Subunit interaction of monomeric alanine racemases from four Shigella species in catalytic reaction.
Bacterial alanine racemases are classified into two types of subunit structure (monomer and homodimer). To clarify the catalytic unit of monomeric alanine racemases, we examined the apparent molecular mass of the monomeric alanine racemases from Shigella dysenteriae, Shigella boydii, Shigella flexneri, and Shigella sonnei by gel filtration in the presence of the substrate and inhibitor. The enzymes were eluted on gel filtration as a monomer of about 39,000 Da at low protein concentration and in the absence of L-alanine and D-cycloserine. An increase in the apparent molecular mass was induced by increasing the protein concentration or by adding the ligands in the elution buffer. The increase ratio depended on the ligand concentration, and the maximum apparent molecular masses of all enzymes were 60,000 and 76,000 Da in the presence of 100 mM L-alanine and 5 mM D-cycloserine, respectively. D-cycloserine may induce an inactive dimer and L-alanine may induce an intermediate between the monomer and dimer because of dynamic equilibrium. The apoenzyme also showed similar behavior in the presence of the ligands, but the increase ratios were lower than those of the holoenzymes. The Bacillus psychrosaccharolyticus alanine racemase, having a dimeric structure, showed a constant molecular mass irrespective of the absence or presence of the ligands. These results suggest that the monomeric Shigella Alr enzymes have a dimeric structure in the catalytic reaction. Substances that inhibit the subunit interaction of monomeric alanine racemases may be useful as a new type of antibacterial.
Department of Food Science and Nutrition, Nara Women's University, Japan. yokoigawa@cc.nara-wu.ac.jp
Kumio
K
Yokoigawa
Yoko
Y
Okubo
Kenji
K
Soda
D000411:Alanine Racemase / Q000235:genetics / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain; D003523:Cycloserine / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D007700:Kinetics; D012761:Shigella boydii / Q000201:enzymology* / Q000235:genetics; D012762:Shigella dysenteriae / Q000201:enzymology* / Q000235:genetics; D012763:Shigella flexneri / Q000201:enzymology* / Q000235:genetics; D012764:Shigella sonnei / Q000201:enzymology* / Q000235:genetics; D013237:Stereoisomerism
D004791:Enzyme Inhibitors; D003523:Cycloserine; D000411:Alanine Racemase