Novel malonamidases in Bradyrhizobium japonicum. Purification, characterization, and immunological comparison.
Three novel malonamidases (E1a, E1b, and E2) occurring constitutively in Bradyrhizobium japonicum were purified to electrophoretic homogeneity. They were found to catalyze one or more of the following three types of reactions: malonyl transfer to hydroxylamine (reaction 1), hydroxaminolysis of malonamate (reaction 2), and hydrolysis of malonamate (reaction 3). The molecular sizes of E1a, E1b, and E2 were 126, 107, and 103 kDa, respectively, and they were each composed of two identical subunits. The pIs of E1a and E1b, 5.5 and 5.0 respectively, were similar, but that of E2 was 7.2. Optimum pH values varied with the type of reactions catalyzed, but among the enzymes they were found to be similar. The affinity of E2 for malonamate was about 30- and 70-fold higher than that of E1a and E1b, respectively. Acetate and propionate inhibited E1a activity competitively, whereas malonate inhibited E2 activity noncompetitively. The amino acid composition and N-terminal amino acid sequence of the three enzymes were found to be different. These enzymes were also immunologically different. E1a was found to form a malonyl-enzyme intermediate during the catalysis through the isolation of [14C]malonyl-enzyme with gel filtration and through isotope exchange experiments with [18O]malonate. These malonamidases may play a role for the self-protection against malonate toxicity in nodule bacteroids and may also be involved in the transport of fixed nitrogen to the plant cell.
Kim
Y S
YS
Department of Biochemistry, College of Science, Yonsei University, Seoul, Korea.
Kang
S W
SW
D000581:Amidohydrolases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism*; D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001426:Bacterial Proteins; D002845:Chromatography; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D012230:Rhizobiaceae / Q000201:enzymology*
D000596:Amino Acids; D001426:Bacterial Proteins; D007527:Isoenzymes; D017886:Durapatite; D000581:Amidohydrolases; C064615:malonamidase E2 protein, Bradyrhizobium japonicum