Molecular Characterization of a Novel N-Acetyltransferase from Chryseobacterium sp.
Molecular Characterization of a Novel N-Acetyltransferase from Chryseobacterium sp.
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金黄杆菌属新型 N-乙酰转移酶的分子表征。
DOI：
10.1128/aem.03449-13
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发表时间：
2014
期刊：
影响因子：
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通讯作者：
and Kenich Yoshida
中科院分区：
文献类型：
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作者：
Shinji Takenaka;Kenji Yoshida;Kosei Tanaka;and Kenich Yoshida
金黄色杆菌N-乙酰转移酶的研究菌株5-3B是一种乙酰辅酶A(乙酰辅酶A)依赖的酶，催化乙酰辅酶A上的乙酰基对映体选择性地转移到1-2-苯甘氨酸的氨基上，生成(2S)-2-乙酰氨基-2-苯基乙酸。我们从菌株5-3B中纯化了该酶，并推导了其N-端氨基酸序列。该基因被命名为NatA，与另外两个假设的蛋白质基因一起被克隆；这三个基因可能形成了一个2.5kb的操纵子。其推导的氨基酸序列与金杆菌N-乙酰转移酶序列高度一致。但不能与其他已知的芳胺和芳基烷基胺N-乙酰基转移酶结合。系统发育分析表明，NatA与已知的N-乙酰转移酶形成了不同的谱系。我们在大肠杆菌中异源表达了重组NatA(RNatA)，并进行了纯化。RNatA对1-2-苯甘氨酸及其氯代和羟基衍生物具有较高的活性。1-2-苯甘氨酸的KmandVmax分别为0.145±0.026 mm和43.6±2.39mmol.minμ−1·mg蛋白−1。该酶对5-氨基水杨酸和5-羟色胺的活性较低，而5-氨基水杨酸和5-羟色胺是已知的芳香胺N-乙酰基转移酶和芳基烷胺N-乙酰基转移酶的良好底物。RNatA具有相对广泛的酰基供体特异性，将酰基转移到L-2-苯甘氨酸并产生相应的2-乙酰氨基-2-苯基乙酸(与乙酰基供体乙酰辅酶A、丙酰辅酶A、丁酰辅酶A、戊酰辅酶A和己酰辅酶A的相对活性，100：108：122：10：<；1)。
N-Acetyltransferase from Chryseobacterium sp. strain 5-3B is an acetyl coenzyme A (acetyl-CoA)-dependent enzyme that catalyzes the enantioselective transfer of an acetyl group from acetyl-CoA to the amino group ofl-2-phenylglycine to produce (2S)-2-acetylamino-2-phenylacetic acid. We purified the enzyme from strain 5-3B and deduced the N-terminal amino acid sequence. The gene, designatednatA, was cloned with two other hypothetical protein genes; the three genes probably form a 2.5-kb operon. The deduced amino acid sequence of NatA showed high levels of identity to sequences of putativeN-acetyltransferases of Chryseobacterium spp. but not to other known arylamine and arylalkylamineN-acetyltransferases. Phylogenetic analysis indicated that NatA forms a distinct lineage from knownN-acetyltransferases. We heterologously expressed recombinant NatA (rNatA) in Escherichia coli and purified it. rNatA showed high activity forl-2-phenylglycine and its chloro- and hydroxyl-derivatives. TheKmandVmaxvalues forl-2-phenylglycine were 0.145 ± 0.026 mM and 43.6 ± 2.39 μmol · min−1· mg protein−1, respectively. The enzyme showed low activity for 5-aminosalicylic acid and 5-hydroxytryptamine, which are reported as good substrates of a known arylamineN-acetyltransferase and an arylalkylamineN-acetyltransferase. rNatA had a comparatively broad acyl donor specificity, transferring acyl groups tol-2-phenylglycine and producing the corresponding 2-acetylamino-2-phenylacetic acids (relative activity with acetyl donors acetyl-CoA, propanoyl-CoA, butanoyl-CoA, pentanoyl-CoA, and hexanoyl-CoA, 100:108:122:10:<1).