Yernool D A, McCarthy J K, Eveleigh D E, Bok J D
Department of Biochemistry and Microbiology, Cook College, Rutgers University, New Brunswick, New Jersey 08901, USA.
J Bacteriol. 2000 Sep;182(18):5172-9. doi: 10.1128/JB.182.18.5172-5179.2000.
Characterization in Thermotoga neapolitana of a catabolic gene cluster encoding two glycosyl hydrolases, 1,4-beta-D-glucan glucohydrolase (GghA) and cellobiose phosphorylase (CbpA), and the apparent absence of a cellobiohydrolase (Cbh) suggest a nonconventional pathway for glucan utilization in Thermotogales. GghA purified from T. neapolitana is a 52.5-kDa family 1 glycosyl hydrolase with optimal activity at pH 6.5 and 95 degrees C. GghA releases glucose from soluble glucooligomers, with a preference for longer oligomers: k(cat)/K(m) values are 155.2, 76.0, and 9.9 mM(-1) s(-1) for cellotetraose, cellotriose, and cellobiose, respectively. GghA has broad substrate specificity, with specific activities of 236 U/mg towards cellobiose and 251 U/mg towards lactose. With p-nitrophenyl-beta-glucoside as the substrate, GghA exhibits biphasic kinetic behavior, involving both substrate- and end product-directed activation. Its capacity for transglycosylation is a factor in this activation. Cloning of gghA revealed a contiguous upstream gene (cbpA) encoding a 93.5-kDa cellobiose phosphorylase. Recombinant CbpA has optimal activity at pH 5.0 and 85 degrees C. It has specific activity of 11.8 U/mg and a K(m) of 1.42 mM for cellobiose, but shows no activity towards other disaccharides or cellotriose. With its single substrate specificity and low K(m) for cellobiose (compared to GghA's K(m) of 28.6 mM), CbpA may be the primary enzyme for attacking cellobiose in Thermotoga spp. By phosphorolysis of cellobiose, CbpA releases one activated glucosyl molecule while conserving one ATP molecule per disaccharide. CbpA is the first hyperthermophilic cellobiose phosphorylase to be characterized.
嗜热栖热菌中一个分解代谢基因簇的特性研究，该基因簇编码两种糖基水解酶，即1,4-β-D-葡聚糖葡糖水解酶（GghA）和纤维二糖磷酸化酶（CbpA），且明显不存在纤维二糖水解酶（Cbh），这表明嗜热栖热菌在利用葡聚糖方面存在一条非常规途径。从嗜热栖热菌中纯化得到的GghA是一种52.5 kDa的1家族糖基水解酶，在pH 6.5和95℃时具有最佳活性。GghA从可溶性葡糖寡聚物中释放葡萄糖，更倾向于较长的寡聚物：纤维四糖、纤维三糖和纤维二糖的k(cat)/K(m)值分别为155.2、76.0和9.9 mM⁻¹ s⁻¹。GghA具有广泛的底物特异性，对纤维二糖的比活性为236 U/mg，对乳糖的比活性为251 U/mg。以对硝基苯基-β-葡糖苷为底物时，GghA表现出双相动力学行为，涉及底物导向和终产物导向的激活。其转糖基化能力是这种激活的一个因素。gghA的克隆揭示了一个相邻的上游基因（cbpA），编码一种93.5 kDa的纤维二糖磷酸化酶。重组CbpA在pH 5.0和85℃时具有最佳活性。它对纤维二糖的比活性为11.8 U/mg，K(m)为1.42 mM，但对其他二糖或纤维三糖无活性。凭借其单一的底物特异性和对纤维二糖较低的K(m)（与GghA的28.6 mM的K(m)相比），CbpA可能是嗜热栖热菌属中攻击纤维二糖的主要酶。通过纤维二糖的磷酸解作用，CbpA释放一个活化的葡糖基分子，同时每分子二糖节省一个ATP分子。CbpA是第一个被表征的嗜热纤维二糖磷酸化酶。
