Characterization of the Bacillus subtilis ywtD gene, whose product is involved in gamma-polyglutamic acid degradation.
The ywtD gene, which codes for an enzyme that degrades gamma-polyglutamic acid (PGA), was cloned from Bacillus subtilis IFO16449. The gene is located immediately downstream of ywsC and ywtABC, a PGA operon involved in PGA biosynthesis, and it showed partial similarity to genes coding for DL-endopeptidase, a peptidoglycan-degrading enzyme. The ywtD gene, from which signal sequence is excised, was inserted into pET15b, and the recombinant plasmid was then transformed into Escherichia coli. Histidine-tagged YwtD was purified from sonicated cells of the transformant. The purified YwtD degraded PGA to yield two hydrolyzed products, a high-molecular-mass product (490 kDa with nearly 100% L-glutamic acid) and an 11-kDa product (with D-glutamic acid and L-glutamic acid in an 80:20 ratio). This finding and results of enzymatic analysis of the two products with carboxypeptidase G suggest that YwtD is a novel enzyme cleaving the gamma-glutamyl bond only between D- and L-glutamic acids of PGA, and it may be designated gamma-DL-glutamyl hydrolase.
Suzuki
Takao
T
Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan.
Tahara
Yasutaka
Y
D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000235:genetics*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D018698:Glutamic Acid / Q000737:chemistry / Q000378:metabolism; D008969:Molecular Sequence Data; D011099:Polyglutamic Acid / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D011623:gamma-Glutamyl Hydrolase / Q000235:genetics* / Q000378:metabolism*
D001426:Bacterial Proteins; D011099:Polyglutamic Acid; D018698:Glutamic Acid; D011623:gamma-Glutamyl Hydrolase