Structure of 2-Methylisoborneol Synthase from <i>Streptomyces coelicolor</i> and Implications for the Cyclization of a Noncanonical <i>C</i>-Methylated Monoterpenoid Substrate

The crystal structure of 2-methylisoborneol synthase (MIBS) from <i>Streptomyces coelicolor</i> A3(2) has been determined in complex with substrate analogues geranyl-<i>S</i>-thiolodiphosphate and 2-fluorogeranyl diphosphate at 1.80 and 1.95 Å resolution, respectively. This terpenoid cyclase catalyzes the cyclization of the naturally occurring, noncanonical <i>C</i>-methylated isoprenoid substrate, 2-methylgeranyl diphosphate, to form the bicyclic product 2-methylisoborneol, a volatile C₁₁ homoterpene alcohol with an earthy, musty odor. While MIBS adopts the tertiary structure of a class I terpenoid cyclase, its dimeric quaternary structure differs from that previously observed in dimeric terpenoid cyclases from plants and fungi. The quaternary structure of MIBS is nonetheless similar in some respects to that of dimeric farnesyl diphosphate synthase, which is not a cyclase. The structures of MIBS complexed with substrate analogues provide insights regarding differences in the catalytic mechanism of MIBS and the mechanisms of (+)-bornyl diphosphate synthase and <i>endo</i>-fenchol synthase, plant cyclases that convert geranyl diphosphate into products with closely related bicyclic bornyl skeletons, but distinct structures and stereochemistries

Unexpected Reactivity of 2‑Fluorolinalyl Diphosphate in the Active Site of Crystalline 2‑Methylisoborneol Synthase

The crystal structure of 2-methylisoborneol synthase (MIBS) from <i>Streptomyces coelicolor</i> A3(2) has been determined in its unliganded state and in complex with two Mg²⁺ ions and 2-fluoroneryl diphosphate at 1.85 and 2.00 Å resolution, respectively. Under normal circumstances, MIBS catalyzes the cyclization of the naturally occurring, noncanonical 11-carbon isoprenoid substrate, 2-methylgeranyl diphosphate, which first undergoes an ionization–isomerization–ionization sequence through the tertiary diphosphate intermediate 2-methyllinalyl diphosphate to enable subsequent cyclization chemistry. MIBS does not exhibit catalytic activity with 2-fluorogeranyl diphosphate, and we recently reported the crystal structure of MIBS complexed with this unreactive substrate analogue [Köksal, M., Chou, W. K. W., Cane, D. E., Christianson, D. W. (2012) Biochemistry 51, 3011–3020]. However, cocrystallization of MIBS with the fluorinated analogue of the tertiary allylic diphosphate intermediate, 2-fluorolinalyl diphosphate, reveals unexpected reactivity for the intermediate analogue and yields the crystal structure of the complex with the primary allylic diphosphate, 2-fluoroneryl diphosphate. Comparison with the structure of the unliganded enzyme reveals that the crystalline enzyme active site remains partially open, presumably due to the binding of only two Mg²⁺ ions. Assays in solution indicate that MIBS catalyzes the generation of (1<i>R</i>)-(+)-camphor from the substrate 2-fluorolinalyl diphosphate, suggesting that both 2-fluorolinalyl diphosphate and 2-methyllinalyl diphosphate follow the identical cyclization mechanism leading to 2-substituted isoborneol products; however, the initially generated 2-fluoroisoborneol cyclization product is unstable and undergoes elimination of hydrogen fluoride to yield (1<i>R</i>)-(+)-camphor

Structure of Geranyl Diphosphate <i>C</i>-Methyltransferase from <i>Streptomyces coelicolor</i> and Implications for the Mechanism of Isoprenoid Modification

Geranyl diphosphate <i>C</i>-methyltransferase (GPPMT) from <i>Streptomyces coelicolor</i> A3(2) is the first methyltransferase discovered that modifies an acyclic isoprenoid diphosphate, geranyl diphosphate (GPP), to yield a noncanonical acyclic allylic diphosphate product, 2-methylgeranyl diphosphate, which serves as the substrate for a subsequent cyclization reaction catalyzed by a terpenoid cyclase, methylisoborneol synthase. Here, we report the crystal structures of GPPMT in complex with GPP or the substrate analogue geranyl <i>S</i>-thiolodiphosphate (GSPP) along with <i>S</i>-adenosyl-l-homocysteine in the cofactor binding site, resulting from <i>in situ</i> demethylation of <i>S</i>-adenosyl-l-methionine, at 2.05 or 1.82 Å resolution, respectively. These structures suggest that both GPP and GSPP can undergo catalytic methylation in crystalline GPPMT, followed by dissociation of the isoprenoid product. <i>S</i>-Adenosyl-l-homocysteine remains bound in the active site, however, and does not exchange with a fresh molecule of cofactor <i>S</i>-adenosyl-l-methionine. These structures provide important clues about the molecular mechanism of the reaction, especially with regard to the face of the 2,3 double bond of GPP that is methylated as well as the stabilization of the resulting carbocation intermediate through cation−π interactions

Substitution of Aromatic Residues with Polar Residues in the Active Site Pocket of <i>epi</i>-Isozizaene Synthase Leads to the Generation of New Cyclic Sesquiterpenes

The sesquiterpene cyclase <i>epi</i>-isozizaene synthase (EIZS) catalyzes the cyclization of farnesyl diphosphate to form the tricyclic hydrocarbon precursor of the antibiotic albaflavenone. The hydrophobic active site pocket of EIZS serves as a template as it binds and chaperones the flexible substrate and carbocation intermediates through the conformations required for a multistep reaction sequence. We previously demonstrated that the substitution of hydrophobic residues with other hydrophobic residues remolds the template and expands product chemodiversity [Li, R., Chou, W. K. W., Himmelberger, J. A., Litwin, K. M., Harris, G. G., Cane, D. E., and Christianson, D. W. (2014) <i>Biochemistry 53</i>, 1155–1168]. Here, we show that the substitution of hydrophobic residuesspecifically, Y69, F95, F96, and W203with polar side chains also yields functional enzyme catalysts that expand product chemodiversity. Fourteen new EIZS mutants are reported that generate product arrays in which eight new sesquiterpene products have been identified. Of note, some mutants generate acyclic and cyclic hydroxylated products, suggesting that the introduction of polarity in the hydrophobic pocket facilitates the binding of water capable of quenching carbocation intermediates. Furthermore, the substitution of polar residues for F96 yields high-fidelity sesquisabinene synthases. Crystal structures of selected mutants reveal that residues defining the three-dimensional contour of the hydrophobic pocket can be substituted without triggering significant structural changes elsewhere in the active site. Thus, more radical nonpolar–polar amino acid substitutions should be considered when terpenoid cyclase active sites are remolded by mutagenesis with the goal of exploring and expanding product chemodiversity

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

Terpenoid synthases catalyze isoprenoid cyclization reactions underlying the generation of more than 80,000 natural products. Such dramatic chemodiversity belies the fact that these enzymes generally consist of only three domain folds designated as α, β, and γ. Catalysis by class I terpenoid synthases occurs exclusively in the α domain, which is found with α, αα, αβ, and αβγ domain architectures. Here, we explore the influence of domain architecture on catalysis by taxadiene synthase from <i>Taxus brevifolia</i> (TbTS, αβγ), fusicoccadiene synthase from <i>Phomopsis amygdali</i> (PaFS, (αα)₆), and ophiobolin F synthase from <i>Aspergillus clavatus</i> (AcOS, αα). We show that the cyclization fidelity and catalytic efficiency of the α domain of TbTS are severely compromised by deletion of the βγ domains; however, retention of the β domain preserves significant cyclization fidelity. In PaFS, we previously demonstrated that one α domain similarly influences catalysis by the other α domain [Chen, M., Chou, W. K. W., Toyomasu, T., Cane, D. E., and Christianson, D. W. (2016) ACS Chem. Biol. 11, 889−899]. Here, we show that the hexameric quaternary structure of PaFS enables cluster channeling. We also show that the α domains of PaFS and AcOS can be swapped so as to make functional chimeric αα synthases. Notably, both cyclization fidelity and catalytic efficiency are altered in all chimeric synthases. Twelve newly formed and uncharacterized C₂₀ diterpene products and three C₂₅ sesterterpene products are generated by these chimeras. Thus, engineered αβγ and αα terpenoid cyclases promise to generate chemodiversity in the greater family of terpenoid natural products

The T296V Mutant of Amorpha-4,11-diene Synthase Is Defective in Allylic Diphosphate Isomerization but Retains the Ability To Cyclize the Intermediate (3<i>R</i>)‑Nerolidyl Diphosphate to Amorpha-4,11-diene

The T296V mutant of amorpha-4,11-diene synthase catalyzes the abortive conversion of the natural substrate (<i>E</i>,<i>E</i>)-farnesyl diphosphate mainly into the acyclic product (<i>E</i>)-β-farnesene (88%) instead of the natural bicyclic sesquiterpene amorphadiene (7%). Incubation of the T296V mutant with (3<i>R</i>,6<i>E</i>)-nerolidyl diphosphate resulted in cyclization to amorphadiene. Analysis of additional mutants of amino acid residue 296 and <i>in vitro</i> assays with the intermediate analogue (2<i>Z</i>,6<i>E</i>)-farnesyl diphosphate as well as (3<i>S</i>,6<i>E</i>)-nerolidyl diphosphate demonstrated that the T296V mutant can no longer catalyze the allylic rearrangement of farnesyl diphosphate to the normal intermediate (3<i>R</i>,6<i>E</i>)-nerolidyl diphosphate, while retaining the ability to cyclize (3<i>R</i>,6<i>E</i>)-nerolidyl diphosphate to amorphadiene. The T296A mutant predominantly retained amorphadiene synthase activity, indicating that neither the hydroxyl nor the methyl group of the Thr296 side chain is required for cyclase activity