Cloning and characterization of a novel 2-ketoisovalerate reductase from the beauvericin producer Fusarium proliferatum LF061

Tao Zhang,Lixin Zhang,Jintao Liu,Xiaopeng Jia,Hong Gao,Ying Zhuo,Mei Liu

doi:10.1186/1472-6750-12-55

Abstract

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.

Highlights

The ketoisovalerate reductase (EC 1.2.7.7 ) is required for the formation of beauvericin via the nonribosomal peptide synthetase biosynthetic pathway
Xu et al, demonstrated the novel kivr-encoding 2-ketoisovalerate reductase is the sole supplier of D-Hiv for both beauvericin and bassianolide biosynthesis in Beauveria bassiana ATCC 7159 [20]
The FpBEAS gene encoding the beauvericin synthetase was found to be clustered with a gene (KivRFp) encoding a deduced putative 2-ketoisovalerate reductase (KivRFp)

Summary

Introduction

The 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. Beauvericin, produced by certain fungi of Beauveria [1], Isaria [6] and Fusarium [7] that exhibits insecticidal, displays antimicrobial, In mammals, the catabolism of the branched-chain amino acids (BCAAs) leucine, isoleucine, and valine proceeds by two-step process, i.e. transamination step (aminotransfer) and oxidation step. D-hydroxyisovalerate dehydrogenase, catalyzes the reversible reduction of 2-ketoisovalertate (2-Kiv), the intermediate of branched-chain amino acid metabolism [16], to D-Hiv in the presence of NADPH, which cannot be replaced by NADH [17,18,19]. Xu et al, demonstrated the novel kivr-encoding 2-ketoisovalerate reductase is the sole supplier of D-Hiv for both beauvericin and bassianolide biosynthesis in Beauveria bassiana ATCC 7159 [20]

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