The crystal structure of (4S)-limonene synthase from Mentha spicata, a metal ion-dependent monoterpene cyclase that catalyzes the coupled isomerization and cyclization of geranyl diphosphate, is reported at 2.7-Å resolution in two forms liganded to the substrate and intermediate analogs, 2-fluorogeranyl diphosphate and 2-fluorolinalyl diphosphate, respectively. The implications of these findings are described for domain interactions in the homodimer and for changes in diphosphate-metal ion coordination and substrate binding conformation in the course of the multistep reaction.crystal structure ͉ geranyl diphosphate ͉ linalyl diphosphate ͉ monoterpene cyclase ͉ monoterpene synthase

Two crystal structures for E. coli thymidylate synthase (TS) bound to the mechanism-based inhibitor 5-fluoro-dUMP (FdUMP) and methylenetetrahydrofolate (CH2THF) have been determined to 2.6 and 2.2 A nominal resolutions, with crystallographic R factors of 0.180 and 0.178, respectively. The inhibitor and cofactor are well ordered in both structures and display covalent links to each other and to Cys 146 in the TS active site. The structures are in general agreement with a previous report for this complex (D. A. Matthews et al. (1990) J. Mol. Biol. 214, 937-948), but differ in two key respects: (i) the methylene bridge linking FdUMP and CH2THF is rotated about 60 degrees to a different position and (ii) the electron density for C6 of FdUMP, which is covalently linked to Cys 146, is more diffuse than for the other atoms in the pyrimidine ring. The ligand arrangement observed in the previous structure led the authors to propose that a large conformational change in ligand geometry must occur in order to facilitate catalysis and yield the correct chirality in the methyl of product dTMP. The new structures suggest a different mechanism for product formation that does not require ligands to greatly alter their conformations during catalysis and which makes use of instability in the nucleotide-Cys 146 thiol adduct to avoid a deep free energy well and assist in proton abstraction from dUMP. All intermediates in the proposed mechanism were modeled and energy minimized in the TS active site, and all can be accommodated in the present structures. The role of ligand-induced conformational change in the TS mechanism and the possibility of Tyr 94 acting as a base during catalysis are also discussed.

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Polymethoxylated flavonoids occur in a number of plant families, including the Lamiaceae. To date, the metabolic pathways giving rise to the diversity of these compounds have not been studied. Analysis of our expressed sequence tag database for four sweet basil (Ocimum basilicum) lines afforded identification of candidate flavonoid O-methyltransferase genes. Recombinant proteins displayed distinct substrate preferences and product specificities that can account for all detected 7-/6-/49-methylated, 8-unsubstituted flavones. Their biochemical specialization revealed only certain metabolic routes to be highly favorable and therefore likely in vivo. Flavonoid O-methyltransferases catalyzing 49-and 6-O-methylations shared high identity (approximately 90%), indicating that subtle sequence changes led to functional differentiation. Structure homology modeling suggested the involvement of several amino acid residues in defining the proteins' stringent regioselectivities. The roles of these individual residues were confirmed by site-directed mutagenesis, revealing two discrete mechanisms as a basis for the switch between 6-and 49-O-methylation of two different substrates. These findings delineate major pathways in a large segment of the flavone metabolic network and provide a foundation for its further elucidation.

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A radiation-induced mutant of Scotch spearmint (Mentha x graclis) was shown to produce an essential oil containing principally C3-oxygenated p-menthane monoterpenes that are typical of peppermint, instead of the CS-oxygenated monoterpene family characteristic of spearmint. In vitro measurement of all of the enzymes responsible for the production of both the C3-oxygenated and CS-oxygenated families of monoterpenes from the common precursor (-)-limonene indicated that a virtually identical complement of enzymes was present in wild type and mutant, with the exception of the microsomal, cytochrome P-450-dependent (-)-limonene hydroxylase; the C6-hydroxylase producing (-)-trans-carveol in the wild type had been replaced by a C3-hydroxylase producing (--trans-isopiperitenol in the mutant. Additionally, the mutant, but not the wild type, could carry out the cytochrome P-450-dependent epoxidation of the ai-unsaturated bond of the ketones formed via C3-hydroxylation. Although present in the wild type, the enzymes of the C3-pathway that convert trans-isopiperitenol to menthol isomers are synthetically inactive because of the absence of the key C3-oxygenated intermediate generated by hydroxylation of limonene. These results, which clarify the origins of the C3-and CS-oxygenation patterns, also allow correction of a number of earlier biogenetic proposals for the formation of monoterpenes in Mentha.The monoterpene constituents of the essential oils of peppermint (Mentha piperita L.) and spearmint (native = Mentha spicata L.; Scotch = Mentha x gracilis [28]) are distinguished by the position of oxygenation on the p-menthane ring3 (18,25). Peppermint produces almost exclusively monoterpenes bearing an oxygen function at C3, such as menthone and menthol, whereas spearmint species produce almost exclusively monoterpenes bearing an oxygen function at C6, typi-

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