Characterization of homoisocitrate dehydrogenase involved in lysine biosynthesis of an extremely thermophilic bacterium, Thermus thermophilus HB27, and evolutionary implication of β-decarboxylating dehydrogenase
Characterization of homoisocitrate dehydrogenase involved in lysine biosynthesis of an extremely thermophilic bacterium, Thermus thermophilus HB27, and evolutionary implication of β-decarboxylating dehydrogenase
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DOI：
10.1074/jbc.m205133200
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发表时间：
2003-01-17
影响因子：
4.8
通讯作者：
Yamane, H
中科院分区：
文献类型：
--
作者：
Miyazaki, J;Kobashi, N;Yamane, H
尽管在赖氨酸生物合成途径中通过a-氨基己二酸（AAA）催化同型异柠檬酸的β -脱羧脱氢合成2-氧己二酸的酶已经被假设存在，但这种酶还没有被分析，因为直到最近才发现编码这种酶的基因。从嗜热热菌HB27中克隆了一个氨基酸序列与异柠檬酸脱氢酶和3-异丙基苹果酸脱氢酶同源的蛋白基因。在重组大肠杆菌细胞中产生的基因产物显示出高异柠檬酸脱氢酶（HICDH）活性。该基因的敲除突变体表现为AAA-营养不良表型，表明该基因产物通过AAA参与赖氨酸的生物合成，因此我们将该基因命名为hicdh。基因产物HICDH没有催化3-异丙基苹果酸转化为2-氧异己酸，这是一种赖氨酸生物合成反应，但它确实识别了三羧酸循环中的相关化合物异柠檬酸，以及作为底物的同型异柠檬酸。令人感兴趣的是，HICDH催化异柠檬酸盐反应的效率比与假定的天然底物同质异柠檬酸盐反应的效率高约20倍。广泛的特异性和可能的双重功能表明，该酶是利用柠檬酸衍生物的途径进化的关键环节。定点诱变研究表明，用Val取代Arg（85）会导致HICDH对异柠檬酸盐的活性完全丧失，但对3-异丙基苹果酸盐的活性显著，并保留对同异柠檬酸盐的活性。这些结果表明，Arg（85）是底物特异性和β -脱羧脱氢酶进化的关键残基。
Although the presence of an enzyme that catalyzes beta-decarboxylating dehydrogenation of homoisocitrate to synthesize 2-oxoadipate has been postulated in the lysine biosynthesis pathway through a-aminoadipate (AAA), the enzyme has not yet been analyzed at all, because no gene encoding the enzyme has been identified until recently. A gene encoding a protein with a significant amino acid sequence identity to both isocitrate dehydrogenase and 3-isopropylmalate dehydrogenase was cloned from Thermus thermophilus HB27. The gene product produced in recombinant Escherichia coli cells demonstrated homoisocitrate dehydrogenase (HICDH) activity. A knockout mutant of the gene showed an AAA-auxotrophic phenotype, indicating that the gene product is involved in lysine biosynthesis through AAA. We therefore named this gene hicdh. HICDH, the gene product, did not catalyze the conversion of 3-isopropylmalate to 2-oxoisocaproate, a leucine biosynthetic reaction, but it did recognize isocitrate, a related compound in the tricarboxylic acid cycle, as well as homoisocitrate as a substrate. It is of interest that HICDH catalyzes the reaction with isocitrate about 20 times more efficiently than the reaction with the putative native substrate, homoisocitrate. The broad specificity and possible dual function suggest that this enzyme represents a key link in the evolution of the pathways utilizing citrate derivatives. Site-directed mutagenesis study reveals that replacement of Arg(85) with Val in HICDH causes complete loss of activity with isocitrate but significant activity with 3-isopropylmalate and retains activity with homoisocitrate. These results indicate that Arg(85) is a key residue for both substrate specificity and evolution of beta-decarboxylating dehydrogenases.