The putative malate/lactate dehydrogenase from Pseudomonas putida is an NADPH-dependent Δ1-piperideine-2-carboxylate/Δ1-pyrroline-2-carboxylate reductase involved in the catabolism of D-lysine and D-proline
The putative malate/lactate dehydrogenase from Pseudomonas putida is an NADPH-dependent Δ1-piperideine-2-carboxylate/Δ1-pyrroline-2-carboxylate reductase involved in the catabolism of D-lysine and D-proline
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DOI：
10.1074/jbc.m411918200
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发表时间：
2005-02-18
影响因子：
4.8
通讯作者：
Esaki, N
中科院分区：
文献类型：
--
作者：
Muramatsu, H;Mihara, H;Esaki, N
通过同源重组破坏恶臭假单胞菌ATCC 12633基因dpkA，其编码在各种序列数据库中注释为苹果酸/L-乳酸脱氢酶的推定蛋白。得到的dpkA（-）突变体被剥夺了使用D-赖氨酸和D-脯氨酸作为唯一碳源的能力。克隆了dpkA基因并在大肠杆菌中过表达，并对基因产物进行了表征。该酶既不表现出苹果酸脱氢酶活性，也不表现出乳酸脱氢酶活性，但催化NADPH依赖性还原环亚胺如δ（1）-哌啶-2-羧酸和δ（1）-吡咯啉-2-羧酸，分别形成L-哌啶酸和L-脯氨酸。NADH也作为两种底物的氢供体，尽管反应速率小于NADPH的1%。逆反应也被酶催化，但速率要低得多。因此，该酶具有双重代谢功能，并且我们将酶Delta（1）-哌啶-2-羧酸/Delta（1）-吡咯啉-2-羧酸还原酶命名为NAD（P）依赖性氧化还原酶大家族中的新亚类的第一个成员。
A Pseudomonas putida ATCC 12633 gene, dpkA, encoding a putative protein annotated as malate/L-lactate dehydrogenase in various sequence data bases was disrupted by homologous recombination. The resultant dpkA(-) mutant was deprived of the ability to use D-lysine and also D-proline as a sole carbon source. The dpkA gene was cloned and overexpressed in Escherichia coli, and the gene product was characterized. The enzyme showed neither malate dehydrogenase nor lactate dehydrogenase activity but catalyzed the NADPH-dependent reduction of such cyclic imines as Delta(1)-piperideine-2-carboxylate and Delta(1)-pyrroline-2-carboxylate to form L-pipecolate and L-proline, respectively. NADH also served as a hydrogen donor for both substrates, although the reaction rates were less than 1% of those with NADPH. The reverse reactions were also catalyzed by the enzyme but at much lower rates. Thus, the enzyme has dual metabolic functions, and we named the enzyme Delta(1)-piperideine-2-carboxylate/Delta(1)-pyrroline-2-carboxylate reductase, the first member of a novel subclass in a large family of NAD(P)-dependent oxidoreductases.