Expression in Escherichia coli, purification and kinetic analysis of the aspartokinase and aspartate semialdehyde dehydrogenase from the rifamycin SV-producing Amycolatopsis mediterranei U32

The operon encoding aspartokinase and aspartate semialdehyde dehydrogenase was cloned and sequenced from rifamycin-SV-producing Amycolatopsis mediterr anei U32 previously. In the present work, these two genes were introduced i nto the auxotrophic Esc-herichia coli strain CGSC5074 (ask(-)) and E. coli X6118 (asd(-)), respectively. The A. mediterranei U32 aspartokinase and asp artate semialdehyde dehydrogenase genes can be functionally expressed in E. coli and the gene products are able to substitute for the E. coli enzymes. Histidine-tagged aspartokinase and aspartate semialdehyde dehydrogenase we re partially purified from E. coli cellular extracts and their kinetic char acteristics were studied. Both aspartokinase and aspartate semialdehyde deh ydrogenase showed typical Michaelis-Menten type substrate saturation patter ns. Aspartokinase has K-m values of 3.4 mM for aspartate and 2.3 mM for ATP , while aspartate semialdehyde dehydrogenase has K-m values of 1.25 mM for or-aspartate semialdehyde and 0.73 mM for NADP, respectively. Aspartokinase was inhibited by L-threonine, L-lysine, and L-methionine, but not by L-iso leucine and diaminopimelate. Aspartate semialdehyde dehydrogenase was not i nhibited by any of the end-product amino acids at a concentration of less t han 5 mM. Hill plot analysis suggested that aspartokinase was subject to al losteric control by L-threonine. Repression of both aspartokinase and aspar tate semialdehyde dehydrogenase gene transcription in A. mediaterranei U32 by L-lysine, L-methionine, L-threonine, and L-isoleucine were found. The ne twork of regulation of aspartokinase and aspartate semialdehyde dehydrogena se in rifamycin SV-producing A. mediterranei U32 is presented.