Maltose Metabolism in the Hyperthermophilic Archaeon Thermococcus litoralis: Purification and Characterization of Key Enzymes
Maltose Metabolism in the Hyperthermophilic Archaeon Thermococcus litoralis: Purification and Characterization of Key Enzymes
复制标题
DOI：
10.1128/jb.181.11.3358-3367.1999
复制
发表时间：
1999-06
影响因子：
3.2
通讯作者：
K. Xavier;R. Peist;M. Kossmann;W. Boos;H. Santos
中科院分区：
文献类型：
--
作者：
K. Xavier;R. Peist;M. Kossmann;W. Boos;H. Santos
摘要研究了极端嗜热古菌滨海热球菌（Thermococcus litoralis）的麦芽糖代谢。麦芽糖的降解是由4-α-葡聚糖转移酶和麦芽糊精磷酸化酶（MalP）协同作用完成的。第一种酶产生葡萄糖和一系列麦芽糖糊精，当葡萄糖残基的链长等于或大于4时，麦芽糖糊精可以被MalP作用，以产生葡萄糖-1-磷酸。在T. litoralis细胞提取物中也检测到磷酸葡萄糖变位酶活性。4-α-葡聚糖转移酶作用产生的葡萄糖随后通过Embden-Meyerhof途径代谢。密切相关的生物激烈火球菌使用了不同的代谢策略，其中麦芽糖主要通过α-葡萄糖苷酶、对硝基苯基-α-d-吡喃葡萄糖苷（PNPG）水解酶的作用被切割，从麦芽糖产生葡萄糖。T. litoralis，但麦芽糖不是这种酶的底物。对T. litoralis纯化到同质和特点;他们组成型合成，虽然磷酸化酶的表达是由麦芽糖糊精或麦芽糖诱导的两倍。通过在大肠杆菌中互补获得编码MalP的基因并测序（计算分子量，96，622 Da）。从生物体纯化的酶在最大活性温度（98°C）下对麦芽七糖具有66 U/mg的比活性。在60°C下，用七糖作为底物测定AKm为0.46 mM。推导的氨基酸序列与超嗜热古菌Pyrococcus horikoshii OT 3的推测酶的氨基酸序列有较高的同源性（66%），与超嗜热细菌Thermotoga maritima的酶的氨基酸序列有较高的同源性（60%），与结核分枝杆菌的酶的氨基酸序列有较高的同源性（31%），而与大肠杆菌的酶的氨基酸序列没有同源性。大肠杆菌（13%）。吡哆醛5′-磷酸的共有结合位点在T. litoralis酶
ABSTRACT Maltose metabolism was investigated in the hyperthermophilic archaeon Thermococcus litoralis. Maltose was degraded by the concerted action of 4-α-glucanotransferase and maltodextrin phosphorylase (MalP). The first enzyme produced glucose and a series of maltodextrins that could be acted upon by MalP when the chain length of glucose residues was equal or higher than four, to produce glucose-1-phosphate. Phosphoglucomutase activity was also detected inT. litoralis cell extracts. Glucose derived from the action of 4-α-glucanotransferase was subsequently metabolized via an Embden-Meyerhof pathway. The closely related organism Pyrococcus furiosus used a different metabolic strategy in which maltose was cleaved primarily by the action of an α-glucosidase, ap-nitrophenyl-α-d-glucopyranoside (PNPG)-hydrolyzing enzyme, producing glucose from maltose. A PNPG-hydrolyzing activity was also detected in T. litoralis, but maltose was not a substrate for this enzyme. The two key enzymes in the pathway for maltose catabolism in T. litoralis were purified to homogeneity and characterized; they were constitutively synthesized, although phosphorylase expression was twofold induced by maltodextrins or maltose. The gene encoding MalP was obtained by complementation in Escherichia coli and sequenced (calculated molecular mass, 96,622 Da). The enzyme purified from the organism had a specific activity for maltoheptaose, at the temperature for maximal activity (98°C), of 66 U/mg. AKm of 0.46 mM was determined with heptaose as the substrate at 60°C. The deduced amino acid sequence had a high degree of identity with that of the putative enzyme from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 (66%) and with sequences of the enzymes from the hyperthermophilic bacteriumThermotoga maritima (60%) and Mycobacterium tuberculosis (31%) but not with that of the enzyme from E. coli (13%). The consensus binding site for pyridoxal 5′-phosphate is conserved in the T. litoralis enzyme.