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首页> 外文期刊>Journal of bacteriology >Cellobiose-6-Phosphate Hydrolase (CelF) ofEscherichia coli: Characterization and Assignment to the Unusual Family 4 of Glycosylhydrolases
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Cellobiose-6-Phosphate Hydrolase (CelF) ofEscherichia coli: Characterization and Assignment to the Unusual Family 4 of Glycosylhydrolases

机译：大肠杆菌的纤维二糖6磷酸水解酶（CelF）：糖基水解酶异常家族4的表征和分配

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摘要

The gene celF of the cryptic cel operon ofEscherichia coli has been cloned, and the encoded 6-phospho-β-glucosidase (cellobiose-6-phosphate [6P] hydrolase; CelF [EC 3.2.1.86]) has been expressed and purified in a catalytically active state. Among phospho-β-glycosidases, CelF exhibits unique requirements for a divalent metal ion and NAD+ for activity and, by sequence alignment, is assigned to family 4 of the glycosylhydrolase superfamily. CelF hydrolyzed a variety of P-β-glucosides, including cellobiose-6P, salicin-6P, arbutin-6P, gentiobiose-6P, methyl-β-glucoside-6P, and the chromogenic analog,p-nitrophenyl-β-d-glucopyranoside-6P. In the absence of a metal ion and NAD+, purified CelF was rapidly and irreversibly inactivated. The functional roles of the cofactors have not been established, but NAD+ appears not to be a reactant and there is no evidence for reduction of the nucleotide during substrate cleavage. In solution, native CelF exists as a homotetramer (M w, ～200,000) composed of noncovalently linked subunits, and this oligomeric structure is maintained independently of the presence or absence of a metal ion. The molecular weight of the CelF monomer (M r, ～50,000), estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is in agreement with that calculated from the amino acid sequence of the polypeptide (450 residues;M r = 50,512). Comparative sequence alignments provide tentative identification of the NAD+-binding domain (residues 7 to 40) and catalytically important glutamyl residues (Glu112 and Glu356) of CelF.
机译：克隆了大肠杆菌的隐秘 cel 操纵子的基因 celF ，并编码了6-磷酸-β-葡萄糖苷酶（cellobiose-6 -磷酸[6P]水解酶; CelF [EC 3.2.1.86]）已以催化活性状态表达和纯化。在磷酸-β-糖苷酶中，CelF对二价金属离子和NAD + 具有独特的活性要求，并通过序列比对将其分配给糖基水解酶超家族的第4族。 CelF水解了各种P-β-葡萄糖苷，包括纤维二糖6P，水杨素6P，熊果苷6P，龙胆二糖6P，甲基-β-葡萄糖苷6P和生色类似物 p -硝基苯基-β-d-吡喃葡萄糖苷-6P。在没有金属离子和NAD + 的情况下，纯化的CelF迅速且不可逆地失活。辅助因子的功能性作用尚未确定，但是NAD + 似乎不是反应物，也没有证据表明在底物切割过程中核苷酸会减少。在溶液中，天然CelF是以非共价连接的亚基组成的同四聚体（ M w ，〜200,000）的形式存在，并且这种寡聚结构的维持与存在或不存在金属离子。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测得的CelF单体分子量（ M r ，〜50,000）与氨基酸序列计算的分子量相符多肽的残基（450个残基; M r = 50,512）。比较的序列比对可初步鉴定NAD + 结合结构域（残基7至40）以及催化上重要的谷氨酰基残基（Glu 112 和Glu 356 ）。

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