Novel poly-gamma-glutamate-processing enzyme catalyzing gamma-glutamyl DD-amidohydrolysis.
The pgdS gene product of Bacillus subtilis, PgdS, cleaves poly-gamma-glutamate (PGA) in an endo-peptidase-like fashion. However, its catalytic property remains obscure. In this study, a simple assay for the PgdS enzyme using 1-fluoro-2,4-dinitrobenzene was developed, and some characteristics of PgdS, such as optimal pH, were examined. The enzyme was strongly inhibited by a thiol-modifying reagent, suggesting that it possesses essential cysteine residue(s) in catalysis. PgdS exhibited a high affinity to PGA that consisted mainly of D-glutamate residues, but no affinity to PGA composed only of L-glutamate residues (L-PGA). The enzyme processed DL-copolymer-type PGA (DL-PGA) with an average molecular mass of 1,000 kDa to a high-molecular-mass L-glutamate-rich fragment (average 200 kDa), the L-rich PGA fragment, and low-molecular-mass fragment composed mostly of D-glutamate residues (average 5 kDa), D-fragment. To deepen our understanding of the catalytic property of the PgdS enzyme, we analyzed the structures of the N- and C-terminal regions and found that D-glutamyl residues successively lie even at both ends of the L-rich PGA fragment. Our observations indicate that PgdS is a novel endo-peptidase that specifically cleaves the gamma-amide linkage between two D-glutamate residues in PGA, i.e., gamma-glutamyl DD-amidohydrolase. The enzyme is possibly useful in the biochemical processing of B. subtilis DL-PGA.
Ashiuchi
Makoto
M
Department of Bioresources Science, Kochi University, Nankoku, Kochi 783-8502, Japan. ashiuchi@cc.kochi-u.ac.jp
Nakamura
Hisaaki
H
Yamamoto
Masayoshi
M
Misono
Haruo
H
D000619:Aminohydrolases / Q000032:analysis* / Q000737:chemistry*; D001412:Bacillus subtilis / Q000201:enzymology*; D002384:Catalysis; D004139:Dinitrofluorobenzene / Q000032:analysis* / Q000737:chemistry*; D004789:Enzyme Activation; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D011099:Polyglutamic Acid / Q000032:analysis* / Q000737:chemistry*; D013379:Substrate Specificity
D011099:Polyglutamic Acid; D004139:Dinitrofluorobenzene; D000619:Aminohydrolases