Cloning and characterization of a novel 2-ketoisovalerate reductase from the beauvericin producer Fusarium proliferatum LF061.
Cloning and characterization of a novel 2-ketoisovalerate reductase from the beauvericin producer Fusarium proliferatum LF061.
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来自白僵菌素生产者增殖镰刀菌 LF061 的新型 2-酮异戊酸还原酶的克隆和表征
DOI：
10.1186/1472-6750-12-55
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发表时间：
2012-08-23
影响因子：
3.5
通讯作者：
Zhang L
中科院分区：
文献类型：
--
作者：
Zhang T;Jia X;Zhuo Y;Liu M;Gao H;Liu J;Zhang L
研究背景：酮基异戊酸还原酶(EC 1.2.7.7)是通过非核糖体肽合成酶生物合成途径合成白藜芦素所必需的。它催化NADPH专一性地将酮基异戊酸还原为羟基异戊酸酯。然而，关于镰刀菌中2-KIV还原酶的生物信息学数据知之甚少。结果利用pET表达系统亚克隆了KivRFp基因，并在大肠杆菌BL21中进行了表达。KivRFp基因含有1,359 的开放阅读框，编码452个氨基酸的多肽，分子质量为52 kDa。序列分析表明，该基因与球孢白僵菌ATCC 7159(ACI30654)和绿僵菌CQMa 102(EFY89891)的酮基异戊酸还原酶的氨基酸同源性分别为61%和52%，并发现了几个保守的区域，包括推测的核苷酸结合签名位点GXGXXG、催化三联体(Glu405、Asn184和Lys285)。通过还原酮基异戊酸酯，KivRFp在35℃和pH 7.5时表现出最高的活性。在较宽的温度和pH范围内，以及在金属离子和洗涤剂的存在下，该酶具有较高的稳定性。结论从产去脂多肽的真菌增殖F.KivRFp已被证明具有中等的热稳定性和广泛的pH最佳值等有用的性质，并可能作为未来蛋白质工程和定向进化的起点，朝着为下游生物技术应用开发高效酶的目标迈进。
BackgroundThe ketoisovalerate reductase (EC 1.2.7.7 ) is required for the formation of beauvericin via the nonribosomal peptide synthetase biosynthetic pathway. It catalyzes the NADPH-specific reduction of ketoisovaleric acid to hydroxyisovalerate. However, little is known about the bioinformatics’ data about the 2-Kiv reductase in Fusarium. To date, heterologous production of the gene KivRFp from Fusarium has not been achieved.ResultsThe KivRFp gene was subcloned and expressed in Escherichia coli BL21 using the pET expression system. The gene KivRFp contained a 1,359 bp open reading frame (ORF) encoding a polypeptide of 452 amino acids with a molecular mass of 52 kDa. Sequence analysis indicated that it showed 61% and 52% amino acid identities to ketoisovalerate reductase from Beauveria bassiana ATCC 7159 (ACI30654) and Metarhizium acridum CQMa 102 (EFY89891), respectively; and several conserved regions were identified, including the putative nucleotide-binding signature site, GXGXXG, a catalytic triad (Glu405, Asn184, and Lys285). The KivRFp exhibited the highest activity at 35°C and pH 7.5 respectively, by reduction of ketoisovalerate. It also exhibited the high level of stability over wide temperature and pH spectra and in the presence of metal ions or detergents.ConclusionsA new ketoisovalerate reductase KivRFp was identified and characterized from the depsipeptide-producing fungus F. proliferatum. KivRFp has been shown to have useful properties, such as moderate thermal stability and broad pH optima, and may serve as the starting points for future protein engineering and directed evolution, towards the goal of developing efficient enzyme for downstream biotechnological applications.
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影响因子：
11.4
作者：
Kato, Masato;Wynn, R. Max;Chuang, David T.
通讯作者：
Chuang, David T.
影响因子：
--
作者：
Luangsa-Ard, J. Jennifer;Berkaew, Pitchapa;Isaka, Masahiko
通讯作者：
Isaka, Masahiko
影响因子：
2.9
作者：
BROWN, RE;JARVIS, KL;HYLAND, KJ
通讯作者：
HYLAND, KJ
影响因子：
10.7
作者：
SAITOU, N;NEI, M
通讯作者：
NEI, M
影响因子：
5.8
作者：
Arnold, K;Bordoli, L;Schwede, T
通讯作者：
Schwede, T