Edeine B1

A gene with a high-nucleotide sequence homology to the edeine B1 amidinohydrolase gene of Bacillus brevis was identified in the database of the Bacillus subtilis genome. The gene was isolated, expressed in Escherichia coli, and the gene product was analyzed with regard to the characteristics of its enzyme activity. A 32-kDa protein encoded by the ywhG gene showed a 69.8% amino acid sequence-homology to the edeine B1 amidinohydrolase of B. brevis. Among various guanidino-compounds, edeine B1 and agmatine were both efficiently hydrolyzed by the protein encoded by the ywhG gene, although edeine B1 was a more potent substrate than agmatine in this assay system. These data indicate that the protein encoded by the ywhG gene is an agmatinase that is essential for polyamine biosynthesis in B. subtilis.

A peptide antibiotic, edeine B(1), exerts a lethal action in Bacillus subtilis causing filamentous morphology. This antibiotic assumes to inhibit cell division by interacting with FtsZ and inhibiting FtsZ polymerization. The temperature-sensitive mutant ftsZ ts1 was shown to be hypersensitive to the antibiotic as compared to the parent 168 with respect to its lethal action and the sensitivity to the antibiotic of the revertant of ftsZ ts1 was shown to be intermediate between those of the parent 168 and the ftsZ ts1. Alteration of FtsZ sequence may be responsible for sensitivity to edeine B(1). The residues at 240, 278, 345 and 346 in the FtsZ sequence of the parent 168 were A240, A278, D345 and A346. Those of ftsZ ts1 were V240, V278, E345 and P346. Those of the revertant of ftsZ ts1 were A240, A278, E345 and P346. The difference in sensitivity to edeine B(1) among these strains is presumably due to the difference in the residues at 240, 278, 345 and 346 in the FtsZ sequence. The sequential events of the inhibition of FtsZ assembly and the inhibition of protein biosynthesis by edeine B(1) may progress synergistically, resulting in cell death.

The edeines analogs were tested in several in vitro and in vivo assays using the mouse model, with edeine B (peptide W1) and cyclosporine A as reference compounds. The peptides displayed moderate, stimulatory effects on concanavalin A-induced (ConA-induced) splenocyte proliferation, whereas their effects on pokeweed mitogen-induced (PWM-induced) splenocyte proliferation were inhibitory. The peptides inhibited lipopolysacharide-induced (LPS-induced) tumor necrosis factor alpha production but had little effect on interleukin 6 production. In the model of the humoral immune response in vitro to sheep red blood cells, peptide 1 was distinctly stimulatory in the investigated concentrations (1-100 microg/ml), whereas peptides 3 and 4 only stimulated the number of antibody-forming cells at the highest concentration (100 microg/ml). In the model of the delayed type hypersensitivity in vivo to ovalbumin, the peptides were moderately suppressive (3 being the most active). The reference peptide W1 stimulated ConA-induced cell proliferation at 1-10 microg/ml but was inhibitory at 100 microg/ml. It also inhibited PWM-induced cell proliferation in a dose-dependent manner. This peptide had no effect on the humoral immune response in vitro or on cytokine production, but inhibited DTH reaction in vivo. The relationship between structure and activity, and a possible mode of action of the peptides, is discussed in this paper.