Chemistry and biosynthesis of the poly( -D-glutamyl) capsule in Bacillus licheniformis. I. Properties of the membrane-mediated biosynthetic reaction.

Abstract A cell envelope particulate fraction from an encapsulated strain of Bacillus licheniformis ATCC 9945A contains a polyglutamyl synthetase which catalyzes the polymerization of l-glutamic acid to form a high molecular weight polymer of γ-d-glutamic acid. The reaction, which is specific for l-glutamic acid, requires ATP and Mg2+ ion and is stimulated by K+ ion and dithiothreitol. A nearly 2-fold increase in activity is observed in the presence of optimal levels of glycerol (0.1 m) and dimethylsulfoxide (0.1 m). The Km for l-glutamic acid is 0.5 mm. d-Glutamic acid is not incorporated nor does it influence the polymerization of the l isomer. Glutamyl dipeptides inhibited polyglutamyl synthetase activity. The cell envelope-associated polyglutamyl synthetase does not require an RNA template since it is refractory to treatment with RNase. It is also not inhibited by chloramphenicol, actinomycin D, puromycin, or rifampicin. Thus, the mechanism of capsular polymer formation is uniquely different from that involved in normal protein synthesis. Pre-treatment of the particulate fraction with Pronase destroyed its ability to synthesize the capsular polymer. Since a molar excess of [12C]α-ketoglutarate had no effect on the incorporation of l-[14C]glutamic acid, the subsequent interconversion of the glutamyl optical antipodes does not involve a series of glutamyl transamination and alanine racemization reactions in which α-ketoglutarate is a key intermediate. These reactions have been demonstrated in soluble extracts from B. licheniformis and have been shown to be one mechanism to account for the formation of d-glutamic acid from the l isomer. The observation that l-glutamine also has no effect on l-glutamic acid incorporation rules out the possible involvement of a transamidation reaction between glutamine and l- or d-glutamic acid. Polymer formation does not involve a series of transpeptidation reactions between glutamyl dipeptides since dipeptides inhibit polymer synthesis. Although the precise mechanism of activation, racemization, and polymerization is not fully understood, the results of these studies establish that the poly(γ-d-glutamyl) capsule is synthesized by a sequence of membrane-associated enzymatic reactions. No evidence for the involvement of small molecular weight oligopeptides as free intermediates has been obtained. The enzymatically synthesized α-d-glutamyl polymers were readily isolated by virtue of their complete resistance to hydrolysis by Pronase. After treatment with this protease, molecular sieve chromatography resulted in the quantitative isolation of the capsular polymers. Glutamic acid was the only labeled amino acid recovered following acid-catalyzed hydrolysis of the purified product. The specific activity of the glutamic acid was 90% that of the initial specific activity which indicates a considerable amount of de novo synthesis. Subsequent chemical, physical, and enzymatic analyses have confirmed that the enzymatically synthesized polymers are structurally identical with the capsular γ-d-glutamyl polymers synthesized in vivo.