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


RESULTS
The 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.


CONCLUSIONS
A 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.
Chinese Academy of Sciences Key Laboratory of Pathogenic Microbiology and Immunology, Institute of Microbiology, Chinese Academy of Sciences, Bei'er Tiao Road, Zhongguancun Haidian District, Beijing 100190, China.
Tao
T
Zhang
Xiaopeng
X
Jia
Ying
Y
Zhuo
Mei
M
Liu
Hong
H
Gao
Jintao
J
Liu
Lixin
L
Zhang
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D047630:Depsipeptides / Q000096:biosynthesis*; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D005670:Fusarium / Q000737:chemistry / Q000145:classification / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D010802:Phylogeny; D016415:Sequence Alignment
D047630:Depsipeptides; D005656:Fungal Proteins; C004456:beauvericin; D000429:Alcohol Oxidoreductases; C021934:2-dehydropantoate 2-reductase