Bacillus ComQ participates in the biosynthesis of a quorum-sensing signaling molecule (ComX pheromone) through catalyzing the prenylation at a Trp residue of the precursor peptide (pre-ComX) with geranyl diphosphate (C10 type) or farnesyl diphosphate (C15 type). We hypothesized that several residues specifically conserved among either type of ComQs are important for their substrate specificities. Using a simple bioassay, we revealed that Phe63, Asn186, and Gly190 in ComQRO-E-2 (C10 type) were nondisplaceable to Ser63, Gly186, and Val190, the corresponding residues in the C15-type ComQ, respectively. A three-dimensional model suggested that the 186th and 190th residues are involved in the pre-ComX binding. In vitro analysis showed that substitution of Phe63 with Ser in ComQRO-E-2 significantly reduced the geranylation activity but substantially enhanced the farnesylation activity, whereas substitution of Ser63 with Phe in ComQ168 (C15 type) reduced the farnesylation activity. Therefore, the 63rd residue was found to be significant for the prenyl-substrate preference.Abbreviations: GPP: geranyl diphosphate; FPP: farnesyl diphosphate; IPP: isopentenyl diphosphate; GGPP: geranylgeranyl diphosphate; FARM: first aspartate-rich motif; SARM: second aspartate-rich motif; β-Gal: β-galactosidase; TBABG: tryptose blood agar base supplemented with glucose; X-gal: 5-bromo-4-chloro-3-indolyl-β-D-galactoside.
Keywords: Bacillus subtilis; post-translational modification; prenylation; quorum sensing.