Thomas J. Savage

Monoterpene Synthases from Common Sage (Salvia officinalis) cDNA ISOLATION, CHARACTERIZATION, AND FUNCTIONAL EXPRESSION OF (+)-SABINENE SYNTHASE, 1,8-CINEOLE SYNTHASE, AND (+)-BORNYL DIPHOSPHATE SYNTHASE

Common sage (Salvia officinalis) produces an extremely broad range of cyclic monoterpenes bearing diverse carbon skeletons, including members of thep-menthane (1,8-cineole), pinane (α- and β-pinene), thujane (isothujone), camphane (camphene), and bornane (camphor) families. An homology-based polymerase chain reaction cloning strategy was developed and used to isolate the cDNAs encoding three multiproduct monoterpene synthases from this species that were functionally expressed in Escherichia coli. The heterologously expressed synthases produce (+)-bornyl diphosphate, 1,8-cineole, and (+)-sabinene, respectively, as their major products from geranyl diphosphate. The bornyl diphosphate synthase also produces significant amounts of (+)-α-pinene, (+)-camphene, and (±)-limonene. The 1,8-cineole synthase produces significant amounts of (+)- and (−)-α-pinene, (+)- and (−)-β-pinene, myrcene and (+)-sabinene, and the (+)-sabinene synthase produces significant quantities of γ-terpinene and terpinolene. All three enzymes appear to be translated as preproteins bearing an amino-terminal plastid targeting sequence, consistent with the plastidial origin of monoterpenes in plants. Deduced sequence analysis and size exclusion chromatography indicate that the recombinant bornyl diphosphate synthase is a homodimer, whereas the other two recombinant enzymes are monomeric, consistent with the size and subunit architecture of their native enzyme counterparts. The distribution and stereochemistry of the products generated by the recombinant (+)-bornyl diphosphate synthase suggest that this enzyme might represent both (+)-bornyl diphosphate synthase and (+)-pinene synthase which were previously assumed to be distinct enzymes.

Isotopically sensitive branching as a tool for evaluating multiple product formation by monoterpene cyclases

Abstract The deuterated substrates [4-2H2,1-3H]geranyl pyrophosphate and [10-2H3,1-3H]geranyl pyrophosphate were employed to examine isotopically sensitive branching in the biosynthesis of monoterpene olefin isomers. By this method, (−)-α-pinene and (−)-β-pinene were shown to be synthesized via a common intermediate by a single cyclization enzyme from grand fir (Abies grandis), as were (−)-(α-phellandrene and (−)-β-phellandrene by a single cyclase from lodgepole pine (Pinus contorta). Kinetic isotope effects were determined for the various deprotonations leading to the pinenes and phellandrenes.

Monoterpene synthases from gymnosperms and angiosperms: stereospecificity and inactivation by cysteinyl- and arginyl-directed modifying reagents.

To further define specific structural and mechanistic differences among monoterpene synthases from divergent plant sources, the stereospecificity of the enzyme-catalyzed isomerization of geranyl pyrophosphate to linalyl pyrophosphate and the subsequent cyclization to monoterpene olefins (which have been well established for monoterpene synthases from herbaceous angiosperms) were examined for monoterpene synthases from a conifer, lodgepole pine (Pinus contorta). The chiral monoterpenes isolated from lodgepole pine oleoresin and the major chiral products from cell-free assays of each of the four lodgepole pine monoterpene synthases belonged to the stereochemical family related by the biosynthetic intermediacy of 3S-linalyl pyrophosphate. Furthermore, both the putative intermediate, 3S-linalyl pyrophosphate, and the natural substrate, geranyl pyrophosphate, were enzymatically converted to the same monoterpene enantiomers. Thus, like monoterpene synthases from herbaceous angiosperms, monoterpene synthases from lodgepole pine appear to catalyze both the stereospecific isomerization of geranyl pyrophosphate to linalyl pyrophosphate and the subsequent cyclization of this enzyme-bound intermediate to multiple, stereochemically related monoterpene olefin isomers. The susceptibility of monoterpene synthases to inactivation by cysteinyl- and arginyl-directed chemical modification reagents was also examined to identify specific structural differences between enzymes from conifers and angiosperms. Like monoterpene synthases from peppermint (Mentha x piperita) and culinary sage (Salvia officinalis), monoterpene synthases from lodgepole pine were inactivated by thiol-directed reagents; however, unlike monoterpene synthases from these herbaceous angiosperms, monoterpene synthases from lodgepole pine were not protected against inactivation by coincubation with substrate and metal ion cofactor. Lodgepole pine monoterpene synthases were also inactivated by the arginyl-directed reagent phenylglyoxal, and coincubation with substrate and cofactor, to effect active-site protection, reduced the rate of inactivation 10-fold. (+)-Pinene synthase and (-)-pinene synthase from sage were also inactivated by phenylglyoxal, but no protection was afforded by coincubation with substrate and cofactor. Thus, monoterpene synthases of conifers appear to have catalytically important arginyl residues specifically located at or near the active site and have at least some catalytically important thiol residues at a non-substrate-protectable region of the enzyme, in contrast to monoterpene synthases from angiosperms which appear to have catalytically important cysteinyl residues at the active site and have catalytically important arginyl residues located at a non-substrate-protectable region of the enzyme.

Evidence for an Elongation/Reduction/C1-Elimination Pathway in the Biosynthesis of n-Heptane in Xylem of Jeffrey Pine

The biosynthetic pathway to n-heptane was investigated by examining the effect of the [beta]-keto acyl-acyl carrier protein synthase inhibitor (2R,3S)-2,3-epoxy-4-oxo-7E,10E-dodecadienamide (cerulenin), a thiol reagent ([beta]-mercaptoethanol), and an aldehydetrapping reagent (hydroxylamine) on the biosynthesis of n-[14C]heptane and putative intermediates in xylem sections of Jeffrey pine (Pinus jeffreyi Grev. & Balf.) incubated with [14C]acetate. Cerulenin inhibited C18 fatty acid biosynthesis but had relatively little effect on radiolabel incorporation into C8 fatty acyl groups and n-heptane. [beta]-Mercaptoethanol inhibited n-heptane biosynthesis, with a corresponding accumulation of radiolabel into both octanal and 1-octanol, whereas hydroxylamine inhibited both n-heptane and 1-octanol biosynthesis, with radiolabel accumulation in octyl oximes. [14C]Octanal was converted to both n-heptane and 1-octanol when incubated with xylem sections, whereas [14C]1-octanol was converted to octanal and n-heptane in a hydroxylamine-sensitive reaction. These results suggest a pathway for the biosynthesis of n-heptane whereby acetate is polymerized via a typical fatty acid synthase reaction sequence to yield a C8 thioester, which subsequently undergoes a two-electron reduction to generate a free thiol and octanal, the latter of which alternately undergoes an additional, reversible reduction to form 1-octanol or loss of C1 to generate n-heptane.

Condensation of the isoprenoid and amino precursors in the biosynthesis of domoic acid

Understanding how environmental signals regulate production of domoic acid in blooms of Pseudo-nitzschia spp. at a molecular level requires description of the biochemical pathway to this kainoid neurotoxin. Precursor feeding studies have suggested domoic acid arises from the condensation of the C(10) isoprenoid geranyl diphosphate with glutamate, but the specific reactions leading to domoic acid from these precursors remain undescribed. Here, we develop a method to derivatize domoic acid with propyl chloroformate that enables gas chromatography-mass spectrometry (GC-MS) analysis to measure incorporation of stable isotopes into domoic acid generated in cultures incubated with isotopically-labeled substrates. We apply this method to demonstrate that both (2)H from [1-(2)H(2)]geraniol are incorporated into domoic acid, suggesting that the condensation of geranyl diphosphate with an amino group occurs by nucleophilic substitution of the diphosphate rather than by oxidation of geraniol to the aldehyde before reaction with an amino group to form an imine. Ultimately, these and similar studies will facilitate the identification of DA biosynthetic enzymes and genes which will enable the study of how environmental factors regulate DA biosynthesis at the molecular level.

Biochemistry of short-chain alkanes : tissue-specific biosynthesis of n-heptane in Pinus jeffreyi

Short-chain (C7-C11) alkanes accumulate as the volatile component of oleoresin (pitch) in several pine species native to western North America. To establish the tissue most amenable for use in detailed studies of short-chain alkane biosynthesis, we examined the tissue specificity of alkane accumulation and biosynthesis in Pinus jeffreyi Grev. & Balf. Short-chain alkane accumulation was highly tissue specific in both 2-year-old saplings and mature trees; heart-wood xylem accumulated alkanes up to 7.1 mg g-1 dry weight, whereas needles and other young green tissue contained oleoresin with monoterpenoid, rather than paraffinic, volatiles. These tissue-specific differences in oleoresin composition appear to be a result of tissue-specific rates of alkane and monoterpene biosynthesis; incubation of xylem tissue with [14C]sucrose resulted in accumulation of radiolabel in alkanes but not monoterpenes, whereas incubation of foliar tissue with 14CO2 resulted in the accumulation of radiolabel in monoterpenes but not alkanes. Furthermore, incubation of xylem sections with [14C]acetate resulted in incorporation of radiolabel into alkanes at rates up to 1.7 nmol h-1 g-1 fresh weight, a rate that exceeds most biosynthetic rates reported with other plant systems for the incorporation of this basic precursor into natural products. This suggests that P. jeffreyi may provide a suitable model for elucidating the enzymology and molecular biology of short-chain alkane biosynthesis.

The effect of disturbance by Phellinus weirii on decomposition and nutrient mineralization in a Tsuga mertensiana forest

SummaryMicrobial biomass in the upper 7 cm of soil and needle decomposition on the forest floor were measured seasonally for 10 months in a mountain hemlock (Tsuga mertensiana) old-growth forest and in a regrowth forest after Phellinus weirii, a root-rot pathogen infection, had caused disturbance. The microbial biomass was higher in the old-growth forest soil than in the regrowth forest soil. However, T. mertensiana needle decomposition rates were higher in the regrowth than in the old-growth forest. Total N, Ca, Fe, Cu, and Zn concentrations in needles increased during the 1st year of decomposition in both the old and the regrowth forests, but P, K, Mg, Mn, and B concentrations decreased. N, P, K, Mg, Cu, and Zn concentrations were lower in regrowth than in old-growth decomposing needles. During mineralization, needles in the regrowth forests released more N, P, and K as a result of higher needle decomposition rates. Our results suggest that higher needle decomposition rates increased the mineralization of N, P, and K, which may lead to increased soil fertility and faster tree growth rates in the regrowth forest.

Biochemistry of Short-Chain Alkanes: Evidence for an Elongation/Reduction/Cl-Elimination Pathway

C7- C11 alkanes accumulate in the oleoresins of several Pinus species native to western North America, most notably in the xylem oleoresin of Jeffrey pine, Pinus jeffreyi Grey. & Half. (Savage et al., 1990a). Biological production of light hydrocarbons is of interest because they possess the same excellent combustion properties as petrochemical hydrocarbons in gasoline. Whereas production levels of short-chain alkanes in plants are insufficient to provide an economically viable fuel source, the genes encoding the alkane biosynthetic pathway may provide a biotechnological resource for engineering fermentation organisms with the capability to convert biomass to an alkane-based fuel. However, the feasibility of transgenic alkane biosynthesis depends upon the complexity of the alkane biosynthetic pathway.