Identification of critical residues for the catalytic activity of ComQ, a Bacillus prenylation enzyme for quorum sensing, by using a simple bioassay system

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 (C-10 type) or farnesyl diphosphate (C-15 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 ComQ(RO-E-2) (C-10 type) were nondisplaceable to Ser63, Gly186, and Val190, the corresponding residues in the C-15-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 ComQ(RO-E-2) significantly reduced the geranylation activity but substantially enhanced the farnesylation activity, whereas substitution of Ser63 with Phe in ComQ(168) (C-15 type) reduced the farnesylation activity. Therefore, the 63rd residue was found to be significant for the prenyl-substrate preference.