Physiological and biochemical characteristics of poly γ‐glutamate synthetase complex of Bacillus subtilis

Abstract

An enzymatic system for poly g-glutamate (PGA) synthesis in Bacillus subtilis, the PgsBCA system, was investigated. The gene-disruption experiment showed that the enzymatic system was the sole machinery of PGA synthesis in B. subtilis. We succeeded in achieving the enzymatic synthesis of elongated PGAs with the cell membrane of the Escherichia coli clone producing PgsBCA in the presence of ATP and D-glutamate. The enzyme preparation solubilized from the membrane with 8 mM Chaps catalyzed ADPforming ATP hydrolysis only in the presence of glutamate; the D-enantiomer was the best cosubstrate, followed by the L-enantiomer. Each component of the system, PgsB, PgsC, and PgsA, was translated in vitro and the glutamatedependent ATPase reaction was kinetically analyzed. The PGA synthetase complex, PgsBCA, was suggested to be an atypical amide ligase.

Poly-γ-glutamic acid: production, properties and applications

“…This reaction continues to polymerize c-PGA at the active site of the synthase complex (PgsBCA). PgsB and PgsC together form most parts of the complex's catalytic site, whereas PgsA seems to remove the elongated chain from the active site so that the next monomer can be added and may also be involved in transporting c-PGA (Ashiuchi et al, 2001b;Urushibata et al, 2002a). Activity of PgsBCA was found to be dependent on Mg 2+ (Ashiuchi et al, 2004).…”

Disruption of the cell wall lytic enzyme CwlO affects the amount and molecular size of poly-γ-glutamic acid produced by Bacillus subtilis (natto)

“…2). The γPGA synthetic enzyme complex was located on the cell membrane (Ashiuchi et al, 2001;Candela and Fouet, 2006), and γPGA accumulated extracellularly. Therefore, γPGA degradation may occur extracellularly.…”

“…1). PgsB is the γPGA synthase dependent on ATP (Ashiuchi et al, 1999(Ashiuchi et al, , 2001Urushibata et al, 2002) and the pgsB gene is expressed in the early stationary phase (Kimura et al, 2009). These results suggested that the accumulation of ATP and/or an intermediate of γPGA are required for γPGA synthesis.…”

“…In B. subtilis (natto), the enzymes required for γPGA synthesis are encoded by the pgsBCA operon, which contains the γPGA synthetase gene pgsB (Ashiuchi et al, 1999(Ashiuchi et al, , 2001Urushibata et al, 2002). γPGA production is under the control of the ComQXPA quorumsensing system Tran et al, 2000) and the DegS DegU two-component system, and DegQ in particular plays an important role in γPGA production (Kimura et al, 2009;Ohsawa et al, 2009;Osera et al, 2009;Stanley and Lazazzera, 2005).…”

Bacteriophages of Bacillus subtilis (natto) and Their Contamination in Natto Factories

“…5C), are synthesized by binding a γ-carboxyl group and an amino group of an adjacent glutamic acid via a hyperphosphorylated intermediate (Fig. 5D) (Ashiuchi et al, 2001). Genes related to the production of PGA were cloned and expressed well in Escherichia coli cells (Ashiuchi et al, 1999).…”