Thermostable alanine racemase of Bacillus stearothermophilus. Construction and expression of active fragmentary enzyme.
Limited proteolysis studies on alanine racemase suggested that the enzyme subunit is composed of two domains (Galakatos, N. G., and Walsh, C. T. (1987) Biochemistry 26, 8475-8480). We have constructed a mutant gene that tandemly encodes the two polypeptides of the Bacillus stearothermophilus enzyme subunit cleaved at the position corresponding to the predicted hinge region. The mutant gene product purified was shown to be composed of two sets of the two polypeptide fragments and was immunologically identical to the wild-type enzyme. The mutant enzyme, i.e. the fragmentary alanine racemase, was active in both directions of the racemization of alanine. The maximum velocity (Vmax) was about half that of the wild-type enzyme, and the Km value was about double. Absorption and circular dichroism spectra of the fragmentary enzyme were similar to those of the wild-type enzyme. An attempt was made to separately express in Escherichia coli a single polypeptide corresponding to each domain, but no protein reactive with the antibody against the wild-type alanine racemase was produced. Therefore, it is suggested that the two polypeptide fragments can fold into an active structure only when they are co-translated and that they correspond to structural folding units in the parental polypeptide chain.
Toyama
H
H
Laboratory of Microbial Biochemistry, Kyoto University, Japan.
Tanizawa
K
K
Yoshimura
T
T
Asano
S
S
Lim
Y H
YH
Esaki
N
N
Soda
K
K
D000411:Alanine Racemase / Q000737:chemistry / Q000235:genetics* / Q000276:immunology; D000595:Amino Acid Sequence; D001483:Base Sequence; D002384:Catalysis; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004252:DNA Mutational Analysis; D005818:Genetic Engineering; D001411:Geobacillus stearothermophilus / Q000201:enzymology* / Q000235:genetics; D006358:Hot Temperature; D005779:Immunodiffusion; D008969:Molecular Sequence Data; D009841:Oligonucleotides / Q000737:chemistry; D013050:Spectrometry, Fluorescence; D013057:Spectrum Analysis; D013329:Structure-Activity Relationship
D009841:Oligonucleotides; D000411:Alanine Racemase