Molecular analysis of maleate cis-trans isomerase from thermophilic bacteria.
Several strains of thermophilic bacteria containing maleate cis-trans isomerase were isolated from soil samples and identified as Bacillus stearothermophilus, Bacillus circulans, Bacillus brevis, and Deleya halophila. The maleate cis-trans isomerase was purified and characterized from one of the isolated strains, B. stearothermophilus MI-102. The purified enzyme of strain MI-102 showed higher thermal stability than the enzyme of a mesophile, Alcaligenes faecalis IFO13111. The seven maleate cis-trans isomerase genes (maiA) of thermophile were cloned and sequenced. B. stearothemophilus MI-102 MaiA has 67% amino acid identity with A. faecalis MaiA. All eight amino acid sequences of maiA gene products had significant conserved regions containing cysteine residues, which were previously suggested to be involved in an active site of the enzyme. To probe the catalytic mechanism, three cysteine residues in the conserved regions of A. faecalis MaiA were replaced with serine by site-directed mutagenesis. The results suggest that Cys80 and Cys198 play important roles in the enzyme activity.
Hatakeyama
K
K
Biochemicals Laboratory, Mitsubishi Chemical Corporation, Inashiki, Ibaraki, Japan.
Goto
M
M
Uchida
Y
Y
Kobayashi
M
M
Terasawa
M
M
Yukawa
H
H
D000421:Alcaligenes / Q000201:enzymology; D000595:Amino Acid Sequence; D001407:Bacillus / Q000145:classification / Q000201:enzymology / Q000302:isolation & purification; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D004269:DNA, Bacterial; D004789:Enzyme Activation; D004795:Enzyme Stability; D005798:Genes, Bacterial; D001411:Geobacillus stearothermophilus / Q000145:classification / Q000201:enzymology* / Q000302:isolation & purification; D008298:Maleates / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016296:Mutagenesis; D016415:Sequence Alignment; D013696:Temperature; D019745:cis-trans-Isomerases / Q000235:genetics* / Q000378:metabolism
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D008298:Maleates; C030272:maleic acid; D019745:cis-trans-Isomerases; C108846:maleate isomerase