Acetylpolyamine amidohydrolase from Mycoplana ramosa: Gene cloning and characterization of the metal-substituted enzyme
Acetylpolyamine amidohydrolase from Mycoplana ramosa: Gene cloning and characterization of the metal-substituted enzyme
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DOI：
10.1128/jb.178.19.5781-5786.1996
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发表时间：
1996-10-01
影响因子：
3.2
通讯作者：
Aisaka, K
中科院分区：
文献类型：
--
作者：
Sakurada, K;Ohta, T;Aisaka, K
我们从M. ramosa ATCC 49678（以前称为Mycoplana bullata）中克隆了编码乙酰多胺酰胺水解酶的基因（aphA），用从M. ramosa纯化的酶的N端氨基酸序列设计的寡核苷酸探针筛选M. ramosa的基因组文库，核苷酸序列分析揭示了1,023 bp的开放阅读框，其编码分子量为36,337 Da，这是乙酰多胺酰胺水解酶结构的首次报道。将aphA基因亚克隆到trc启动子的控制下，并在大肠杆菌MM294中表达。纯化重组酶，并对酶性质进行表征，底物特异性、K-m 值和 V-max 值与从 M. ramosa 中纯化的天然酶相同。在对金属取代酶的分析中，我们发现 pH 速率曲线的酸性部分从原始锌酶的 7.2 变为钴酶的 6.6，这一变化表明锌原子对于酶的催化活性至关重要，类似于羧肽酶 A 中的锌原子。
We have cloned a gene (aphA) encoding acetylpolyamine amidohydrolase from Mycoplana ramosa ATCC 49678 (previously named Mycoplana bullata), A genomic library of M. ramosa was screened with an oligonucleotide probe designed from a N-terminal amino acid sequence of the enzyme purified from M. ramosa, Nucleotide sequence analysis revealed an open reading frame of 1,023 bp which encodes a polypeptide with a molecular mass of 36,337 Da, This is the first report of the structure of acetylpolyamine amidohydrolase. The aphA gene was subcloned under the control of the trc promoter and was expressed in Escherichia coli MM294. The recombinant enzyme was purified, and the enzymatic properties were characterized, Substrate specificities, K-m values, and V-max values were identical to those of the native enzyme purified from M. ramosa. In the analysis of the metal-substituted enzymes, we found that the acid limb of pH rate profiles shifts from 7.2 for the original zinc enzyme to 6.6 for the cobalt enzyme, This change suggests that the zinc atom is essential for the catalytic activity of the enzyme similarly to the zinc atom in carboxypeptidase A.