Characterization and structural analyses of a novel glycosyltransferase acting on the β-1,2-glucosidic linkages

Abstract

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Phytopathogenic cyclic glucohexadecaose from an inverting transglycosylase

“…2e (see Methods for details). Although the kcat value of XccOpgD was slightly lower than expected for a GH enzyme, the Km value of XccOpgD was much lower than those of EcOpgD (GH186) and comparable to other β-1,2-glucan-related GHs, i.e., SGL from Chitinophaga pinensis and SGL from Talaromyces funiculosus (GH144 and GH162, respectively) 12,14,15 , leading to a kcat/Km indicative of GH enzymes 16 (Table 1).…”

“…Colour development of the reaction mixtures was performed using the GOPOD method 16 to quantify the concentration of glucose derived from the products released by XccOpgD. Molar concentrations of linear β-1,2-glucan substrates were calculated based on the Mn of the substrates.…”

“…BtBGL and bis-Tris-HCl (pH 5.5) were added (final concentrations were 1 mg/mL and 83 mM, respectively) to convert non-cyclized β-1,2-glucan into glucose. The amounts of produced glucose were measured by the GOPOD method 16 to determine the amounts of residual linear β-1,2-glucan. The conversion rates were calculated as subtracts between weights of the initial and residual substrates in the reaction by XccOpgD.…”

“…The samples were then treated with 0.6 mg/mL of BtBGL and 0.15 mg/mL CpSGL at 30 °C for 24 h to convert CβG16α into 11 glucoses and a non-degradable glucopentaose. The main residual oligosaccharide was identified to be glucopentaose by ESI-MS.Colour development of the reaction mixtures was performed using the GOPOD method16 to quantify the concentration of glucose derived from the products released by XccOpgD. Molar concentrations of linear β-1,2-glucan substrates were calculated based on the Mn of the substrates.…”

Functional and structural analysis of a cyclization domain in a cyclic β-1,2-glucan synthase

“…Consequently, a bacterial SGL from Chitinophaga pinensis (CpSGL) and a fungal SGL from Talaromyces funiculosus (TfSGL) were sequentially identified for the first time, leading to creation of new families (GH144 and GH162), respectively (Abe et al 2017; Tanaka et al 2019). Subsequently, functions and structures of several β-1,2-glucan-associated enzymes including the ones that are given new EC numbers were reported (Nakajima et al 2016; Nakajima et al 2017; Shimizu et al 2018; Kobayashi et al 2022). In addition, another SGL which neither belongs to GH144 nor GH162 has been identified from Escherichia coli recently, leading to foundation of a new family, GH186 (Motouchi et al 2023).…”