Characterization of the Bacillus subtilis ywtD gene, whose product is involved in gamma-polyglutamic acid degradation

Abstract

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.

FIG. 1.

Gene organization of ywtD and its upstream region (A), catalytic domains of YwtD and dl-endopeptidases of LytF and LytE (B), and amino acid sequence alignment of the catalytic domains (C). Numbers are amino acid residue numbers. Black shading indicates identity to YwtD.

FIG. 2.

SDS-PAGE of YwtD produced in E. coli BL21(DE3) harboring pYWTD and the purified YwtD protein. SDS-PAGE was done with a 12% polyacrylamide gel at pH 7.0 by the method of Laemmli (13). The gel was stained with Coomassie brilliant blue R-250. Lane 1, marker proteins; lane 2, cell extracts without IPTG induction; lane 3, cell extracts with 0.5 mM IPTG induction; lane 4, purified YwtD after nickel-nitrilotriacetic acid column chromatography.

FIG. 3.

HPLC analysis of hydrolyzed products from PGA by YwtD. PGA and the hydrolyzed products were analyzed by HPLC using an Asahipack GF-7 M HQ (300 by 7.6 mm; Showa Denko Co., Tokyo, Japan) with a refractive index detector. Samples were eluted with 50 mM phosphate buffer (pH 6.8) at a flow rate of 0.6 ml/min at 32°C. Peaks were assigned by coelution with authentic pullulan with a molecular mass of 200 kDa (Tokyo Kasei Co, Tokyo, Japan) and α-l-PGAs with molecular masses of 58, 32, and 14 kDa (Sigma).