Switching on a Nontraditional Enzymatic Base—Deprotonation by Serine in the ent-Kaurene Synthase from Bradyrhizobium japonicum

Abstract

Terpene synthases often catalyze complex carbocation cascade reactions. It has been previously shown that single-residue switches involving replacement of a key aliphatic residue with serine or threonine can "shortcircuit" such reactions that are presumed to act indirectly via dipole stabilization of intermediate carbocations. Here a similar switch was found in the structurally characterized ent-kaurene synthase from Bradyrhizobium japonicum. Application of a recently developed computational approach to terpene synthases, TerDockin, surprisingly indicates direct action of the introduced serine hydroxyl as a catalytic base. Notably, this model suggests an alternative interpretation of previous results and potential routes toward reengineering terpene synthase activity more generally.

Probing Enzymatic Structure and Function in the Dihydroxylating Sesquiterpene Synthase ZmEDS

“…While it has been suggested that the G1/2 helix break is important for the initial 1,10-ring closure, 20 these mutants do not seem to affect this first (macro)cyclization, but instead the subsequent protonation-initiated (bi)cyclization and/or rearrangement, as well as the second addition of water. Although the ability of hydroxylated amino acid side chains to serve as catalytic bases deprotonating carbocations in TPSs has been shown, 16 the effects of the T409 and S410 mutants do not seem to indicate such a role for these residues in ZmEDS either. On the other hand, it could be argued that T409 might play a role in positioning the water to be added to the eudesmolyl carbocation.…”

“…The resulting complexes were visualized with PyMOL 1.8.x. A Mg 2+ –diphosphate complex was placed in ZmEDS by modeling with a distinct TEAS structure (PDB entry 4RNQ) as the template and then placing the Mg 2+ –diphosphate complex from this into the modeled ZmEDS structure, much as previously described for other TPSs …”

“…The G1/2 helix break is conserved in TPSs; however, the amino acid residues that make up this kink are diverse, and changes to these residues have been shown to have profound effects on product outcome. , The ZmEDS model indicates that the corresponding residues are T 409 S 410 G 411 (Figure ). A number of mutants were made for each of these residues.…”

“…A Mg 2+ − diphosphate complex was placed in ZmEDS by modeling with a distinct TEAS structure (PDB entry 4RNQ) 15 as the template and then placing the Mg 2+ −diphosphate complex from this into the modeled ZmEDS structure, much as previously described for other TPSs. 16 Purification and Identification of Products. To obtain sufficient amounts of terpenoid products for chemical structure elucidation, the culture scale was increased to 6 L. Terpenoids were extracted as described above and isolated through silica gel chromatography.…”

“…The resulting complexes were visualized with PyMOL 1.8.x. A Mg 2+ -diphosphate complex was placed into ZmEDS by modeling with a distinct TEAS structure(PDB ID: 4RNQ) 15 as the template, and then placing the Mg 2+ -diphosphate complex from this into the modeled ZmEDS structure, much as previously described for other TPSs 16 . NMR analysis-The purified terpenoid products were dissolved in 0.5 ml CDCl 3 and transferred into a 2.5 mm × 100 mm NMR tube for NMR analysis, which was carried out on a Bruker Avance 600 MHz instrument with 1 H and 13 C spectroscopic analysis at 22°C using a 5-mm TXI CryoProbe.…”