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

• Published in: Tetrahedron Vol. 67; no. 35; pp. 6627 - 6632
• Main Authors: Chou, Wayne K.W., Ikeda, Haruo, Cane, David E.
• Format: Journal Article
• Language: English
• Published: Kidlington Elsevier Ltd 02.09.2011; Elsevier
• Subjects: Alicyclic compounds, terpenoids, prostaglandins, steroids; Bioconversions. Hemisynthesis; Biological and medical sciences; Biotechnology; Chemistry; Cloning; Diphosphates; Escherichia coli; Exact sciences and technology; Fundamental and applied biological sciences. Psychology; Genes; Genome mining; Methods. Procedures. Technologies; Monoterpenes; Organic chemistry; Preparations and properties; Proteins; Pseudomonas fluorescens; Recombinant; Terpene biosynthesis; Terpenes; Terpenoids; Volatile organics; ZASFWGOMAIPHLN-UHFFFAOYSA-N; IWWRARFUNWPJJJ-UHFFFAOYSA-N; Volatile organics; Terpene biosynthesis; Monoterpenes; Pseudomonas fluorescens; NLEXDUMLYCKKMI-MQIHADEYSA-N; Genome mining; Pseudomonadales; Escherichia coli; Micromonospora; Alcohol; Molecular interaction; Biosynthesis; Synthase; Cyclases; Actinomycetales; Characterization; Bacterium; Gene; Organic diphosphate; Bacteria; Pseudomonadaceae; Chemical synthesis; Enterobacteriaceae; Gram negative bacteria; Terpenoid; Catalytic reaction; Enzyme; Micromonosporaceae; Cloning; Prediction; Steady state; Protein; Anionic surfactant; Terpene; Monoterpene; Actinomycetes; Kinetic parameter; Genome; Divalent metal
• ISSN: 0040-4020; 1464-5416
• DOI: 10.1016/j.tet.2011.05.084

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 P. 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-methylcamphene 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. [Display omitted]