Malate Synthase and β-Methylmalyl Coenzyme A Lyase Reactions in the Methylaspartate Cycle in Haloarcula hispanica
Malate Synthase and β-Methylmalyl Coenzyme A Lyase Reactions in the Methylaspartate Cycle in Haloarcula hispanica
复制标题
Haloarcula hispanica 甲基天冬氨酸循环中的苹果酸合酶和 β-甲基苹果酰辅酶 A 裂解酶反应。
DOI：
10.1128/jb.00657-16
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发表时间：
2017-02-01
影响因子：
3.2
通讯作者：
Berg, Ivan A.
中科院分区：
文献类型：
--
作者：
Borjian, Farshad;Han, Jing;Berg, Ivan A.
盐古菌属是极端嗜盐的异养微生物，属于盐菌纲（广古菌门）。几乎一半的盐古菌拥有编码甲基天冬氨酸循环酶的基因，这是最近发现的回补乙酸同化途径。在这个循环中，三羧酸循环的酶与甲基天冬氨酸循环的专用酶一起将两个乙酰辅酶A（乙酰辅酶A）分子转化为苹果酸。甲基天冬氨酸循环涉及由属于CitE样酶超家族的同源酶催化的两个反应，即苹果酰辅酶A裂解酶/硫酯酶（盐古菌苹果酸合酶[hMS]; Hah_2476，在Haloarcula hispanica中）和β-甲基苹果酰辅酶A裂解酶（盐古菌β-甲基苹果酰辅酶A裂解酶[hMCL]; Hah_1341）。尽管两种酶催化相同的反应，但先前提出hMS优先催化由乙酰辅酶A和乙醛酸形成苹果酸（苹果酸合酶活性），并且提出hMCL主要将β-甲基苹果酰辅酶A裂解为丙酰辅酶A和乙醛酸。本文研究了这些酶在H.通过使用野生型和缺失突变体的生物化学测定，我们的研究结果表明，hMS的主要生理功能是苹果酰辅酶A（而不是苹果酸）的形成和hMCL催化的β-甲基苹果酰辅酶A裂解酶反应在体内。这两种酶的苹果酰辅酶A硫酯酶活性似乎对乙酸盐的生长不是必需的。有趣的是，尽管hMS和hMCL的生理功能不同，但结构比较预测这两种蛋白质具有几乎相同的活性位点，因此突出了对其催化功能进行实验验证的必要性。我们的研究结果进一步证明了甲基天冬氨酸循环的运作，并表明存在一个独特的，尚未被发现的苹果酰辅酶A硫酯酶在盐古菌。重要的是乙酸是在自然环境中最重要的物质之一。乙酸的活化形式，乙酰辅酶A（乙酰辅酶A），是中枢代谢十字路口的高能中间体：它的氧化为细胞产生能量，大约三分之一的生物合成通量直接从乙酰辅酶A开始。许多有机化合物通过这个关键分子进入中心碳代谢。为了维持乙酰辅酶A生成化合物的生长，需要专门的同化（回补）途径。回补途径的存在是在许多环境中生长的先决条件，对于环境、工业和临床上重要的微生物是重要的。在这里，我们研究了最近发现的乙酸同化途径，甲基天冬氨酸循环，在极端嗜盐古菌发挥作用的具体反应。
Haloarchaea are extremely halophilic heterotrophic microorganisms belonging to the class Halobacteria (Euryarchaeota). Almost half of the haloarchaea possesses the genes coding for enzymes of the methylaspartate cycle, a recently discovered anaplerotic acetate assimilation pathway. In this cycle, the enzymes of the tricarboxylic acid cycle together with the dedicated enzymes of the methylaspartate cycle convert two acetyl coenzyme A (acetyl-CoA) molecules to malate. The methylaspartate cycle involves two reactions catalyzed by homologous enzymes belonging to the CitE-like enzyme superfamily, malyl-CoA lyase/thioesterase (haloarchaeal malate synthase [hMS]; Hah_2476 in Haloarcula hispanica) and beta-methylmalyl-CoA lyase (haloarchaeal beta-methylmalyl-CoA lyase [hMCL]; Hah_1341). Although both enzymes catalyze the same reactions, hMS was previously proposed to preferentially catalyze the formation of malate from acetyl-CoA and glyoxylate (malate synthase activity) and hMCL was proposed to primarily cleave beta-methylmalyl-CoA to propionyl-CoA and glyoxylate. Here we studied the physiological functions of these enzymes during acetate assimilation in H. hispanica by using biochemical assays of the wild type and deletion mutants. Our results reveal that the main physiological function of hMS is malyl-CoA (not malate) formation and that hMCL catalyzes a beta-methylmalyl-CoA lyase reaction in vivo. The malyl-CoA thioesterase activities of both enzymes appear to be not essential for growth on acetate. Interestingly, despite the different physiological functions of hMS and hMCL, structural comparisons predict that these two proteins have virtually identical active sites, thus highlighting the need for experimental validation of their catalytic functions. Our results provide further proof of the operation of the methylaspartate cycle and indicate the existence of a distinct, yet-to- be-discovered malyl-CoA thioesterase in haloarchaea.IMPORTANCE Acetate is one of the most important substances in natural environments. The activated form of acetate, acetyl coenzyme A (acetyl-CoA), is the high-energy intermediate at the crossroads of central metabolism: its oxidation generates energy for the cell, and about a third of all biosynthetic fluxes start directly from acetyl-CoA. Many organic compounds enter the central carbon metabolism via this key molecule. To sustain growth on acetyl-CoA-generating compounds, a dedicated assimilation (anaplerotic) pathway is required. The presence of an anaplerotic pathway is a prerequisite for growth in many environments, being important for environmentally, industrially, and clinically important microorganisms. Here we studied specific reactions of a recently discovered acetate assimilation pathway, the methylaspartate cycle, functioning in extremely halophilic archaea.