Molecular Cloning and Characterization of a Large Subunit of Salmonella typhimurium Glutamate Synthase (GOGAT) Gene in Escherichia coli

Tae-Wook Chung; Dong-Ick Lee; Dong-Soo Kim; Un-Ho Jin; Chun Park; Jong-Guk Kim; Min-Gon Kim; Sang-Do Ha; Keun-Sung Kim; Kyu-Ho Lee; Kwang-Yup Kim; Duck Hwa Chung; Cheorl-Ho Kim

doi:2382 [pii]

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Research Support, Non-U.S. Gov't
Molecular Cloning and Characterization of a Large Subunit of Salmonella typhimurium Glutamate Synthase (GOGAT) Gene in Escherichia coli: Tae-Wook Chung ¹, Dong-Ick Lee ², Dong-Soo Kim ², Un-Ho Jin ¹, Chun Park ¹, Jong-Guk Kim ³, Min-Gon Kim ⁴, Sang-Do Ha ⁵, Keun-Sung Kim ⁵, Kyu-Ho Lee ⁶, Kwang-Yup Kim ⁷, Duck Hwa Chung ⁸, Cheorl-Ho Kim ¹; Journal of Microbiology 2006;44(3):301-310
DOI: https://doi.org/2382 [pii]

¹Department of Biological Sciences, Sungkyunkwan University, Chunchun-Dong 300, Jangan-Gu, Suwon City, Kyunggi-Do 440-746, Republic of Korea, ²Department of Food Science and Technology, Kyungsung University, Pusan 608-736, Republic of Korea, ³Department of Microbiology, Kyungpook National University, Daegu, Republic of Korea, ⁴NanoBiotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejon 305-600, Republic of Korea, ⁵Department of Food Science & Technology, Chung-Ang University, Ansong, Republic of Korea, ⁶Environmental Science, Hankuk University of Foreign Studies, Gyungki, Republic of Korea, ⁷Department of Food Science & Technology, Chungbuk National University, Cheongju, Republic of Korea, ⁸Division of Applied life science, Gyeongsang National University, Jinju, Republic of Korea¹Department of Biological Sciences, Sungkyunkwan University, Chunchun-Dong 300, Jangan-Gu, Suwon City, Kyunggi-Do 440-746, Republic of Korea, ²Department of Food Science and Technology, Kyungsung University, Pusan 608-736, Republic of Korea, ³Department of Microbiology, Kyungpook National University, Daegu, Republic of Korea, ⁴NanoBiotechnology Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejon 305-600, Republic of Korea, ⁵Department of Food Science & Technology, Chung-Ang University, Ansong, Republic of Korea, ⁶Environmental Science, Hankuk University of Foreign Studies, Gyungki, Republic of Korea, ⁷Department of Food Science & Technology, Chungbuk National University, Cheongju, Republic of Korea, ⁸Division of Applied life science, Gyeongsang National University, Jinju, Republic of Korea

Corresponding author: Cheorl-Ho Kim , Tel: 82-31-290-7002,

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Abstract

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%.

Keywords: glutamate synthase; large subunit; molecular cloning; Salmonella typhimurium

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Molecular Cloning and Characterization of a Large Subunit of Salmonella typhimurium Glutamate Synthase (GOGAT) Gene in Escherichia coli

J. Microbiol. 2006;44(3):301-310.

DOI: https://doi.org/2382 [pii]

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