Cloning and characterization of Pfl_1841, a 2-methylenebornane synthase in Pseudomonas fluorescens PfO-1.

The pfl_1841 gene from Pseudomonas fluorescens PfO-1 is the only gene in any of the three sequenced genomes of the Gram-negative bacterium Pseudomonas fluorescens that is annotated as a putative terpene synthase. The predicted Pfl_1841 protein, which harbors the two strictly conserved divalent metal binding domains found in all terpene cyclases, is closely related to several known or presumed 2-methylisoborneol synthases, with the closest match being to the MOL protein of Micromonaspora olivasterospora KY11048 that has been implicated as a 2-methylenebornane synthase. A synthetic gene encoding P. fluorescens Pfl_1841 and optimized for expression in Escherichia coli was expressed and purified as an N-terminal His(6)-tagged protein. Incubation of recombinant Pfl_1841 with 2-methylgeranyl diphosphate produced 2-methylenebornane as the major product accompanied by 1-methyl camphene as well as other minor, monomethyl-homomonoterpene hydrocarbons and alcohols. The steady-state kinetic parameters for the Pfl_1841-catalyzed reaction were K(M) = 110 ± 13 nM and k(cat) = 2.4 ± 0.1 × 10(-2) s(-1). Attempts to identify the P. fluorescens SAM-dependent 2-methylgeranyl diphosphate synthase have so far been unsuccessful.