Structural Studies of Geosmin Synthase, a Bifunctional Sesquiterpene Synthase with αα Domain Architecture That Catalyzes a Unique Cyclization–Fragmentation Reaction Sequence

Abstract

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 Mg2+ 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 3 Mg2+ 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 based on ~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.

Multi-domain terpenoid cyclase architecture and prospects for proximity in bifunctional catalysis

“…The N-terminal α domain of geosmin synthase catalyzes the cyclization of FPP to form germacradienol, which subsequently undergoes a retro-Prins fragmentation in the C-terminal α domain to generate the 12-carbon product geosmin and the 3-carbon product acetone [34,35]. Recently, the crystal structure of the N-terminal α domain and a homology model of the C-terminal α domain were used to fit the ab initio molecular envelope calculated from small-angle X-ray scattering (SAXS) data [36•]. These structural studies revealed a monomeric architecture in which the active site of one domain was not oriented directly toward the active site of the second domain.…”

Exploring the Influence of Domain Architecture on the Catalytic Function of Diterpene Synthases

“…48–50 The N-terminal α domain converts FPP into germacradienol, which undergoes further cyclization and fragmentation in the C-terminal α domain to form acetone plus the earthy odorant geosmin. 51,52 The N-terminal α domain in isolation can convert FPP to germacradienol, and the C-terminal α domain in isolation can convert germacradienol to geosmin and acetone.…”

“…It has also been shown that the germacradienol intermediate does not channel from one active site to the other, but instead dissociates into the medium and re-associates with the active site in the C-terminal α domain to complete the reaction sequence. 52 Given that ScGS is an αα monomer in solution as determined by small-angle X-ray scattering, 50 there is no possibility of cluster channeling similar to that implicated in (αα) 6 PaFS.…”

“…The domain assembly shown is based on the best fit of small-angle X-ray scattering data (the 41-residue interdomain linker is not shown). 50 …”

Die Enzymologie organischer Umwandlungen: Namensreaktionen in biologischen Systemen

“…Die C‐terminale Domäne bewirkt dagegen die anschließende Cyclisierung von 157 zu Geosmin ( 158 ) unter Bildung von Aceton über eine Retro‐Prins‐Reaktion. Beide Domänen wirken unabhängig voneinander, es gibt keinen erkennbaren Reaktionsweg zwischen den beiden aktiven Zentren . Daher bleibt unklar, ob das an der N‐terminalen Domäne gebildete intermediäre Germacradienol ( 157 ) an der C‐terminalen Domäne desselben oder eines anderen Proteinmoleküls weiterreagiert.…”