Cloning of Alanine Racemase Genes from Pseudomonas fluorescens Strains and Oligomerization States of Gene Products Expressed in Escherichia coli(GENETICS, MOLECULAR BIOLOGY, AND GENE ENGINEERING)
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概要
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Bacterial alanine racemase (EC 5.1.1.1) is a pyridoxal 5'-phosphate-dependent enzyme. Almost all eubacteria known to date possess a biosynthetic alr gene and some bacteria have an additional catabolic dadX gene. On the basis of the subunit structure, alanine racemases are classified into two types, monomeric and homodimeric. Alanine racemase genes were cloned from two distinct Pseudomonas fluorescens strains, the psychrotrophic TM5-2 strain and the soil-borne LRB3W1 strain, by means of complementing an Escherichia coli alanine racemase-deficient mutant. From the cloning results, both strains are likely to possess only one alanine racemase gene, dadX, in the same manner as the other P. fluorescens strains. Gene organization surrounding the dadX gene is highly conserved among Pseudomonas strains. The gene for D-amino acid dehydrogenase is located adjacent to the dadX gene in both strains. The DadX alanine racemases were expressed in E. coli as C-terminal His-tagged fusion proteins and purified to homogeneity. The catalytic activity of LRB3W1 DadX was higher than that of TM5-2 DadX. The association states of P. fluorescens DadX subunits in the E. coli alanine racemase-deficient mutant were analyzed by gel filtration chromatography. Alanine racemase subunits were demonstrated to exist as both monomers and dimers. The enzyme was in a monomer-dimer equilibrium, and the catalytic activity of the enzyme was proportional to the equilibrium association constant for dimerization.
- 社団法人日本生物工学会の論文
- 2005-10-25
著者
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MISONO Haruo
Department of Bioresources Science, Kochi University
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OHNISHI Kouhei
Research Institute of Molecular Genetics, Kochi University
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Meguro H
Tohoku Fukushi University
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Misono H
Kochi Univ. Kochi Jpn
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Misono Haruo
Department Of Bioresources Science Faculty Of Agriculture Kochi University
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Misono Haruo
Department Of Bioresource Science Kochi University
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Misono Haruo
Laboratory Of Applied Microbiology Department Of Agricultural Chemistry Kochi University
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Ohnishi Kouhei
Research Institute Of Molecular Genetics Kochi University
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Ohnishi Kouhei
Res. Inst. Of Molecular Genetics Kochi Univ. Kochi 783-8502 Jpn
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YOKOIGAWA Kumio
Department of Food Science and Nutrition, Nara Women's University
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Ju Jiansong
Department of Applied Bioresource Science, The United Graduate School of Agricultural Sciences, Ehim
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Yokoigawa Kumio
Department Of Mathematical And Natural Sciences Faculty Of Integrated Arts And Sciences The Universi
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Yokoigawa Kumio
Department Of Food Science And Nutrition Nara Women's University
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Ju Jiansong
Life Coll. Of Sci. And Engineering Shaanxi Univ. Of Sci. And Technol. Xi'an 710021 Chn
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Ju Jiansong
State Key Laboratory Of Microbial Resources Institute Of Microbiology Chinese Academy Of Sciences:li
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Ohnishi K
Research Institute of Molecular Genetics, Kochi University
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MISONO Haruo
Department of Agricultural and Biological Chemistry, Kochi University
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