Molecular analysis of the Corynebacterium glutamicum gdh gene encoding glutamate dehydrogenase

Abstract

The Corynebacterium glutamicum gdh gene encoding NADP-dependent glutamate dehydrogenase (GDH) has been isolated by complementation of the Escherichia coli gdh mutant PA340. The gdh gene was subcloned into the E. coli/C. glutamicum shuttle vector pEK0 and introduced into C. glutamicum. Recombinant strains showed approximately eightfold higher specific GDH activity (15U mg protein-1) relative to the wild type (1.8U mg protein-1). Physiological studies with wild-type and recombinant C. glutamicum strains revealed no indication of significant regulation of gdh expression. The DNA sequence of 2082 bp, including the gdh gene, 5'-, and 3'-flanking regions, was determined. The structural gene consists of 1344 bp and codes for a polypeptide of 448 amino acid residues (Mr 49,152) showing up to 53.6% identity with reported amino acid sequences of glutamate dehydrogenases from other organisms. Northern blot hybridization revealed a 1.65kb mRNA transcript, indicating that the gdh gene of C. glutamicum is monocistronic. Transcription occurred from a G residue located 284 bp upstream of the AUG considered to be the translational initiation codon.