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

ABSTRACT: 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.

SUBMITTER: Chou WK

PROVIDER: S-EPMC3144570 | biostudies-literature | 2011 Sep

REPOSITORIES: biostudies-literature

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

Chou Wayne K W WK Ikeda Haruo H Cane David E DE

Tetrahedron 20110901 35

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 olivaste ...[more]

PMID: 21804650

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Project description:Pseudomonas fluorescens CY091 cultures produce an extracellular protease with an estimated molecular mass of 50 kDa. Production of this enzyme (designated AprX) was observed in media containing CaCl2 or SrCl2 but not in media containing ZnCl2, MgCl2, or MnCl2. The requirement of Ca2+ (or Sr2+) for enzyme production was concentration dependent, and the optimal concentration for production was determined to be 0.35 mM. Following ammonium sulfate precipitation and ion-exchange chromatography, the AprX in the culture supernatant was purified to near electrophoretic homogeneity. Over 20% of the enzyme activity was retained in the AprX sample which had been heated in boiling water for 10 min, indicating that the enzyme is highly resistant to heat inactivation. The enzyme activity was almost completely inhibited in the presence of 1 mM 1,10-phenanthroline, but only 30% of the activity was inhibited in the presence of 1 mM EGTA. The gene encoding AprX was cloned from the genome of P. fluorescens CY091 by isolating cosmid clones capable of restoring the protease production in a nonproteolytic mutant of strain CY091. The genomic region of strain CY091 containing the aprX gene was located within a 7.3-kb DNA fragment. Analysis of the complete nucleotide sequence of this 7.3-kb fragment revealed the presence of a cluster of genes required for the production of extracellular AprX in P. fluorescens and Escherichia coli. The AprX protein showed 50 to 60% identity in amino acid sequence to the related proteases produced by Pseudomonas aeruginosa and Erwinia chrysanthemi. Two conserved sequence domains possibly associated with Ca2+ and Zn2+ binding were identified. Immediately adjacent to the aprX structural gene, a gene (inh) encoding a putative protease inhibitor and three genes (aprD, aprE, and aprF), possibly required for the transport of AprX, were also identified. The organization of the gene cluster involved in the synthesis and secretion of AprX in P. fluorescens CY091 appears to be somewhat different from that previously demonstrated in P. aeruginosa and E. chrysanthemi.

Project description:The secondary metabolite hydrogen cyanide (HCN) is produced by Pseudomonas fluorescens from glycine, essentially under microaerophilic conditions. The genetic basis of HCN synthesis in P. fluorescens CHA0 was investigated. The contiguous structural genes hcnABC encoding HCN synthase were expressed from the T7 promoter in Escherichia coli, resulting in HCN production in this bacterium. Analysis of the nucleotide sequence of the hcnABC genes showed that each HCN synthase subunit was similar to known enzymes involved in hydrogen transfer, i.e., to formate dehydrogenase (for HcnA) or amino acid oxidases (for HcnB and HcnC). These similarities and the presence of flavin adenine dinucleotide- or NAD(P)-binding motifs in HcnB and HcnC suggest that HCN synthase may act as a dehydrogenase in the reaction leading from glycine to HCN and CO2. The hcnA promoter was mapped by primer extension; the -40 sequence (TTGGC ... ATCAA) resembled the consensus FNR (fumarate and nitrate reductase regulator) binding sequence (TTGAT ... ATCAA). The gene encoding the FNR-like protein ANR (anaerobic regulator) was cloned from P. fluorescens CHA0 and sequenced. ANR of strain CHA0 was most similar to ANR of P. aeruginosa and CydR of Azotobacter vinelandii. An anr mutant of P. fluorescens (CHA21) produced little HCN and was unable to express an hcnA-lacZ translational fusion, whereas in wild-type strain CHA0, microaerophilic conditions strongly favored the expression of the hcnA-lacZ fusion. Mutant CHA21 as well as an hcn deletion mutant were impaired in their capacity to suppress black root rot of tobacco, a disease caused by Thielaviopsis basicola, under gnotobiotic conditions. This effect was most pronounced in water-saturated artificial soil, where the anr mutant had lost about 30% of disease suppression ability, compared with wild-type strain CHA0. These results show that the anaerobic regulator ANR is required for cyanide synthesis in the strictly aerobic strain CHA0 and suggest that ANR-mediated cyanogenesis contributes to the suppression of black root rot.

Dataset Information

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

Publications

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

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