Cloning, expression, and characterization of bacterial L-arabinose 1-dehydrogenase involved in an alternative pathway of L-arabinose metabolism
Cloning, expression, and characterization of bacterial L-arabinose 1-dehydrogenase involved in an alternative pathway of L-arabinose metabolism
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DOI：
10.1074/jbc.m506477200
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发表时间：
2006-02-03
影响因子：
4.8
通讯作者：
Makino, K
中科院分区：
文献类型：
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作者：
Watanabe, S;Kodak, T;Makino, K
巴西固氮螺菌通过5个假设酶步骤将L-阿拉伯糖转化为α-酮戊二酸。我们纯化并鉴定了L-阿拉伯糖1-脱氢酶(EC1.1.1.46)，该酶催化L-阿拉伯糖转化为L-阿拉伯-伽马内酯，是L-阿拉伯糖代谢这一交替途径的第一步。纯化后的酶以NADP(+)为主，而以NAD(+)为主。动力学分析表明，该酶对L-阿拉伯糖和D-半乳糖均有较高的催化效率。用纯化的L-阿拉伯糖1-脱氢酶的部分肽序列克隆了该酶的编码基因，并在大肠杆菌中进行了高效表达。该酶由308个氨基酸组成，计算相对分子质量为33663.92Da。推导的氨基酸序列与葡萄糖-果糖氧化还原酶、D-木糖1-脱氢酶和D-半乳糖1-脱氢酶有一定的相似性。定点突变表明，该酶具有独特的催化氨基酸残基。Northern杂交分析表明，该基因受L-阿拉伯糖诱导，但不受D-半乳糖诱导。此外，L-阿拉伯糖1-脱氢酶基因的一个干扰物不能在L-阿拉伯糖上生长，而在D-半乳糖上以与野生型菌株相同的生长速度生长。在L-阿拉伯糖1-脱氢酶基因的侧翼区域存在一个与L-阿拉伯糖转运有关的部分基因。这些结果表明，该酶参与了L-阿拉伯糖的代谢，但不参与D-半乳糖的代谢。这是首次在细菌中鉴定出参与L-阿拉伯糖代谢的另一条途径的基因。
Azospirillum brasiliense converts L-arabinose to alpha-ketoglutarate via five hypothetical enzymatic steps. We purified and characterized L-arabinose 1-dehydrogenase (EC 1.1.1.46), catalyzing the conversion of L-arabinose to L-arabino-gamma-lactone as an enzyme responsible for the first step of this alternative pathway of L-arabinose metabolism. The purified enzyme preferred NADP(+) to NAD(+) as a coenzyme. Kinetic analysis revealed that the enzyme had high catalytic efficiency for both L-arabinose and D-galactose. The gene encoding L-arabinose 1-dehydrogenase was cloned using a partial peptide sequence of the purified enzyme and was overexpressed in Escherichia coli as a fully active enzyme. The enzyme consists of 308 amino acids and has a calculated molecular mass of 33,663.92 Da. The deduced amino acid sequence had some similarity to glucose-fructose oxidoreductase, D-xylose 1-dehydrogenase, and D-galactose 1-dehydrogenase. Site-directed mutagenesis revealed that the enzyme possesses unique catalytic amino acid residues. Northern blot analysis showed that this gene was induced by L-arabinose but not by D-galactose. Furthermore, a disruptant of the L-arabinose 1-dehydrogenase gene did not grow on L-arabinose but grew on D-galactose at the same growth rate as the wild-type strain. There was a partial gene for L-arabinose transport in the flanking region of the L-arabinose 1-dehydrogenase gene. These results indicated that the enzyme is involved in the metabolism of L-arabinose but not D-galactose. This is the first identification of a gene involved in an alternative pathway of L-arabinose metabolism in bacterium.