A C-terminal deletion in Corynebacterium glutamicum homoserine dehydrogenase abolishes allosteric inhibition by l-threonine

In Escherichia coli, Bacillus subtilis and Corynebacterium glutamicum, homoserine dehydrogenase (HD), the enzyme after the branch point of the threonine/methionine and lysine biosynthetic pathways, is allosterically inhibited by l-threonine. To investigate the regulation of the C. glutamicum HD enzyme by l-threonine, the structural gene, hom, was mutated by UV irradiation of whole cells to obtain a deregulated allele, hom^dr. l-Threonine inhibits the wild-type (wt) enzyme with a K_i of 0.16 mM. The deregulated enzyme remains 80% active in the presence of 50 mM l-threonine. The hom^dr gene mutant was isolated and cloned in E. coli. In a C. glutamicum wt host background, but not in E. coli, the cloned hom^dr gene is genetically unstable. The cloned hom^dr gene is overexpressed tenfold in C. glutamicum and is active in the presence of over 60 mM l-threonine. Sequence analysis revealed that the hom^dr mutation is a single nucleotide (G¹⁹⁶⁴) deletion in codon 429 within the hom reading frame. The resulting frame-shift mutation radically alters the structure of the C terminus, resulting in ten amino acid (aa) changes and a deletion of the last 7 aa relative to the wt protein. These observations suggest that the C terminus may be associated with the l-threonine allosteric response. The hom^dr mutation is unstable and probably deleterious to the cell. This may explain why only one mutation was obtained despite repeated mutagenesis.