Cloning, sequencing, heterologous expression, purification, and characterization of adenosylcobalamin-dependent D-lysine 5,6-aminomutase from Clostridium sticklandii
Cloning, sequencing, heterologous expression, purification, and characterization of adenosylcobalamin-dependent D-lysine 5,6-aminomutase from Clostridium sticklandii
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DOI：
10.1074/jbc.275.1.106
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发表时间：
2000-01-07
影响因子：
4.8
通讯作者：
Frey, PA
中科院分区：
文献类型：
--
作者：
Chang, CH;Frey, PA
d -赖氨酸5,6-氨基交换酶催化d -赖氨酸ε -氨基的1,2移位和C5(H)的反向迁移。编码5,6-氨基转氨酶的两个基因已被克隆、测序并在大肠杆菌中表达。它们在梭状染色体上相邻，编码57.3和29.2千道尔顿的多肽。预测的氨基酸序列包括一个保守的碱基5'-脱氧腺苷钴胺结合基序和一个小亚基的3-半胱氨酸基团，以及一个大亚基的p环序列。重组酶的活性比从c.s sticklandii纯化的5,6-氨基转氨酶高6倍，可能是由于重组酶中没有结合的、无活性的corrinoids。腺苷钴胺素和吡哆醛5′-磷酸的K-m值分别为6.6 μ M和1.0 μ M。ATP对重组蛋白没有调节作用。从梭状芽胞杆菌中纯化的酶的快速周转相关失活也见于重组形式。氨基转氨酶的活性不依赖于结构或催化金属离子。[n -15-二甲基苯-咪唑]腺苷钴胺素的电子顺磁共振实验证明了碱基结合，与其他b -12依赖性酶破坏未激活的C-H键一致。
D-Lysine 5,6-aminomutase from Clostridium sticklandii catalyzes the 1,2-shift of the epsilon-amino group of D-lysine and reverse migration of C5(H). The two genes encoding 5,6-aminomutase have been cloned, sequenced, and expressed in Escherchia coli. They are adjacent on the Clostridial chromosome and encode polypeptides of 57.3 and 29.2 kilodaltons. The predicted amino acid sequence includes a conserved base-off 5'-deoxyadenosylcobalamin binding motif and a 3-cysteine cluster in the small subunit, as well as a P-loop sequence in the large subunit, Activity of the recombinant enzyme exceeds that of the 5,6-aminomutase purified from C. sticklandii by 6-fold, presumably due to the absence of bound, inactive corrinoids in the recombinant enzyme. The K-m values for adenosylcobalamin and pyridoxal 5'-phosphate are 6.6 and 1.0 mu M, respectively. ATP does not have a regulatory effect on the recombinant protein. The rapid turnover associated inactivation reported for the enzyme purified from Clostridium is also seen with the recombinant form. Aminomutase activity does not depend on structural or catalytic metal ions. Electron paramagnetic resonance experiments with [N-15-dimethylbenz-imidazole]adenosylcobalamin demonstrate base-off binding, consistent with other B-12-dependent enzymes that break unactivated C-H bonds.