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 mediterranei U32 previously. In the present work, these two genes were introduced into the auxotrophic Escherichia coli strain CGSC5074 (ask-) and E. coli X6118 (asd-), respectively. The A. mediterranei U32 asparto-kinase and aspartate 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 were partially purified from E. coli cellular extracts and their kinetic characteristics were studied. Both aspartokinase and aspartate semialdehyde dehydrogenase showed typical Michaelis-Menten type substrate saturation patterns. Aspartokinase has Km values of 3.4 mM for aspartate and 2.3 mM for ATP, while aspartate semialdehyde dehydrogenase has Km values of 1.25 mM for DL-aspartate semialdehyde and 0.73 mM for NADP, respectively. Aspartokinase was inhibited by L-threonine, L-lysine, and L-methionine, but not by L-isoleucine and diaminopimelate. Aspartate semialdehyde dehydrogenase was not inhibited by any of the end-product amino acids at a concentration of less than 5 mM. Hill plot analysis suggested that asparto-kinase was subject to allosteric control by L-threonine. Repression of both aspartokinase and aspartate semi-aldehyde dehydrogenase gene transcription in A. mediterranei U32 by L-lysine, L-methionine, L-threonine, and L-isoleucine were found. The network of regulation of aspartokinase and aspartate semialdehyde dehydrogenase in rifamycin SV-producing A. mediterranei U32 is presented.
Zhang
W W
WW
Department of Microbiology, Shanghai Institute of Plant Physiology, Academia Sinica, PR China. wzhang@hsc.vcu.edu
Jiang
W H
WH
Zhao
G P
GP
Yang
Y L
YL
Chiao
J S
JS
D000192:Actinomycetales / Q000378:metabolism*; D000596:Amino Acids / Q000378:metabolism; D001222:Aspartate Kinase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001223:Aspartate-Semialdehyde Dehydrogenase / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics*; D007700:Kinetics; D012294:Rifamycins / Q000096:biosynthesis*
D000596:Amino Acids; D017931:DNA Primers; D012294:Rifamycins; D001223:Aspartate-Semialdehyde Dehydrogenase; D001222:Aspartate Kinase