Literature summary for 4.1.99.16 extracted from

Harris, G.G.; Lombardi, P.M.; Pemberton, T.A.; Matsui, T.; Weiss, T.M.; Cole, K.E.; Koeksal, M.; Murphy, F.V.; Vedula, L.S.; Chou, W.K.; Cane, D.E.; Christianson, D.W.
Structural studies of geosmin synthase, a bifunctional sesquiterpene synthase with alpha-alpha domain architecture that catalyzes a unique cyclization fragmentation reaction sequence (2015), Biochemistry, 54, 7142-7155 .

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Cloned(Commentary)

Cloned (Comment)	Organism
gene cyc2, recombinant expression of C-terminally His6-tagged full-length enzyme and of the N-terminal domain of ScGS (residues 1-366) in Escherichia coli strain BL21(DE3)pLysS , subcloning in Escherichia coli strain XL-1 Blue	Streptomyces coelicolor

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant N-terminal domain of ScGS, unliganded and in complex with 3 Mg2+ ions and alendronate, mixing of 600 nl of 7 mg/ml protein in 25 mM Tris, pH 8.2, 5 mM MgCl2, 10 mM BME, and 1.5 mM sodium alendronate, with 600 nl of precipitant solution containing 0.2 M sodium acetate trihydrate, pH 7.0, and 20% w/v PEG 3350, and equilibration against 0.1 ml of reservoir solution at room temperature, X-ray diffraction structure determination and analysis at 2.4 A resolution. Neither full-length ScGS nor constructs of the C-terminal domain can be crystallized, but homology models of the C-terminal domain are constructed based on about 36% sequence identity with the N-terminal domain	Streptomyces coelicolor

Crystallization (Comment)

Organism

purified recombinant N-terminal domain of ScGS, unliganded and in complex with 3 Mg2+ ions and alendronate, mixing of 600 nl of 7 mg/ml protein in 25 mM Tris, pH 8.2, 5 mM MgCl2, 10 mM BME, and 1.5 mM sodium alendronate, with 600 nl of precipitant solution containing 0.2 M sodium acetate trihydrate, pH 7.0, and 20% w/v PEG 3350, and equilibration against 0.1 ml of reservoir solution at room temperature, X-ray diffraction structure determination and analysis at 2.4 A resolution. Neither full-length ScGS nor constructs of the C-terminal domain can be crystallized, but homology models of the C-terminal domain are constructed based on about 36% sequence identity with the N-terminal domain

Streptomyces coelicolor

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	each enzyme domain requires Mg2+ for catalysis, binding structure analysis at the N-terminal domain, overview	Streptomyces coelicolor

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.
(1E,4S,5E,7R)-germacra-1(10),5-dien-11-ol + H2O	Streptomyces coelicolor	-	(-)-geosmin + acetone	-	?
(1E,4S,5E,7R)-germacra-1(10),5-dien-11-ol + H2O	Streptomyces coelicolor ATCC BAA-471 / A3(2) / M145	-	(-)-geosmin + acetone	-	?
additional information	Streptomyces coelicolor	geosmin synthase is a bifunctional sesquiterpene synthase with alpha-alpha domain architecture that catalyzes a unique cyclization fragmentation reaction sequence	?	-	?
additional information	Streptomyces coelicolor ATCC BAA-471 / A3(2) / M145	geosmin synthase is a bifunctional sesquiterpene synthase with alpha-alpha domain architecture that catalyzes a unique cyclization fragmentation reaction sequence	?	-	?

Organism

Organism	UniProt	Comment	Textmining
Streptomyces coelicolor	Q9X839	-	-
Streptomyces coelicolor ATCC BAA-471 / A3(2) / M145	Q9X839	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant C-terminally His6-tagged full-length enzyme from Escherichia coli strain BL21(DE3)pLysS by nickel affinity chromatography, recombinant N-terminal domain of ScGS (residues 1-366) from Escherichia coli strain BL21(DE3)pLysS by ammonium sulfate fractionation, adsorption chromatography on methyl resin, gel filtration, and ultrafiltartion	Streptomyces coelicolor

Reaction

Reaction	Comment	Organism	Reaction ID
(1E,4S,5E,7R)-germacra-1(10),5-dien-11-ol + H2O = (-)-geosmin + acetone	the active site in the N-terminal domain of enzyme ScGS catalyzes the ionization-dependent cyclization of FPP to form diphosphate and two cyclic products: germacradienol (major product, 85%) and germacrene D (minor product, 15%). After dissociation from the N-terminal domain, germacradienol is rebound to the active site of the C-terminal domain where it is converted to geosmin in a protonation-dependent cyclization reaction accompanied by the elimination of acetone through a retro-Prins reaction. The tandem cyclization-fragmentation reactions catalyzed by ScGS require two distinct active sites, a unique alphaalpha domain architecture is predicted for ScGS based on primary structure analysis	Streptomyces coelicolor	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.
(1E,4S,5E,7R)-germacra-1(10),5-dien-11-ol + H2O	-	Streptomyces coelicolor	(-)-geosmin + acetone	-	?
(1E,4S,5E,7R)-germacra-1(10),5-dien-11-ol + H2O	-	Streptomyces coelicolor ATCC BAA-471 / A3(2) / M145	(-)-geosmin + acetone	-	?
additional information	geosmin synthase is a bifunctional sesquiterpene synthase with alpha-alpha domain architecture that catalyzes a unique cyclization fragmentation reaction sequence	Streptomyces coelicolor	?	-	?
additional information	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. The enzyme has an alpha,alpha domain architecture, each domain contains the metal-binding motifs typical of a class I terpenoid cyclase, and each domain requires Mg2+ for catalysis. There is no direct channel for transfer of the intermediate from the active site of the N-terminal domain to that of the C-terminal domain. It is a diiffusive transfer of the germacradienol intermediate	Streptomyces coelicolor	?	-	?
additional information	geosmin synthase is a bifunctional sesquiterpene synthase with alpha-alpha domain architecture that catalyzes a unique cyclization fragmentation reaction sequence	Streptomyces coelicolor ATCC BAA-471 / A3(2) / M145	?	-	?
additional information	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. The enzyme has an alpha,alpha domain architecture, each domain contains the metal-binding motifs typical of a class I terpenoid cyclase, and each domain requires Mg2+ for catalysis. There is no direct channel for transfer of the intermediate from the active site of the N-terminal domain to that of the C-terminal domain. It is a diiffusive transfer of the germacradienol intermediate	Streptomyces coelicolor ATCC BAA-471 / A3(2) / M145	?	-	?

Subunits

Subunits	Comment	Organism
More	geosmin synthase has alpha-alpha domain architecture. The N-terminal domain and the C-terminal domain are separated by a 41-residue linker and share 28% and 29% amino acid sequence identity, respectively, with pentalenene synthase. Each domain contains characteristic metal ion-binding motifs of class I terpenoid cyclases. The aspartate-rich motif is found as D86DHFLE91 and D455DYYP459, and the NSE/DTE motif is found as N229DLFSYQRE237 and N598DVFSYQKE606. The C-terminal domain is also predicted to adopt an alpha fold homologous to that of the N-terminal domain based on approximately 36% amino acid sequence identity between these domains. Primary, secondary and quarternary enzyme structrue analysis, and homology modeling, overview	Streptomyces coelicolor

Synonyms

Synonyms	Comment	Organism
Cyc2	-	Streptomyces coelicolor
ScGS	-	Streptomyces coelicolor

General Information

General Information	Comment	Organism
additional information	neither full-length ScGS nor constructs of the C-terminal domain can be crystallized, but homology models of the C-terminal domain are constructed based on about 36% sequence identity with the N-terminal domain, analysis of the crystal structure of the N-terminal domain in unliganded or liganded form, overview. Possible alpha,alpha domain architectures as frameworks for bifunctional catalysis	Streptomyces coelicolor
physiological function	geosmin is a powerful odorant with an extremely low human detection threshold of less than 10 parts-per-trillion, and is mainly responsible for the characteristic odor of freshly turned earth. Although geosmin contributes to the pleasant, earthy flavor of beets, it is also a commonly occurring contaminant of musty-tasting water, wine, and fish. Geosmin is not known to cause human disease, but its detection and elimination from potable water sources is a critical environmental and water quality issue	Streptomyces coelicolor