Purification and characterization of a malic enzyme from the ruminal bacterium Streptococcus bovis ATCC 15352 and cloning and sequencing of its gene
Purification and characterization of a malic enzyme from the ruminal bacterium Streptococcus bovis ATCC 15352 and cloning and sequencing of its gene
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DOI：
10.1128/aem.62.8.2692-2700.1996
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发表时间：
1996-08-01
影响因子：
4.4
通讯作者：
Kumagai, H
中科院分区：
文献类型：
--
作者：
Kawai, S;Suzuki, H;Kumagai, H
苹果酸酶（EC 1.1.1.39）催化L-苹果酸氧化脱羧和丙酮酸还原羧化，从牛链球菌ATCC 15352中纯化至均一，并测定该酶的性质。克隆了含有苹果酸酶基因的2.9kb片段，并测定和分析了序列。研究了S.牛苹果酸酶与以往报道的苹果酸酶几乎相同，但我们发现，S.牛苹果酸酶催化未知的酶促反应，包括还原2-氧代异戊酸、还原2-氧代异己酸、氧化D-2-羟基异戊酸和氧化D-2-羟基异己酸。这些独特活性对阳离子的需求和最适pH与L-苹果酸氧化脱羧活性对阳离子的需求和最适pH不同。对克隆片段的序列分析表明存在两个开放阅读框，分别为1，299和1，170个核苷酸。从1，170个核苷酸的开放阅读框推导出的389个氨基酸的多肽被鉴定为苹果酸酶;该酶与嗜热脂肪芽孢杆菌和流感嗜血杆菌的苹果酸酶表现出高度的相似性，酶和乳酸乳球菌的苹果酸-乳酸酶。
Malic enzyme (EC 1.1.1.39), which catalyzes L-malate oxidative decarboxylation and pyruvate reductive carboxylation, was purified to homogeneity from Streptococcus bovis ATCC 15352, and properties of this enzyme were determined, The 2.9-kb fragment containing the malic enzyme gene was cloned, and the sequence was determined and analyzed. The enzymatic properties of the S. bovis malic enzyme were almost identical to those of other malic enzymes previously reported, However, we found that the S. bovis malic enzyme catalyzed unknown enzymatic reactions, including reduction of 2-oxoisovalerate, reduction of 2-oxoisocaproate, oxidation of D-2-hydroxyisovalerate, and oxidation of D-2-hydroxyisocaproate. The requirement for cations and the optimum pH of these unique activities were different from the requirement for cations and the optimum pH of the L-malate oxidative decarboxylating activity. A sequence analysis of the cloned fragment revealed the presence of two open reading frames that sere 1,299 and 1,170 nucleotides long, The 389-amino-acid polypeptide deduced from the 1,170-nucleotide open reading frame was identified as the malic enzyme; this enzyme exhibited high levels of similarity to malic enzymes of Bacillus stearothermophilus and Haemophilus influenzae and was also similar to other malic enzymes and the malolactic enzyme of Lactococcus lactis.