Alanine dehydrogenases from two Bacillus species with distinct thermostabilities: molecular cloning, DNA and protein sequence determination, and structural comparison with other NAD(P)(+)-dependent dehydrogenases.
The gene encoding alanine dehydrogenase (EC 1.4.1.1) from a mesophile, Bacillus sphaericus, was cloned, and its complete DNA sequence was determined. In addition, the same gene from a moderate thermophile, B. stearothermophilus, was analyzed in a similar manner. Large parts of the two translated amino acid sequences were confirmed by automated Edman degradation of tryptic peptide fragments. Each alanine dehydrogenase gene consists of a 1116-bp open reading frame and encodes 372 amino acid residues corresponding to the subunit (Mr = 39,500-40,000) of the hexameric enzyme. The similarity of amino acid sequence between the two alanine dehydrogenases with distinct thermostabilities is very high (greater than 70%). The nonidentical residues are clustered in a few regions with relatively short length, which may correlate with the difference in thermal stability of the enzymes. Homology search of the primary structures of both alanine dehydrogenases with those of other pyridine nucleotide-dependent oxidoreductases revealed significant sequence similarity in the regions containing the coenzyme binding domain. Interestingly, several catalytically important residues in lactate and malate dehydrogenases are conserved in the primary structure of alanine dehydrogenases at matched positions with similar mutual distances.
Kuroda
S
S
Institute for Chemical Research, Kyoto University, Japan.
Tanizawa
K
K
Sakamoto
Y
Y
Tanaka
H
H
Soda
K
K
D050788:Alanine Dehydrogenase; D000594:Amino Acid Oxidoreductases / Q000235:genetics* / Q000302:isolation & purification; D000595:Amino Acid Sequence; D001665:Binding Sites; D003001:Cloning, Molecular; D003067:Coenzymes; D004795:Enzyme Stability; D001411:Geobacillus stearothermophilus; D006358:Hot Temperature; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010088:Oxidoreductases
D003067:Coenzymes; D010088:Oxidoreductases; D000594:Amino Acid Oxidoreductases; D050788:Alanine Dehydrogenase