Gabaculine resistance of Synechococcus glutamate 1-semialdehyde aminotransferase.
Glutamate 1-semialdehyde aminotransferase (GSA-AT) catalyzes the transfer of the C2 amino group of glutamate 1-semialdehyde (GSA) to the C1 position. Nucleic acid sequences encoding this enzyme from wild type and a gabaculine (GAB) resistant strain of Synechococcus have been cloned and overexpressed in Escherichia coli. Tolerance to GAB of the mutant GSA-AT resulted from a point mutation, Met-248-Ile, in the middle of the polypeptide chain accompanied by a deletion of three amino acids close to the NH2 terminus but can also be effected by the point mutation alone. Purified enzymes from these two strains contain vitamin B6 and use a typical ping-pong Bi-Bi mechanism, in which 4,5-diaminovalerate (DAVA) is a likely intermediate. The catalytic efficiency (Kcat/Km) of wild-type GSA-AT for GSA is about 3 times larger than that of the mutant enzyme. Comparison of substrate specificities (kmax/Km) for GSA and various analogues reveals that wild-type GSA-AT has values that are about 2-20 times larger than those of the mutant enzyme, except in the case of GAB for which the specificity is 2-3 orders of magnitude larger. These differences are attributed to impaired prototropic rearrangement and transaldimination by mutant GSA-AT. They lead to accumulation of quinonoid and other intermediates upon addition of various substrates such as ALA and DOVA, as well as to instability of their aldimines (418 nm) upon Sephadex gel filtration.
Smith
M A
MA
Carlsberg Laboratory, Department of Physiology, Copenhagen Valby, Denmark.
Grimm
B
B
D000622:Aminolevulinic Acid / Q000378:metabolism; D002850:Chromatography, Gel; D000458:Cyanobacteria / Q000187:drug effects / Q000201:enzymology*; D003509:Cyclohexanecarboxylic Acids / Q000494:pharmacology*; D004351:Drug Resistance; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D007097:Imines / Q000378:metabolism; D019751:Intramolecular Transferases; D007535:Isomerases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D016296:Mutagenesis; D011994:Recombinant Proteins / Q000378:metabolism; D013053:Spectrophotometry
D003509:Cyclohexanecarboxylic Acids; D007097:Imines; D011994:Recombinant Proteins; C012987:gabaculine; D000622:Aminolevulinic Acid; D007535:Isomerases; D019751:Intramolecular Transferases; C061049:glutamate-1-semialdehyde 2,1-aminomutase