germacradienol and geosmin

Geosmin synthase from Streptomyces coelicolor (ScGS) catalyzes an unusual, metal-dependent terpenoid cyclization and fragmentation reaction sequence. Two distinct active sites are required for catalysis: the N-terminal domain catalyzes the ionization and cyclization of farnesyl diphosphate to form germacradienol and inorganic pyrophosphate (PPi), and the C-terminal domain catalyzes the protonation, cyclization, and fragmentation of germacradienol to form geosmin and acetone through a retro-Prins reaction. A unique αα domain architecture is predicted for ScGS based on amino acid sequence: each domain contains the metal-binding motifs typical of a class I terpenoid cyclase, and each domain requires Mg(2+) for catalysis. Here, we report the X-ray crystal structure of the unliganded N-terminal domain of ScGS and the structure of its complex with three Mg(2+) ions and alendronate. These structures highlight conformational changes required for active site closure and catalysis. Although neither full-length ScGS nor constructs of the C-terminal domain could be crystallized, homology models of the C-terminal domain were constructed on the basis of ∼36% sequence identity with the N-terminal domain. Small-angle X-ray scattering experiments yield low-resolution molecular envelopes into which the N-terminal domain crystal structure and the C-terminal domain homology model were fit, suggesting possible αα domain architectures as frameworks for bifunctional catalysis.

Topics: Alendronate; Bacterial Proteins; Crystallography, X-Ray; Cyclization; Magnesium; Models, Molecular; Naphthols; Polyisoprenyl Phosphates; Protein Structure, Tertiary; Sesquiterpenes; Streptomyces coelicolor

Topics: Bacterial Proteins; Naphthols; Polyisoprenyl Phosphates; Sesquiterpenes; Streptomyces coelicolor

Geosmin is responsible for the characteristic odor of moist soil. Incubation of recombinant germacradienol synthase, encoded by the SCO6073 (SC9B1.20) gene of the Gram-positive soil bacterium Streptomyces coelicolor, with farnesyl diphosphate (2, FPP) in the presence of Mg2+ gave a mixture of (4S,7R)-germacra-1(10)E,5E-diene-11-ol (3) (74%), (-)-(7S)-germacrene D (4) (10%), geosmin (1) (13%), and a hydrocarbon, tentatively assigned the structure of octalin 5 (3%). Individual incubations of recombinant germacradienol synthase with [1,1-2H2]FPP (2a), (1R)-[1-2H]-FPP (2b), and (1S)-[1-2H]-FPP (2c), as well as with FPP (2) in D2O, and GC-MS analysis of the resulting deuterated products supported a mechanism of geosmin formation involving proton-initiated cyclization and retro-Prins fragmentation of the initially formed germacradienol to give intermediate 5, followed by protonation of 5, 1,2-hydride shift, and capture of water.

Topics: Alkyl and Aryl Transferases; Cyclization; Mass Spectrometry; Molecular Structure; Naphthols; Polyisoprenyl Phosphates; Recombinant Proteins; Sesquiterpenes; Streptomyces coelicolor

Geosmin (1) is responsible for the characteristic odor of moist soil. The Gram-positive soil bacterium Streptomyces avermitilis produces geosmin (1) as well as its precursor germacradienol (3). The S. avermitilis gene SAV2163 (geoA) is extremely similar to the S. coelicolor A3(2) SCO6073 gene that encodes a germacradienol/geosmin synthase. S. avermitilis mutants with a deleted geoA were unable to produce either germacradienol (3) or geosmin (1). Biosynthesis of both compounds was restored by introducing an intact geoA gene into the mutants. Incubation of recombinant GeoA, encoded by the SAV2163 gene of S. avermitilis, with farnesyl diphosphate (2) in the presence of Mg2+ gave a mixture of (4S,7R)-germacra-1(10)E,5E-diene-11-ol (3) (66%), (7S)-germacrene D (4) (24%), geosmin (1) (8%), and a hydrocarbon, tentatively assigned the structure of octalin 5 (2%). Incubation of this germacradienol/geosmin synthase with [1,1-(2)H2] FPP (2a) gave geosmin-d1 (1a), as predicted. When recombinant GeoA from either S. avermitilis or S. coelicolor A3(2) was incubated with nerolidyl diphosphate (8), only the acyclic elimination products beta3-farnesene (10), (Z)-alpha-farnesene (11), and (E)-alpha-farnesene (12) were formed, thereby ruling out nerolidyl diphosphate as an intermediate in the conversion of farnesyl diphosphate to geosmin, germacradienol, and germacrene D.

Topics: Cloning, Molecular; Gene Expression Regulation, Bacterial; Naphthols; Polyisoprenyl Phosphates; Sesquiterpenes; Soil; Streptomyces