Homoserine Dehydrogenase Genetically Desensitized to the Feedback Inhibition in Brevibacterium flavum

Publisher Website
Google Scholar
Abstract
Homoserine dehydrogenase [L-homoserine: NADP oxidoreductase, EC 1. 1. 1. 3] from Brevibacterium flavum and from its mutants which were resistant to DL-α-amino-β-hydroxyvaleric acid (AHV) * a threonine analogue, and which produced a large amount of threonine, were partially purified and their properties were compared with each other. AHV inhibited the parental enzyme, similarly to the end products, threonine and isoleucine. The types of the inhibition were competitive to both of the two substrates. In the presence of AHV, homotropic cooperativity of NADPH was observed, as in the presence of threonine or isoleucine. In contrast to the parental enzyme, the mutant enzymes were scarcely inhibited by these amino acids. Concentration of threonine giving 50% inhibition was above 80 mM with the mutant enzymes while 0.06 mM with the parental enzyme. On the other hand, the inhibition by the reaction products, homoserine and NADP+, were of similar degree with the two enzymes. In the absence of KC1, homoserine dehydrogenase of the parental strain was rapidly inactivated, which was protected by the addition of threonine, while the mutant enzymes were stable without both KC1 and threonine. Molecular weight of homoserine dehydrogenase, which was determined to be about 2.5×105 by the gel filtration experiments, was not different between the parental and the mutant strains. Kms for the substrates, aspartic-β-semialdehyde (ASA)* and NADPH, as well as optimum pH for the reaction were similar with these enzymes. Repression of homoserine dehydrogenase by methionine was similarly observed with the two mutants. Aspartate kinase [EC 2. 7. 2. 4] from one of the mutant strains was concertedly inhibited by lysine plus threonine, similarly to that from the parental strain. From the gel filtration experiments, it was confirmed that aspartate kinase and homoserine dehydrogenase of B. flavum were not aggregated with each other in contrast to threonine-sensitive enzymes of Escherichia coli. It is likely from these results that resistance of the mutants to AHV as well as threonine overproduction in the mutants are due to the lack of the feedback inhibition in the mutant homoserine dehydrogenase. This supports the previous suggestion that homoserine dehydrogenase may be the primary control site in the threonine biosynthesis in B. flavum.

This publication has 0 references indexed in Scilit: