2-Methylisocitrate lyases from the bacterium Escherichia coli and the filamentous fungus Aspergillus nidulans -: Characterization and comparison of both enzymes
2-Methylisocitrate lyases from the bacterium Escherichia coli and the filamentous fungus Aspergillus nidulans -: Characterization and comparison of both enzymes
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DOI：
10.1046/j.1432-1327.2001.02262.x
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发表时间：
2001-06-01
期刊：
影响因子：
--
通讯作者：
Buckel, W
中科院分区：
文献类型：
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作者：
Brock, M;Darley, D;Buckel, W
在大肠杆菌和尼杜拉曲霉中，丙酸通过柠檬酸甲酯循环氧化成丙酮酸。在这个循环的最后一步，2-甲基异柠檬酸裂解酶催化2-甲基异柠檬酸裂解为丁二酸和丙酮酸。这两种生物的酶都是用化学合成的苏氨酸-2-甲基异柠檬酸测定的；红血球非对映异构体没有活性。来自大肠杆菌的2-甲基异柠檬酸裂解酶对应于参与丙酸氧化的PRP操纵子的PRPB蛋白。纯化的酶每个亚基的相对分子质量约为32 kDa，低于细菌来源的异柠檬酸裂解酶的相对分子质量(约48 kDa)。2-甲基异柠檬酸裂解酶的表观分子质量为66 kDa/亚基，与同一种微生物的异柠檬酸裂解酶的表观分子质量几乎相等。两种2-甲基异柠檬酸裂解酶均具有天然的同源四聚体结构。这些酶在异柠檬酸中没有可测量的活性。以250 mM丙酮酸、150 mM琥珀酸根和10um丙酮酸为起始原料，以1%的产率合成了具有酶活性的立体异构体。手性高效液相色谱分析表明，产物由单一对映体组成。这一异构体被2-甲基异柠檬酸裂解酶从嗜酸杆菌和大肠杆菌中裂解。PrPB蛋白与化学计量比的3-溴丙酮酸反应，失去活性，每个亚基有一个氨基酸残基被修饰，很可能是半胱氨酸，如大肠杆菌异柠檬酸裂解酶所示。PrPB与吸水链霉菌的羧基磷酸烯醇式丙酮酸磷酸变位酶有34%的序列同源性，其中必需的半胱氨酸残基是保守的。
In Escherichia coli and Aspergillus nidulans, propionate is oxidized to pyruvate via the methylcitrate cycle. The last step of this cycle, the cleavage of 2-methylisocitrate to succinate and pyruvate is catalysed by 2-methylisocitrate lyase. The enzymes from both organisms were assayed with chemically synthesized threo-2-methylisocitrate; the erythro-diastereomer was not active. 2-Methylisocitrate lyase from E. coli corresponds to the PrpB protein of the prp operon involved in propionate oxidation. The purified enzyme has a molecular mass of approximately 32 kDa per subunit, which is lower than those of isocitrate lyases from bacterial sources (approximate to 48 kDa). 2-Methylisocitrate lyase from A. nidulans shows an apparent molecular mass of 66 kDa per subunit, almost equal to that of isocitrate lyase of the same organism. Both 2-methylisocitrate lyases have a native homotetrameric structure as identified by size-exclusion chromatography. The enzymes show no measurable activity with isocitrate. Starting from 250 mm pyruvate, 150 mm succinate and 10 mum PrpB, the enzymatically active stereoisomer could be synthesized in 1% yield. As revealed by chiral HPLC, the product consisted of a single enantiomer. This isomer is cleaved by 2-methylisocitrate lyases from A. nidulans and E. coli. The PrpB protein reacted with stoichiometric amounts of 3-bromopyruvate whereby the activity was lost and one amino-acid residue per subunit became modified, most likely a cysteine as shown for isocitrate lyase of E. coli. PrpB exhibits 34% sequence identity with carboxyphosphoenolpyruvate phosphonomutase from Streptomyces hygroscopicus, in which the essential cysteine residue is conserved.