Characterization of an archaeal malic enzyme from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1
Characterization of an archaeal malic enzyme from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1
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DOI：
10.1155/2005/250757
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发表时间：
2005-05-01
期刊：
影响因子：
2.4
通讯作者：
Imanaka, Tadayuki
中科院分区：
文献类型：
--
作者：
Fukuda, Wakao;Ismail, Yulia Sari;Imanaka, Tadayuki
尽管C4和C3化合物之间的相互转化在超嗜热古菌的整体代谢中起重要作用，但对这些代谢物作用的酶的性质和调控的信息有限。苹果酸酶是参与这种相互转化的酶之一，它催化苹果酸氧化脱羧生成丙酮酸以及与NAD(P)H偶联的还原性羧化。本研究的重点是在异养、超嗜热古细菌kodakaraensis Thermococcus KOD1 （Tk-Mae）基因组中鉴定的一种未表征的苹果酸酶同源物的酶学特性和表达谱。Tk-Mae的氨基酸序列与细菌中苹果酸酶的相似度为52-58%，而与真核生物同源物的相似度较低。在Tk-Mae的一级结构中，有几个重要的催化区域和残基是保守的。重组蛋白形成同二聚体，表现出严格的二价阳离子依赖的耐热苹果酸酶活性。该酶更倾向于NADP（+）而不是NAD(+)，但不催化草酰乙酸的脱羧，这与通常依赖NADP的苹果酶不同。Tk-Mae对苹果酸盐的表观米切里斯常数（K-m）为16.9 mM，远远大于已知酶的表观米切里斯常数（K-m），导致其对反应方向没有强烈的偏好。无论生长基质如何，柯达卡拉白T的Tk-Mae基因转录和细胞内苹果酸酶活性均呈弱结构。在此基础上讨论了Tk-Mae可能的作用，并根据基因组序列推断出了柯达卡拉白T的代谢途径。
Although the interconversion between C4 and C3 compounds has an important role in overall metabolism, limited information is available on the properties and regulation of enzymes acting on these metabolites in hyperthermophilic archaea. Malic enzyme is one of the enzymes involved in this interconversion, catalyzing the oxidative decarboxylation of malate to pyruvate as well as the reductive carboxylation coupled with NAD(P)H. This study focused on the enzymatic properties and expression profile of an uncharacterized homolog of malic enzyme identified in the genome of a heterotrophic, hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 (Tk-Mae). The amino acid sequence of Tk-Mae was 52-58% identical to those of malic enzymes from bacteria, whereas the similarities to the eukaryotic homologs were lower. Several catalytically important regions and residues were conserved in the primary structure of Tk-Mae. The recombinant protein, which formed a homodimer, exhibited thermostable malic enzyme activity with strict divalent cation dependency. The enzyme preferred NADP(+) rather than NAD(+), but did not catalyze the decarboxylation of oxaloacetate, unlike the usual NADP-dependent malic enzymes. The apparent Michaelis constant (K-m) of Tk-Mae for malate (16.9 mM) was much larger than those of known enzymes, leading to no strong preference for the reaction direction. Transcription of the gene encoding Tk-Mae and intracellular malic enzyme activity in T kodakaraensis were constitutively weak, regardless of the growth substrates. Possible roles of Tk-Mae are discussed based on these results and the metabolic pathways of T kodakaraensis deduced from the genome sequence.