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Identification and sequence determination of the acetohydroxy acid isomeroreductase gene from Brevibacterium flavum
M Inui1, A A Vertès, M Kobayashi
1Tsukuba Research Center, Mitsubishi Petrochemical Co., Ltd, Ibaraki, Japan.
Abstract:
The enzyme acetohydroxy acid isomeroreductase (AHAIR) is the second enzyme in the parallel isoleucine-valine biosynthetic pathway. We previously reported the cloning and sequencing of the acetohydroxy acid synthase (AHAS) genes from Brevibacterium flavum MJ233. Analysis of the sequence downstream of the AHAS genes identified another open reading frame highly homologous at the amino acid level to the AHAIR gene from Escherichia coli (ilvC). We subcloned the B. flavum AHAIR gene on a 2.1 kb Bg/II-EcoRI fragment by complementation of an E. coli ilvC mutant. The nucleotide sequence of the B. flavum AHAIR gene consists of 338 codons (molecular weight of 36158). Comparison of the deduced protein sequence revealed a high degree of identity with the sequences of ilvC genes from other organisms. Disruption of the B. flavum ilvC gene by a kanamycin resistance cassette resulted in L-isoleucine and L-valine auxotrophy.