Biosynthesis of the earthy odorant geosmin by a bifunctional Streptomyces coelicolor enzyme

Abstract

Geosmin is responsible for the characteristic odor of moist soil, as well as off-flavors in drinking water and foodstuffs. Geosmin is generated from farnesyl diphosphate (FPP, 2) by an enzyme that is encoded by the SCO6073 gene in the soil organism Streptomyces coelicolor A32 (ref. 3). We have now shown that the recombinant N-terminal half of this protein catalyzes the Mg2+-dependent cyclization of FPP to germacradienol and germacrene D, while the highly homologous C-terminal domain, previously thought to be catalytically silent, catalyzes the Mg2+-dependent conversion of germacradienol to geosmin. Site-directed mutagenesis confirmed that the N- and C-terminal domains each harbor a distinct, independently functioning active site. A mutation in the N-terminal domain of germacradienol-geosmin synthase of a catalytically essential serine to alanine results in the conversion of FPP to a mixture of sesquiterpenes that includes an aberrant product identified as isolepidozene, which was previously suggested to be an enzyme-bound intermediate in the cyclization of FPP to germacradienol.

Figure 1

Organization of protein domains and conserved Mg²⁺-binding motifs in S. coelicolor germacradienol–geosmin synthase. The N-terminal domain, corresponding to aa 1–319, based on sequence alignments with known Streptomyces terpene synthases is shown in red, while the C-terminal domain, aa 374–726, is highlighted in blue. T366 and M327 are the N- and C-termini, respectively, of the recombinant N-terminal and C-terminal truncation proteins encoded by pRW22 and pJJ3.

Scheme 1

Mechanism and stereochemistry of the cyclization of FPP (2) to germacradienol (3), germacrene D (4), octalin 5, and geosmin (1), catalyzed by S. coelicolor germacradienol–geosmin synthase (GS), showing branchpoint germacradienyl cation A and proposed intermediacy of isolepidozene (6).

Scheme 2

Role of the N-terminal and C-terminal domains of germacradienol–geosmin synthase. a. Incubation of FPP with recombinant N-terminal domain (pRW22p). b. Incubation of FPP with a mixture of recombinant N-terminal domain (pRW22p) and C-terminal domain (pJJ3p). c. Incubation of FPP with recombinant C-terminal domain (pJJ3p). d. Incubation of germacradienol (3) with recombinant C-terminal domain (pJJ3p).

Scheme 3

Formation of aberrant sesquiterpene products by incubation of FPP with germacradienol–geosmin synthase (GS) mutants D86E, L90D, and S233A.