Crystal structure of PM1Pgh, a poly-γ-glutamate hydrolase from <i>Bacillus</i> phage PM1

Poly-γ-glutamate (γ-PGA) is the key biopolymer responsible for the characteristic viscosity of natto, a traditional Japanese food produced by Bacillus subtilis var. natto. Phage infection frequently leads to γ-PGA degradation and loss of viscosity, a longstanding problem in natto production. While the γ-PGA hydrolase PghP from phage ΦNIT1 has been well characterized, the homologous enzyme encoded by B. subtilis var. natto phage PM1 has remained unstudied and is currently annotated only as ORF34; this protein is referred to here as PM1Pgh. Two catalytic models have been proposed for γ-PGA-degrading metallopeptidases: a direct anhydride mechanism involving the catalytic glutamate and a zinc-dependent, water-mediated mechanism. However, no definitive structural evidence has been obtained to distinguish between these mechanisms. Here, recombinant PM1Pgh was shown to possess γ-PGA hydrolase activity in vitro, and its crystal structures were determined in both zinc-free and zinc-bound states. The zinc-bound structure revealed a Zn²⁺ ion specifically coordinated within the catalytic pocket together with a well ordered water molecule positioned for nucleophilic attack, supporting a zinc-dependent, water-mediated mechanism for γ-PGA hydrolysis, consistent with the canonical mechanism of metallopeptidases.