Structure and properties of malic enzyme from Bacillus stearothermophilus.
The malic enzyme (EC 1.1.1.38) gene of Bacillus stearothermophilus was cloned in Escherichia coli, and the enzyme was purified to homogeneity from the E. coli clone. In addition to the NAD(P)-dependent oxidative decarboxylation of L-malate, the enzyme catalyzes the decarboxylation of oxalacetate. The enzyme is a tetramer of Mr 200,000 consisting of four identical subunits of Mr 50,000. The pH optima for malate oxidation and pyruvate reduction are 8.0 and 6.0, respectively; and the optimum temperature is 55 degrees C. The enzyme strictly requires divalent metal cations for its activity, and the activity is enhanced 5-7 times by NH4+ and K+. Kinetic study shows that the values of the dissociation constant of the enzyme-coenzyme complex are 77 microM for NAD and 1.0 mM for NADP, indicating that the enzyme has a higher affinity for NAD than for NADP. The nucleotide sequence of the gene and its flanking regions was also found. A single open reading frame of 1434 base pairs encoding 478 amino acids was concluded to be that for the malic enzyme gene because the amino acid composition of the enzyme and the sequence of 16 amino acids from the amino terminus of the enzyme agreed well with those deduced from this open reading frame.
Kobayashi
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Department of Fermentation Technology, Osaka University, Japan.
Doi
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Negoro
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Urabe
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Okada
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H
D000595:Amino Acid Sequence; D001483:Base Sequence; D002413:Cations, Divalent; D003001:Cloning, Molecular; D003062:Codon; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D046911:Macromolecular Substances; D008291:Malate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D008293:Malates / Q000378:metabolism; D008969:Molecular Sequence Data; D009243:NAD / Q000378:metabolism / Q000494:pharmacology; D009249:NADP / Q000378:metabolism / Q000494:pharmacology; D010071:Oxaloacetates / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011773:Pyruvates / Q000378:metabolism; D019289:Pyruvic Acid; D013696:Temperature
D002413:Cations, Divalent; D003062:Codon; D004269:DNA, Bacterial; D046911:Macromolecular Substances; D008293:Malates; D010071:Oxaloacetates; D011773:Pyruvates; D009243:NAD; D009249:NADP; C030298:malic acid; D019289:Pyruvic Acid; D008291:Malate Dehydrogenase