,3-galactanase from Clostridium thermocellum BT3683, β-1,3-galactosidase from Bacteroides thetaiotaomicron VPI-5482 PcCel45A, endoglucanase V from P. Pc1,3Gal43A WT and its mutants at residues involved in substrate recognition are expected to be useful tools for structural analysis of AGPs. Some of the residues involved in ligand recognition differ from those of galactomannan-binding CBM35, including substitution of Trp for Gly, which affects pyranose stacking, and substitution of Asn for Asp in the lower part of the binding pocket. Furthermore, the galactan-binding domain in CBM35 has a different ligand interaction mechanism from other sugar-binding CBM35s. Subsite -1 contains a space that can accommodate the C-6 methylol of Gal, enabling the enzyme to bypass the β-1,6-linked galactan side chains of AGPs. Pc1,3Gal43A has three subsites that continue from the bottom of the catalytic pocket to the solvent. Crystal structure and kinetic analyses indicated that the tautomerized imidic acid function of Gln263 serves instead as the catalytic base residue. GH43_sub24 lacks the catalytic base Asp that is conserved among other GH43 subfamilies. It is composed of a glycoside hydrolase family 43 subfamily 24 (GH43_sub24) catalytic domain together with a carbohydrate-binding module family (CBM) 35 binding domain. In this study, we solved the apo and liganded structures of exo-β-1,3-galactanase from the basidiomycete Phanerochaete chrysosporium ( Pc1,3Gal43A), which specifically cleaves AGPs. Arabinogalactan proteins (AGPs) are functional plant proteoglycans, but their functions are largely unexplored, mainly because of the complexity of the sugar moieties, which are generally analyzed with the aid of glycoside hydrolases.
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