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, homdr. L-Threonine inhibits the wild-type (wt) enzyme with a Ki of 0.16 mM. The deregulated enzyme remains 80% active in the presence of 50 mM L-threonine. The homdr gene mutant was isolated and cloned in E. coli. In a C. glutamicum wt host background, but not in E. coli, the cloned homdr gene is genetically unstable. The cloned homdr gene is overexpressed tenfold in C. glutamicum and is active in the presence of over 60 mM L-threonine. Sequence analysis revealed that the homdr 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 homdr mutation is unstable and probably deleterious to the cell. This may explain why only one mutation was obtained despite repeated mutagenesis.
Archer
J A
JA
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Solow-Cordero
D E
DE
Sinskey
A J
AJ
D000494:Allosteric Regulation / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D003352:Corynebacterium / Q000201:enzymology* / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics*; D006715:Homoserine Dehydrogenase / Q000037:antagonists & inhibitors / Q000235:genetics*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009154:Mutation / Q000235:genetics; D015183:Restriction Mapping; D013912:Threonine / Q000494:pharmacology*
D013912:Threonine; D006715:Homoserine Dehydrogenase