Molecular cloning and characterization of a large subunit of Salmonella typhimurium glutamate synthase (GOGAT) gene in Escherichia coli.
Two pathways of ammonium assimilation and glutamate biosynthesis have been identified in microorganisms. One pathway involves the NADP-linked glutamate dehydrogenase, which catalyzes the amination of 2-oxoglutarate to form glutamate. An alternative pathway involves the combined activities of glutamine synthetase, which aminates glutamate to form glutamine, and glutamate synthase, which transfers the amide group of glutamine to 2-oxoglutarate to yield two molecules of glutamate. We have cloned the large subunit of the glutamate synthase (GOGAT) from Salmonella typhimurium by screening the expression of GOGAT and complementing the gene in E. coli GOGAT large subunit-deficient mutants. Three positive clones (named pUC19C12, pUC19C13 and pUC19C15) contained identical Sau3AI fragments, as determined by restriction mapping and Southern hybridization, and expressed GOGAT efficiently and constitutively using its own promoter in the heterologous host. The coding region expressed in Escherichia coli was about 170 kDa on SDS-PAGE. This gene spans 4,732 bases, contains an open reading frame of 4,458 nucleotides, and encodes a mature protein of 1,486 amino acid residues (Mr = 166,208). The FMN-binding domain of GOGAT contains 12 glycine residues, and the 3Fe-4S cluster has 3 cysteine residues. The comparison of the translated amino acid sequence of the Salmonella GOGAT with sequences from other bacteria such as Escherichia coli, Salmonella enterica, Shigella flexneri, Yersinia pestis, Vibrio vulnificus and Pseudomonas aeruginosa shows sequence identity between 87 and 95%.
Chung
Tae-Wook
TW
Department of Biological Sciences, Sungkyunkwan University, Chunchun-Dong 300, Jangan-Gu, Suwon City, Kyunggi-Do 440-746, Republic of Korea.
Lee
Dong-Ick
DI
Kim
Dong-Soo
DS
Jin
Un-Ho
UH
Park
Chun
C
Kim
Jong-Guk
JG
Kim
Min-Gon
MG
Ha
Sang-Do
SD
Kim
Keun-Sung
KS
Lee
Kyu-Ho
KH
Kim
Kwang-Yup
KY
Chung
Duck Hwa
DH
Kim
Cheorl-Ho
CH
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D005816:Genetic Complementation Test; D005970:Glutamate Synthase / Q000235:genetics* / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D012486:Salmonella typhimurium / Q000201:enzymology / Q000235:genetics*
D005970:Glutamate Synthase