2-O-α-D-Glucosylglycerol Phosphorylase from Bacillus selenitireducens MLS10 Possessing Hydrolytic Activity on β-D-Glucose 1-Phosphate

The glycoside hydrolase family (GH) 65 is a family of inverting phosphorylases that act on α-glucosides. A GH65 protein (Bsel_2816) from Bacillus selenitireducens MLS10 exhibited inorganic phosphate (Pi)-dependent hydrolysis of kojibiose at the rate of 0.43 s−1. No carbohydrate acted as acceptor for the reverse phosphorolysis using β-d-glucose 1-phosphate (βGlc1P) as donor. During the search for a suitable acceptor, we found that Bsel_2816 possessed hydrolytic activity on βGlc1P with a kcat of 2.8 s−1; moreover, such significant hydrolytic activity on sugar 1-phosphate had not been reported for any inverting phosphorylase. The H218O incorporation experiment and the anomeric analysis during the hydrolysis of βGlc1P revealed that the hydrolysis was due to the glucosyl-transferring reaction to a water molecule and not a phosphatase-type reaction. Glycerol was found to be the best acceptor to generate 2-O-α-d-glucosylglycerol (GG) at the rate of 180 s−1. Bsel_2816 phosphorolyzed GG through sequential Bi-Bi mechanism with a kcat of 95 s−1. We propose 2-O-α-d-glucopyranosylglycerol: phosphate β-d-glucosyltransferase as the systematic name and 2-O-α-d-glucosylglycerol phosphorylase as the short name for Bsel_2816. This is the first report describing a phosphorylase that utilizes polyols, and not carbohydrates, as suitable acceptor substrates.

糖苷水解酶（glycoside hydrolase, GH）家族65是一类作用于α-葡萄糖苷的反转型磷酸化酶。来自脱硒芽孢杆菌（Bacillus selenitireducens）MLS10的GH65家族蛋白Bsel_2816，可依赖无机磷酸（inorganic phosphate, Pi）催化曲二糖水解，催化速率为0.43 s⁻¹。以β-D-葡萄糖1-磷酸（β-d-glucose 1-phosphate, βGlc1P）为供体的逆磷酸解反应中，未发现可作为受体的碳水化合物。在筛选适宜受体的过程中，我们发现Bsel_2816对βGlc1P具有水解活性，其催化速率常数（kcat）为2.8 s⁻¹；此外，此前未见任何反转型磷酸化酶被报道具备对糖1-磷酸的此类显著水解活性。通过H₂¹⁸O掺入实验及βGlc1P水解过程中的端基异构分析，证实该水解反应为糖基向水分子的转移反应，而非磷酸酶型反应。研究发现甘油为最佳受体，可用于合成2-O-α-D-葡萄糖基甘油（2-O-α-d-glucosylglycerol, GG），合成速率达180 s⁻¹。Bsel_2816通过序列双底物-双产物（Bi-Bi）反应机制催化GG的磷酸解反应，其kcat为95 s⁻¹。我们将Bsel_2816的系统名称拟定为2-O-α-D-吡喃葡萄糖基甘油：磷酸β-D-葡萄糖基转移酶，简称为2-O-α-D-葡萄糖基甘油磷酸化酶。本研究首次报道了一类以多元醇而非碳水化合物作为适宜受体底物的磷酸化酶。