Analysis of oxidation sensitivity of maleate cis-trans isomerase from Serratia marcescens.
The maleate cis-trans isomerase gene (maiA) from Serratia marcescens IFO3736 was cloned and sequenced. Serratia MaiA has 62.4% amino acid identity with Alcaligenes faecalis IFO13111 MaiA and 64.9% with Bacillus stearothermophilus MI-102 MaiA. All known ten amino acid sequences of MaiA had significant conserved regions containing cysteine residues, which were previously suggested to be involved in an active site of the enzyme. The maiA gene was expressed in Escherichia coli, and expressed products MaiA was purified and characterized. The purified enzyme of strain IFO3736 showed high activity at room temperature and high heat stability. It also showed higher activity in the presence of high concentration of aspartic acid than the enzyme of A. faecalis IFO13111, but it was also sensitive to chemical oxidation. By amino acid composition analysis, cysteine, methionine, and tyrosine residues were suggested to be oxidized to inactivate the enzyme by chemical oxidation. To investigate the mechanism of chemical oxidation of the enzyme, six methionine residues in the conserved regions of S. marcescens MaiA were replaced with cysteine residues by site-directed mutagenesis. The analysis of the constructed mutants suggested that the Met201 residue near the Cys198 residue is involved in the sensitivity of the enzyme to chemical oxidation.
Hatakeyama
K
K
Biochemicals Laboratory, Tsukuba Research Center, Mitsubishi Chemical Corporation, Inashiki, Ibaraki, Japan.
Goto
M
M
Kobayashi
M
M
Terasawa
M
M
Yukawa
H
H
D000595:Amino Acid Sequence; D001224:Aspartic Acid; D001426:Bacterial Proteins; D001483:Base Sequence; D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D004795:Enzyme Stability; D004926:Escherichia coli; D015870:Gene Expression; D005798:Genes, Bacterial; D006861:Hydrogen Peroxide / Q000378:metabolism; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012706:Serratia marcescens / Q000201:enzymology* / Q000235:genetics; D013696:Temperature; D019745:cis-trans-Isomerases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D001224:Aspartic Acid; D006861:Hydrogen Peroxide; D019745:cis-trans-Isomerases; C108846:maleate isomerase