pyrophosphate and labdane

Labdane-related diterpenoids are natural products with potential pharmaceutical applications that are rarely found in bacteria. Here, a putative class I labdane-related diterpene synthase (LrdC) identified by genome mining in a streptomycete was successfully crystallized using the microbatch method. Crystals of the LrdC enzyme were obtained in a holo form with its natural cofactor Mg(2+) (LrdC-Mg(2+)) and in complex with inorganic pyrophosphate (PPi) (LrdC-Mg(2+)-PPi). Crystals of native LrdC-Mg(2+) diffracted to 2.50 Å resolution and belonged to the trigonal space group P3221, with unit-cell parameters a = b = 107.1, c = 89.2 Å. Crystals of the LrdC-Mg(2+)-PPi complex grown in the same conditions as the native enzyme with PEG 8000 diffracted to 2.36 Å resolution and also belonged to the trigonal space group P3221. Crystals of the LrdC-Mg(2+)-PPi complex grown in a second crystallization condition with PEG 3350 diffracted to 2.57 Å resolution and belonged to the monoclinic space group P21, with unit-cell parameters a = 49.9, b = 104.1, c = 66.5 Å, β = 111.4°. The structure was determined by the single-wavelength anomalous dispersion (SAD) technique using the osmium signal from a potassium hexachloroosmate (IV) derivative.

Topics: Alkyl and Aryl Transferases; Amino Acid Sequence; Chromatography, Gel; Crystallization; Diphosphates; Diterpenes; Magnesium; Molecular Sequence Data; Streptomyces; X-Ray Diffraction

Terpene synthases catalyze complex reactions, often forming multiple chiral centers in cyclized olefin products from acyclic allylic diphosphate precursors, yet have been suggested to largely control their reactions via steric effects, serving as templates. However, recent results highlight electrostatic effects also exerted by these enzymes. Perhaps not surprisingly, the pyrophosphate co-product released in the initiating and rate-limiting chemical step provides an obvious counter-ion that may steer carbocation migration towards itself. This is emphasized by the striking effects of a recently uncovered single residue switch for diterpene synthase product outcome, whereby substitution of hydroxyl residues for particular aliphatic residues has been shown to be sufficient to "short-circuit" complex cyclization and/or rearrangement reactions, with the converse change further found to be sufficient to increase reaction complexity. The mechanistic hypothesis for the observed effects is hydroxyl dipole stabilization of the specific carbocation formed by initial cyclization, enabling deprotonation of this early intermediate, whereas the lack of such stabilization (i.e. in the presence of an aliphatic side chain) leads to carbocation migration towards the pyrophosphate co-product, resulting in a more complex reaction. This is further consistent with the greater synergy exhibited between pyrophosphate and aza-analogs of late, relative to early, stage carbocation intermediates, and crystallographic analysis of the monoterpene cyclase bornyl diphosphate synthase wherein mechanistically non-relevant counter-ion pairing between aza-analogs of early stage carbocation intermediates and pyrophosphate is observed. Thus, (di)terpene synthases seem to mediate specific reaction outcomes, at least in part, by providing electrostatic effects to counteract those exerted by the pyrophosphate co-product.

Topics: Alkyl and Aryl Transferases; Biocatalysis; Catalytic Domain; Crystallography, X-Ray; Cyclization; Diphosphates; Diterpenes; Kinetics; Static Electricity; Stereoisomerism