Thermostable alanine racemase. Its structural stability.
The gene encoding thermostable alanine recemase from Bacillus stearothermophilus was cloned and expressed in E. coli. The enzyme was purified to homogeneity from cell extracts of E. coli carrying a plasmid designated pICR4. The alanine racemase gene sequenced was found to contain an open reading frame of 1158 nucleotides. The molecular weight of the enzyme subunit was estimated to be 43,341. The alpha-helical and beta-structure contents were calculated to be about 34 and 26%, respectively, from CD data. CD measurements of the denaturation process of enzyme by guanidine hydrochloride showed the presence of a stable intermediate during the denaturation. Limited proteolysis with subtilisin resulted in the formation of two dissimilar peptide fragments with molecular weights of about 28,000 and 13,000 in the early stage of the digestion. These suggest that the enzyme subunit is composed of two structurally dissimilar domains connected by a short polypeptide (residues 258-266), which first suffers the limited proteolysis. However, the enzyme retained almost full activity and the conformation indistinguishable from the intact protein even when it was proteolytically hydrolyzed to more than 10 fragments.
Institute for Chemical Research, Kyoto University, Japan.
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Soda
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Tanizawa
D000411:Alanine Racemase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000591:Amino Acid Isomerases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D004355:Drug Stability; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics; D006358:Hot Temperature; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism; D011487:Protein Conformation
D010447:Peptide Hydrolases; D000591:Amino Acid Isomerases; D000411:Alanine Racemase