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Increasing β-carotene (a vitamin A precursor) content in Triticum turgidum L. ssp. durum (durum wheat) grains is important to improve pasta nutritional quality. Studies in other species show that altering the expression of LCYE genes increases the flux towards the β-β branch, accumulating higher β-carotene levels. Durum wheat is a tetraploid species that has two LCYE genes (LCYE-A and LCYE-B) associated to the A and B genomes. The objective of this work was to produce durum wheat LCYE mutants through EMS to potentially increase β-carotene content. The LCYE point mutations created with EMS were identified using a Kronos TILLING (Targeting Induced Local Lesion IN Genomes) mutant population. Specific primers that amplified exons 3 through 10 of the LCYE genes were designed and validated. To simplify the TILLING procedure, fragments were digested with CJE (Celery Juice Extract) and visualized on 2% agarose gels. 6X mutant pools were identified, which showed cleavage products and then made into 2X pools to identify mutant individuals. LCYE mutants were then sequenced and evaluated with BLOSUM62, SIFT and PSSM algorithms. Mutants with substitutions W437*, P334L and G368R in LCYE-A and P405L, G352R and T393I in LCYE-B predicted to affect protein function were selected. Substitution W437* increased β-carotene in 75% and overall total carotenoids content in leaves of the mutant 2426 (A1 mutant line), but no significant differences relative to the control were found in grains through HPLC. Finally, the increased levels of β-carotene on leaves have potential applications to improving plant resistance under contaminated environmental conditions., Competing Interests: The authors have declared that no competing interests exist.

2-Chloromuconate cycloisomerase from the Gram-positive bacterium Rhodococcus opacus 1CP (Rho-2-CMCI) is an enzyme of a modified ortho-pathway, in which 2-chlorophenol is degraded using 3-chlorocatechol as the central intermediate. In general, the chloromuconate cycloisomerases catalyze not only the cycloisomerization, but also the process of dehalogenation of the chloromuconate to dienelactone. However Rho-2-CMCI, unlike the homologous enzymes from the Gram-negative bacteria, is very specific for only one position of the chloride on the substrate chloromuconate. Furthermore, Rho-2-CMCI is not able to dehalogenate the 5-chloromuconolactone and therefore it cannot generate the dienelactone. The crystallographic structure of the homooctameric Rho-2-CMCI was solved by molecular replacement using the coordinates of the structure of chloromuconate cycloisomerase from Pseudomonas putida PRS2000. The structure was analyzed and compared to the other already known structures of (chloro)muconate cycloisomerases. In addition to this, molecular docking calculations were carried out, which allowed us to determine the residues responsible for the high substrate specificity and the lack of dehalogenation activity of Rho-2-CMCI. Our studies highlight that a histidine, located in a loop that closes the active site cavity upon the binding of the substrate, could be related to the dehalogenation inability of Rho-2-CMCI and in general of the muconate cycloisomerases., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The chemical composition of the coffee beverage is extremely complex, being made up of hundreds of volatile and non-volatile compounds, many of which are generated in the thermal reactions that occur during the roasting process. However, in the raw coffee bean there are also compounds that survive roasting and are therefore extracted into the beverage. Monoterpenes are an example of this category, as their presence has been reported in the coffee flower, fruit, seed, roasted bean and in the beverage aroma. The present work describes the isolation, heterologous expression and functional characterization of three Coffea arabica cDNAs coding for monoterpene synthases. RNA was purified from C. arabica (cv. Catuai Red) flowers, seeds and fruits at 4 successive ripening stages. Degenerate primers were designed on the most conserved regions of the monoterpene synthase gene family, and then used to isolate monoterpene synthase-like sequences from the cDNA libraries. After 5'- and 3'-RACE, the complete transcripts of 4 putative C. arabica monoterpene synthases (CofarTPS) were obtained. Gene expression in different tissues and developmental stages was analysed. After heterologous expression in Escherichia coli, enzyme activity and substrate specificity were evaluated in vitro by incubation of the recombinant proteins with geranyl pyrophosphate (GPP), geranylgeranyl pyrophosphate (GGPP) and farnesyl pyrophosphate (FPP), precursors respectively of mono-, di- and sesquiterpenes. The reaction products were characterized by HS-SPME GC-MS. CofarTPS1 was classified as a limonene synthase gene, while CofarTPS2 and 3 showed lower activity with the production of linalool and β-myrcene., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Lycopene cyclase converts lycopene to beta-carotene by catalyzing the formation of two beta-rings at each end of the linear carotene structure. This reaction takes place as a two-step reaction in which both sides of of the lycopene molecule are cyclized into beta-carotene rings via the monocyclic gamma-carotene as an intermediate. The crtY gene coding for lycopene cyclase from Paracoccus haeundaensis consists of 1,158 base pairs encoding 386 amino acids residues. An expression plasmid containing the crtY gene (pET44a-CrtY) was constructed and expressed in Escherichia coli, and produced a recombinant protein of approximately 43 kDa, corresponding to the molecular mass of lycopene cyclase. The expressed protein was purified to homogeneity by His-tag affinity chromatography and showed enzymatic activity corresponding to lycopene cyclase. We also determined the lycopene substrate specificity and NADPH cofactor requirements of the purified protein. The Km values for lycopene and NADPH were 3.5 microM and 2 mM, respectively. The results obtained from this study will provide a wider base of knowledge on the enzyme characterization of lycopene cyclase at the molecular level.

Plant roots react to pathogen attack by the activation of general and systemic resistance, including the lignification of cell walls and increased release of phenolic compounds in root exudate. Some fungi have the capacity to degrade lignin using ligninolytic extracellular peroxidases and laccases. Aromatic lignin breakdown products are further catabolized via the β-ketoadipate pathway. In this study, we investigated the role of 3-carboxy-cis,cis-muconate lactonizing enzyme (CMLE), an enzyme of the β-ketoadipate pathway, in the pathogenicity of Fusarium oxysporum f. sp. lycopersici towards its host, tomato. As expected, the cmle deletion mutant cannot catabolize phenolic compounds known to be degraded via the β-ketoadipate pathway. In addition, the mutant is impaired in root invasion and is nonpathogenic, even though it shows normal superficial root colonization. We hypothesize that the β-ketoadipate pathway in plant-pathogenic, soil-borne fungi is necessary to degrade phenolic compounds in root exudate and/or inside roots in order to establish disease., (© 2012 THE AUTHORS. MOLECULAR PLANT PATHOLOGY © 2012 BSPP AND BLACKWELL PUBLISHING LTD.)

Nicotiana species of the section Alatae characteristically emit the floral scent compounds of the 'cineole cassette' comprising 1,8-cineole, limonene, myrcene, α-pinene, β-pinene, sabinene, and α-terpineol. We successfully isolated genes of Nicotiana alata and Nicotiana langsdorfii that encoded enzymes, which produced the characteristic monoterpenes of this 'cineole cassette' with α-terpineol being most abundant in the volatile spectra. The amino acid sequences of both terpineol synthases were 99% identical. The enzymes cluster in a monophyletic branch together with the closely related cineole synthase of Nicotiana suaveolens and monoterpene synthase 1 of Solanum lycopersicum. The cyclization reactions (α-terpineol to 1,8-cineole) of the terpineol synthases of N. alata and N. langsdorfii were less efficient compared to the 'cineole cassette' monoterpene synthases of Arabidopsis thaliana, N. suaveolens, Salvia fruticosa, Salvia officinalis, and Citrus unshiu. The terpineol synthases of N. alata and N. langsdorfii were localized in pistils and in the adaxial and abaxial epidermis of the petals. The enzyme activities reached their maxima at the second day after anthesis when flowers were fully opened and the enzyme activity in N. alata was highest at the transition from day to night (diurnal rhythm)., (© The Author 2011. Published by the Molecular Plant Shanghai Editorial Office in association with Oxford University Press on behalf of CSPB and IPPE, SIBS, CAS.)

Myo-inositol is an important constituent of membrane phospholipids and is a precursor for the phosphoinositide signaling pathway. It is synthesized from glucose 6-phosphate by myo-inositol-3-phosphate synthase (IP synthase), a homotrimer composed of a 68-kDa polypeptide in most mammalian tissues. It is a putative target for mood-stabilizing drugs such as lithium and valproate. Here, we show that the rat gene (Isyna1) encoding this enzyme generates a number of alternatively spliced transcripts in addition to the fully spliced form that encodes the 68-kDa subunit (the alpha isoform). Specifically, we identify a small 16-kDa subunit (the gamma(c) isoform) derived by an intron retention mechanism and provide evidence for its existence in rat tissues. The gamma(c) isoform is highly conserved in mammals, but it lacks the catalytic domain while retaining the NAD(+) binding domain. Both alpha and gamma(c) isoforms are predominantly expressed in many rat tissues and display apparent stoichiometry in purified enzyme preparations. An IP synthase polyclonal antibody not only detects the alpha and gamma(c) isoforms but also several other isoforms in pancreas, intestine, and testis suggesting that the holoenzyme is composed of unique subunits in various tissues. Interestingly, the alpha isoform is not expressed in the intestine. IP synthase activity assays using purified alpha and gamma(c) isoforms indicate that the latter negatively modulates alpha isoform activity, possibly by competing for NAD(+) molecules. Our findings have important ramifications for understanding the mood stabilization process and suggest that inositol biosynthesis is a highly regulated and dynamic process.

A fourth and large family of lycopene cyclases was identified in photosynthetic prokaryotes. The first member of this family, encoded by the cruA gene of the green sulfur bacterium Chlorobium tepidum, was identified in a complementation assay with a lycopene-producing strain of Escherichia coli. Orthologs of cruA are found in all available green sulfur bacterial genomes and in all cyanobacterial genomes that lack genes encoding CrtL- or CrtY-type lycopene cyclases. The cyanobacterium Synechococcus sp. PCC 7002 has two homologs of CruA, denoted CruA and CruP, and both were shown to have lycopene cyclase activity. Although all characterized lycopene cyclases in plants are CrtL-type proteins, genes orthologous to cruP also occur in plant genomes. The CruA- and CruP-type carotenoid cyclases are members of the FixC dehydrogenase superfamily and are distantly related to CrtL- and CrtY-type lycopene cyclases. Identification of these cyclases fills a major gap in the carotenoid biosynthetic pathways of green sulfur bacteria and cyanobacteria.

Pseudomonas sp. strain MT1 has recently been reported to degrade 4- and 5-chlorosalicylate by a pathway assumed to consist of a patchwork of reactions comprising enzymes of the 3-oxoadipate pathway. Genes encoding the initial steps in the degradation of salicylate and substituted derivatives were now localized and sequenced. One of the gene clusters characterized (sal) showed a novel gene arrangement, with salA, encoding a salicylate 1-hydroxylase, being clustered with salCD genes, encoding muconate cycloisomerase and catechol 1,2-dioxygenase, respectively, and was expressed during growth on salicylate and chlorosalicylate. A second gene cluster (cat), exhibiting the typical catRBCA arrangement of genes of the catechol branch of the 3-oxoadipate pathway in Pseudomonas strains, was expressed during growth on salicylate. Despite their high sequence similarities with isoenzymes encoded by the cat gene cluster, the catechol 1,2-dioxygenase and muconate cycloisomerase encoded by the sal cluster showed unusual kinetic properties. Enzymes were adapted for turnover of 4-chlorocatechol and 3-chloromuconate; however, 4-methylcatechol and 3-methylmuconate were identified as the preferred substrates. Investigation of the substrate spectrum identified 4- and 5-methylsalicylate as growth substrates, which were effectively converted by enzymes of the sal cluster into 4-methylmuconolactone, followed by isomerization to 3-methylmuconolactone. The function of the sal gene cluster is therefore to channel both chlorosubstituted and methylsubstituted salicylates into a catechol ortho cleavage pathway, followed by dismantling of the formed substituted muconolactones through specific pathways.

Chalcone isomerase (CHI; EC 5.5.1.6) is a key enzyme in the flavonoid biosynthesis pathway. We isolated a CHI gene (SmCHI) from a cDNA library derived from Saussurea medusa (Asteraceae) cell cultures. The cDNA and genomic sequences of SmCHI are the same; in other words, this gene is intronless. The coding region of the gene is 699 bp long, and its deduced protein consists of 232 amino acids with a predicted molecular mass of 24 kDa and a pI of 4.7. The deduced amino acid sequence of SmCHI shares 79.3% identity with CHI from Callistephus chinensis, a familial relative to S. medusa; this homology is higher than those with CHI's from any other plant species. A functional bioassay for SmCHI was performed by transforming Nicotiana tabacum plants in the sense or antisense orientation under the regulation of the cauliflower mosaic virus (CaMV) 35S promoter. Transgenic tobacco plants overexpressing sense SmCHI produced up to fivefold total flavonoids over wild-type tobacco plants, mainly due to an enhanced accumulation of rutin. Transgenic tobacco plants with antisense SmCHI accumulated smaller amounts of flavonoids; this is apparently brought about by suppressed expression of the endogenous CHI gene. CHI activities also positively correlated with the amounts of total flavonoids accumulated in the transgenic plants. It is concluded that overexpression of SmCHI can be used as a useful approach to increase flavonoid production in transgenic plants.

The Bahamian octocoral Pseudopterogorgia elisabethae is the source of pseudopterosins, diterpene glycosides with potent anti-inflammatory activity. The first committed step in pseudopterosin biosynthesis comprises the cyclisation of the universal diterpene precursor geranylgeranyl diphosphate to elisabethatriene. This reaction is catalysed by elisabethatriene synthase, which was purified to homogeneity from a crude coral extract. This represents the first purification to apparent homogeneity of a terpene cyclase from any marine source. The reaction kinetics of elisabethatriene synthase was examined using a steady state approach with (3)H-labelled isoprenyldiphosphates varying in carbon chain length (C(10), C(15), C(20)). For the reaction of elisabethatriene synthase with its natural substrate geranylgeranyl diphosphate, values of K(m) (2.3 x 10(-6) M), V(max) (3.4 x 10(4) nM elisabethatriene x s(-1)) and the specificity constant (k(cat)/K(m)= 1.8 x 10(-10) M(-1) x s(-1)) were comparable with diterpene cyclases from terrestrial plants. Elisabethatriene synthase also catalysed the conversion of C(15) and C(10) isoprenyldiphosphate analogues to monoterpene and sesquiterpene olefins, respectively. Kinetic parameters indicated that substrate specificity and K(m) of elisabethatriene synthase decreased with decreasing isoprenoid carbon chain length. Furthermore, GC-MS analysis showed increased product diversity with decreasing isoprenoid carbon chain length.

All known pseudouridine synthases have a conserved aspartic acid residue that is essential for catalysis, Asp-48 in Escherichia coli TruB. To probe the role of this residue, inactive D48C TruB was oxidized to generate the sulfinic acid cognate of aspartic acid. The oxidation restored significant but reduced catalytic activity, consistent with the proposed roles of Asp-48 as a nucleophile and general base. The family of pseudouridine synthases including TruB also has a nearly invariant histidine residue, His-43 in the E. coli enzyme. To examine the role of this conserved residue, site-directed mutagenesis was used to generate H43Q, H43N, H43A, H43G, and H43F TruB. Except for phenylalanine, the substitutions seriously impaired the enzyme, but all of the altered TruB retained significant activity. To examine the roles of Asp-48 and His-43 more fully, the pH dependences of wild-type, oxidized D48C, and H43A TruB were determined. The wild-type enzyme displays a typical bell-shaped profile. With oxidized D48C TruB, logk(cat) varies linearly with pH, suggesting the participation of specific rather than general base catalysis. Substitution of His-43 perturbs the pH profile, but it remains bell-shaped. The ascending limb of the pH profile is assigned to Asp-48, and the descending limb is tentatively ascribed to an active site tyrosine residue, the bound substrate uridine, or the bound product pseudouridine.

cDNA cloning of a monoterpene synthase from Perilla frutescens whose steam-distilled oil contains 92.9% perillaketone, was performed by the PCR method using primers designed based on limonene synthase. The full-length nucleotide sequence of this cDNA consisted of 1978 bp including a 1827-bp translational region encoding a deduced protein of 608 amino acids, which was similar to that of limonene synthase from P. frutescens (85% identity). Functional expression of this clone in Escherichia coli yielded an active monoterpene synthase enzyme, which converted geranyl diphosphate into 53.8% myrcene, 20.9% sabinene, 19.8% linalool and 5.5% limonene. As for the extraction of reaction products, we performed SPME (solid phase micro extraction) as well as conventional solvent extraction, and compared these two extraction methods.

Melaleuca alternifolia (Cheel) is an Australia native tree harvested for its monoterpene-rich, essential oil. Monoterpene synthases (E.C. 4.2.3.20) were partially purified from the flush growth of the commercially important, high terpinen-4-ol chemotype of M. alternifolia. The purified fractions produced an acyclic monoterpene, linalool that is not present in the essential oil. To further characterise the monoterpene synthase, a cDNA library was constructed and 500 expressed sequence tags (ESTs) were sequenced to isolate putative terpene synthases. A single clone with similarity to the TspB gene sub-family of angiosperm monoterpene and isoprene synthases was isolated but was truncated at the 5' end. This single clone was used to design a probe for a cDNA library and was applied to isolate a full-length clone. This gene encoded a polypeptide 583 amino acids in length (67 kDa) including a putative transit peptide. Heterologous expression of the gene in Escherichia coli and subsequent assay of the recombinant enzyme did not result in the production of terpinen-4-ol, the major constituent of tea tree oil, or of its precursor sabinene hydrate. Significant quantities of linalool were observed in these assays, and in the assays of monoterpene synthase activity of a native enzyme in vitro, but the racemic nature of the linalool means that it may have a non-enzymatic origin.

The human fecal anaerobe Eubacterium ramulus is capable of degrading various flavonoids, including the flavone naringenin. The first step in the proposed degradation pathway is the isomerization of naringenin to the corresponding chalcone. Cell-free extracts of E. ramulus displayed chalcone isomerase activity. The enzyme from E. ramulus was purified to homogeneity. Its apparent molecular mass was estimated to be 136 and 129 kDa according to gel filtration and native polyacrylamide gel electrophoresis, respectively. Chalcone isomerase is composed of one type of subunit of 30 kDa. The purified enzyme catalyzed the isomerization of naringenin chalcone, isoliquiritigenin, and butein, three chalcones that differ in their hydroxylation pattern. N-bromosuccinimide, but also naringenin and phloretin, inhibited the purified enzyme considerably. This is the first report on a bacterial chalcone isomerase. The physiological function of the purified enzyme is unclear, but an involvement in the conversion of the flavanone naringenin to the chalcone is proposed.

The pseudopterosins are diterpene glycosides isolated from the marine gorgonian, Pseudopterogorgia elisabethae, which exhibit anti-inflammatory and analgesic activity greater than the industry standard, indomethacin. Previously, we isolated the pseudopterosin diterpene cyclase product, elisabethatriene, using a radioactivity-guided isolation. Identification of this metabolite, and the conversion of labeled geranylgeranyl diphosphate to elisabethatriene, provided us with an assay to guide the isolation of the enzyme responsible for this cyclization. The soluble protein preparation from P. elisabethae has been partially purified (approximately 15,000-fold) using a combination of low-resolution anion-exchange, low-resolution hydrophobic interaction, high-resolution hydroxyapatite, and high-resolution anion-exchange chromatography. The diterpene cyclase was identified by comparing the molecular weight from gel permeation chromatography (approximately 47,000Da) with those of protein bands from purified fractions using SDS-PAGE gel electrophoresis. Kinetic analysis and evaluation of amino acid inhibition studies indicated that the enzyme displays similar characteristics to other terpenoid cyclases isolated from terrestrial sources. This report represents the first purification and characterization of a terpene biosynthetic enzyme from a marine invertebrate.

Carotenoids have important functions in photosynthesis, nutrition, and protection against oxidative damage. Some natural carotenoids are asymmetrical molecules that are difficult to produce chemically. Biological production of carotenoids using specific enzymes is a potential alternative to extraction from natural sources. Here we report the isolation of lycopene beta-cyclases that selectively cyclize only one end of lycopene or neurosporene. The crtLm genes encoding the asymmetrically acting lycopene beta-cyclases were isolated from non-photosynthetic bacteria that produced monocyclic carotenoids. Co-expression of these crtLm genes with the crtEIB genes from Pantoea stewartii (responsible for lycopene synthesis) resulted in the production of monocyclic gamma-carotene in Escherichia coli. The asymmetric cyclization activity of CrtLm could be inhibited by the lycopene beta-cyclase inhibitor 2-(4-chlorophenylthio)-triethylamine (CPTA). Phylogenetic analysis suggested that bacterial CrtL-type lycopene beta-cyclases might represent an evolutionary link between the common bacterial CrtY-type of lycopene beta-cyclases and plant lycopene beta- and epsilon-cyclases. These lycopene beta-cyclases may be used for efficient production of high-value asymmetrically cyclized carotenoids.

cis,cis-Muconate cycloisomerase (MC) was purified to homogeneity from benzamide-assimilating Arthrobacter sp. BA-5-17. The purified enzyme showed high activities for cis,cis-muconate and 3-methyl-cis,cis-muconate, and preferred the 3-substituted derivatives over the derivatives with the same substituent at the 2 position as a substrate. A gene encoding MC of strain BA-5-17 was cloned and named catB. The catB gene was clustered with catR encoding a putative LysR-type regulator, catC encoding a putative muconolactone isomerase, and catA-II encoding the catechol 1,2-dioxygenase isozymes CD-III-1 and III-2. These genes showed the same orientation in transcriptional direction and the organization of cloned genes was catRBCA-II. In the phylogenetic analysis of MCs and chloro-MCs, the BA-5-17 and Streptomyces setonii MCs formed a subfamily, clearly distinguished from those of other MCs.

Monoterpene cyclization reactions are initiated by ionization and isomerization of geranyl diphosphate, and proceed, via cyclization of bound linalyl diphosphate, through a series of carbocation intermediates with ultimate termination of the multistep cascade by deprotonation or nucleophile capture. Three structurally and mechanistically related monoterpene cyclases from Salvia officinalis, (+)-sabinene synthase (deprotonation to olefin), 1,8-cineole synthase (water capture), and (+)-bornyl diphosphate synthase (diphosphate capture), were employed to explore the structural determinants of these alternative termination chemistries. Results with chimeric recombinant enzymes, constructed by reciprocally substituting regions of sabinene synthase with the corresponding sequences from bornyl diphosphate synthase or 1,8-cineole synthase, demonstrated that exchange of the C-terminal catalytic domain is sufficient to completely switch the resulting product profile. Exchange of smaller sequence elements identified a region of roughly 70 residues from 1,8-cineole synthase that, when substituted into sabinene synthase, conferred the ability to produce 1,8-cineole. A similar strategy identified a small region of bornyl diphosphate synthase important in conducting the anti-Markovnikov addition to the bornane skeleton. Observations made with these chimeric monoterpene cyclases are discussed in the context of the recently determined crystal structure for bornyl diphosphate synthase.

The present study describes the (19)F nuclear magnetic resonance analysis of the conversion of 3-halocatechols to lactones by purified chlorocatechol 1,2-dioxygenase (ClcA2), chloromuconate cycloisomerase (ClcB2), and chloromuconolactone dehalogenase (ClcF) from Rhodococcus opacus 1cp grown on 2-chlorophenol. The 3-halocatechol substrates were produced from the corresponding 2-halophenols by either phenol hydroxylase from Trichosporon cutaneum or 2-hydroxybiphenyl 3-mono-oxygenase from Pseudomonas azelaica. Several fluoromuconates resulting from intradiol ring cleavage by ClcA2 were identified. ClcB2 converted 2-fluoromuconate to 5-fluoromuconolactone and 2-chloro-4-fluoromuconate to 2-chloro-4-fluoromuconolactone. Especially the cycloisomerization of 2-fluoromuconate is a new observation. ClcF catalyzed the dehalogenation of 5-fluoromuconolactone to cis-dienelactone. The ClcB2 and ClcF-mediated reactions are in line with the recent finding of a second cluster of chlorocatechol catabolic genes in R. opacus 1cp which provides a new route for the microbial dehalogenation of 3-chlorocatechol.

Chlorophyll- b-possessing cyanobacteria of the genus Prochlorococcus share the presence of high amounts of alpha- and beta-carotenoids with green algae and higher plants. The branch point in carotenoid biosynthesis is the cyclization of lycopene, for which in higher plants two distinct enzymes are required, epsilon- and beta-lycopene cyclase. All cyanobacteria studied so far possess a single beta-cyclase. Here, two different Prochlorococcus sp. MED4 genes were functionally identified by heterologous gene complementation in Escherichia coli to encode lycopene cyclases. Whereas one is both functionally and in sequence highly similar to the beta-cyclase of Synechococcus sp. strain PCC 7942 and other cyanobacteria, the other showed several intriguing features. It acts as a bifunctional enzyme catalyzing the formation of epsilon- as well as of beta-ionone end groups. Expression of this cyclase in E. coli resulted in the simultaneous accumulation of alpha- beta-, delta-, and epsilon-carotene. Such an activity is in contrast to all lycopene epsilon-cyclases known so far, including those of the higher plants. Thus, for the first time among prokaryotes, two individual enzymes were identified in one organism that are responsible for the formation of cyclic carotenoids with either beta- or epsilon-end groups. These two genes are suggested to be designated as crtL-b and crtL-e. The results indicate that both enzymes might have originated from duplication of a single gene. Consequently, we suggest that multiple gene duplications followed by functional diversification resulted several times, and in independent lineages, in the appearance of enzymes for the biosynthesis of cyclic carotenoids.

In flavonoid, isoflavonoid, and anthocyanin biosynthesis, chalcone isomerase (CHI) catalyzes the intramolecular cyclization of chalcones into (S)-flavanones with a second-order rate constant that approaches the diffusion-controlled limit. The three-dimensional structures of alfalfa CHI complexed with different flavanones indicate that two sets of hydrogen bonds may possess critical roles in catalysis. The first set of interactions includes two conserved amino acids (Thr48 and Tyr106) that mediate a hydrogen bond network with two active site water molecules. The second set of hydrogen bonds occurs between the flavanone 7-hydroxyl group and two active site residues (Asn113 and Thr190). Comparison of the steady-state kinetic parameters of wild-type and mutant CHIs demonstrates that efficient cyclization of various chalcones into their respective flavanones requires both sets of contacts. For example, the T48A, T48S, Y106F, N113A, and T190A mutants exhibit 1550-, 3-, 30-, 7-, and 6-fold reductions in k(cat) and 2-3-fold changes in K(m) with 4,2',4'-trihydroxychalcone as a substrate. Kinetic comparisons of the pH-dependence of the reactions catalyzed by wild-type and mutant enzymes indicate that the active site hydrogen bonds contributed by these four residues do not significantly alter the pK(a) of the intramolecular cyclization reaction. Determinations of solvent kinetic isotope and solvent viscosity effects for wild-type and mutant enzymes reveal a change from a diffusion-controlled reaction to one limited by chemistry in the T48A and Y106F mutants. The X-ray crystal structures of the T48A and Y106F mutants support the assertion that the observed kinetic effects result from the loss of key hydrogen bonds at the CHI active site. Our results are consistent with a reaction mechanism for CHI in which Thr48 polarizes the ketone of the substrate and Tyr106 stabilizes a key catalytic water molecule. Hydrogen bonds contributed by Asn113 and Thr190 provide additional stabilization in the transition state. Conservation of these residues in CHIs from other plant species implies a common reaction mechanism for enzyme-catalyzed flavanone formation in all plants.

The acyclic monoterpene myrcene is the likely progenitor of the unusual cytotoxic halogenated monoterpenes that are found in marine algae and that function as feeding deterrents to herbivores. Myrcene synthase was isolated from suspension cultures of the marine red alga Ochtodes secundiramea, representing the first enzyme of this type from a marine organism. The algal myrcene synthase produces exclusively myrcene from the natural substrate geranyl diphosphate (GDP), utilizes Mg(+2) as the required divalent metal ion cofactor, has a molecular mass of about 69 kDa, and exhibits a pH optimum near 7.2. These features are similar to those of monoterpene synthases from terrestrial organisms. When incubated with neryl diphosphate (the cis-isomer of GDP), the O. secundiramea myrcene synthase produces the cyclic monoterpene limonene, whereas incubation with (+/-)linalyl diphosphate (the tertiary allylic isomer of geranyl diphosphate) yields both acyclic and cyclic monoterpenes. These results suggest that the enzyme is incapable of isomerizing geranyl diphosphate to linalyl diphosphate, a feature common to all monoterpene cyclases from terrestrial sources. The limited catalytic capability of the myrcene synthase may reflect the ancient evolutionary origin of the producing organism. The ability to assay this enzyme in cultured algae, grown under strictly defined conditions, provides an unparalleled opportunity to delineate factors eliciting the biosynthesis of this class of secondary metabolites, to investigate the metabolic pathway leading to the halogenated monoterpenes, and to determine their role in the chemical ecology of marine algae.

An 859-bp cDNA segment of a terpene synthase gene was amplified by PCR from the evergreen sclerophyllous holm oak (Quercus ilex L.) using heterologous primers for conserved regions of terpene synthase genes (TPS) in dicotyledonous plants. Based on the sequence of this segment, homologous primers were designed for amplification by RACE-PCR of a cDNA segment carrying the monoterpene synthase gene myrS. The gene encodes a protein of 597 amino acids including an N-terminal putative plastid transit peptide. The gene without the segment encoding the transit peptide was cloned by PCR into a bacterial expression vector. Expression in Escherichia coli yielded an active monoterpene synthase, which converted geranyl diphosphate (GDP) predominantly into the acyclic monoterpene myrcene and to a very small extent into cyclic monoterpenes. Sequence comparison with previously cloned monoterpene synthases revealed that the myrcene synthase from Q. ilex belongs to the TPSb subfamily.

Muconate cycloisomerase (MCI) was purified from Rhodococcus rhodochrous 89 grown on phenol. The enzyme appears to contain two different type subunits with molecular masses 35.5 and 37 kD. The N-terminal amino acid sequence of both subunits showed more similarity to corresponding enzymes from gram-negative bacteria than to one from Rhodococcus opacus 1CP. MCI from R. rhodochrous 89, like analogous enzymes from gram-negative bacteria, can convert 2-chloromuconate (2-CM) with the formation of both, 2- and 5-chloromuconolactones (CML) as intermediates. Nevertheless, its unique ability to convert 5-CML to cis- but not to trans-dienelactone sets it apart from all known chloromuconate cycloisomerases from gram-negative and gram-positive bacteria.

Chlorocatechol 1,2-dioxygenase (CC 1,2-DO), chloromuconate cycloisomerase (CMCI), chloromuconolactone isomerase (CMLI), and dienolactone hydrolase (DELH), the key enzymes of a new modified ortho-pathway in Rhodococcus opacus 1CP cells utilizing 2-chlorophenol via a 3-chlorocatechol branch of a modified ortho-pathway, were isolated and characterized. CC 1,2-DO showed the maximum activity with 3-chlorocatechol; its activity with catechol and 4-chlorocatechol was 93 and 50%, respectively. The enzyme of the studied pathway had physicochemical properties intermediate between the pyrocatechase of ordinary and chlorocatechase of modified pathways described earlier for this strain. In contrast to the enzymes investigated earlier, CMCI of the new pathway exhibited high substrate specificity. The enzyme had Km for 2-chloromuconate of 142.86 microM, Vmax = 71.43 U/mg, pH optimum around 6.0, and temperature optimum at 65 degrees C. CMCI converted 2-chloromuconate into 5-chloromuconolactone. CMLI converted 5-chloromuconolactone into cis-dienolactone used as a substrate by DELH; this enzyme did not convert trans-dienolactone. DELH had Km for cis-dienolactone of 200 microM, Vmax = 167 U/mg, pH optimum of 8.6, and temperature optimum of 40 degrees C. These results confirm the existence of a new modified ortho-pathway for utilization of 2-chlorophenol by R. opacus 1CP.

The nature of isoprenoids synthesized in plants is primarily determined by the specificity of prenyltransferases. Several of these enzymes have been characterized at the molecular level. The compartmentation and molecular regulation of geranyl diphosphate (GPP), the carbon skeleton that is the backbone of myriad monoterpene constituents involved in plant defence, allelopathic interactions and pollination, is poorly understood. We describe here the cloning and functional expression of a GPP synthase (GPPS) from Arabidopsis thaliana. Immunohistological analyses of diverse non-secretory and secretory plant tissues reveal that GPPS and its congeners, monoterpene synthase, deoxy-xylulose phosphate synthase and geranylgeranyl diphosphate synthase, are equally compartmentalized and distributed in non-green plastids as well in chloroplasts of photosynthetic cells. This argues that monoterpene synthesis is not solely restricted to specialized secretory structures but can also occur in photosynthetic parenchyma. These data provide new information as to how monoterpene biosynthesis is compartmentalized and induced de novo in response to biotic and abiotic stress in diverse plants.

In addition to direct ecological functions in the interaction of plants with the environment, the emission of monoterpenes, especially from the foliage of evergreen trees, is of great importance for the production of ozone and photochemical oxidants in the troposphere. In the present work, we established a reproducible non-radioactive standard enzyme assay and characterized monoterpene synthase activities in needles of Norway spruce (Picea abies (L.) Karst.) and in leaves of holm oak (Quercus ilex L.). In Norway spruce, the dominant monoterpenes formed were alpha-pinene, camphene, and to a lesser extent beta-pinene and limonene. In holm oak, alpha-pinene, sabinene, and beta-pinene were the main products, while limonene was a minor component. Under optimum conditions, in both Norway spruce and holm oak, monoterpene formation remained constant up to 180 min and 90 min, respectively, and varied with the buffer and Mg2+ and Mn2+ concentrations used. Optimum temperature for monoterpene synthase activity was 40 degrees C in both species; optimal pH ranged between 6.5 and 7.5 in both species. Apparent Michaelis-constants for the substrate GDP were ca. 17.9 +/- 5.1 microM for Norway spruce and ca. 69.4 +/- 22.1 microM for holm oak. Molecular weight determination by FPLC indicated that the monoterpene synthases in Norway spruce and holm oak have native molecular weights of ca. 59 and 50 kDa, respectively.

(-)-Sabinene is the major monoterpene produced by a European strain of the liverwort Conocephalum conicum. A cell-free extract from in vitro cultured plants catalysed the cyclization of geranyl diphosphate to sabinene. The responsible monoterpene cyclase was partially purified and characterized as an operationally soluble enzyme of M(r) 65,000, with a pH optimum at 7.5 and a requirement for a divalent metal ion as the only cofactor, with Mg2+ preferred. The general properties of the sabinene synthase from C. conicum resemble those of other monoterpene cyclases from gymnosperms and angiosperms. A North American strain of the liverwort produces (+)-bornyl acetate as the major monoterpene and it was demonstrated that bornane-type monoterpenes are derived from geranyl diphosphate in this liverwort, as in higher plants, by the action of bornyl diphosphate synthase.

Muconate cycloisomerases play a crucial role in the bacterial degradation of aromatic compounds by converting cis,cis-muconate, the product of catechol ring cleavage, to (4S)-muconolactone. Chloromuconate cycloisomerases catalyze both the corresponding reaction and a dehalogenation reaction in the transformation of chloroaromatic compounds. This study reports the first thorough examination of the substrate specificity of the muconate cycloisomerases from Pseudomonas putida PRS2000 and Acinetobacter "calcoaceticus" ADP1. We show that they transform, in addition to cis,cis-muconate, 3-fluoro-, 2-methyl-, and 3-methyl-cis, cis-muconate with high specificity constants but not 2-fluoro-, 2-chloro-, 3-chloro-, or 2,4-dichloro-cis,cis-muconate. Based on known three-dimensional structures, variants of P. putida muconate cycloisomerase were constructed by site-directed mutagenesis to contain amino acids found in equivalent positions in chloromuconate cycloisomerases. Some of the variants had significantly increased specificity constants for 3-chloro- or 2,4-dichloromuconate (e.g., A271S and I54V showed 27- and 22-fold increases, respectively, for the former substrate). These kinetic improvements were not accompanied by a change from protoanemonin to cis,cis-dienelactone as the product of 3-chloro-cis,cis-muconate conversion. The rate of 2-chloro-cis,cis-muconate turnover was not significantly improved, nor was this compound dehalogenated to any significant extent. However, the direction of 2-chloro-cis,cis-muconate cycloisomerization could be influenced by amino acid exchange. While the wild-type enzyme discriminated only slightly between the two possible cycloisomerization directions, some of the enzyme variants showed a strong preference for either (+)-2-chloro- or (+)-5-chloromuconolactone formation. These results show that the different catalytic characteristics of muconate and chloromuconate cycloisomerases are due to a number of features that can be changed independently of each other.

Two muconate cycloisomerases (MC I and MC II, EC 5.5.1.1) were purified to homogeneity from an aniline-grown Frateuria sp. ANA-18. MC I and MC II were similar in molecular mass, optimal pH, and pH stability but different in thermostability, and some other enzymatic properties. NH2-terminal amino acid sequences were different between the two isozymes, indicated that these are encoded by different genes. Different inducible production of MC I and MC II suggested that two catechol branches involved in the beta-ketoadipate pathway function in Frateuria sp. ANA-18.

tRNA pseudouridine 55 (psi 55) synthase, the enzyme that is specific for the conversion of U55 to psi 55 in the m5U psi CG loop in most tRNAs, has been purified from Escherichia coli and cloned. On SDS gels, a single polypeptide chain with a mass of 39.7 kDa was found. The gene is a previously described open reading frame, p35, located at 68.86 min on the E. coli chromosome between the infB and rpsO genes. The proposed name for this gene is truB. There is very little protein sequence homology between the truB gene product and the hisT (truA) product, which forms psi in the anticodon arm of tRNAs. However, there was high homology with a fragment of a Bacillus subtilis gene that may produce the analogous enzyme in that species. The cloned gene was fused to a 5'-leader coding for a (His)6 tract, and the protein was overexpressed > 400-fold in E. coli. The recombinant protein was purified to homogeneity in one step from a crude cell extract by affinity chromatography using a Ni(2+)-containing matrix. The SDS mass of the recombinant protein was 41.5 kDa, whereas that calculated from the gene was 37.3. The recombinant protein was specific for U55 in tRNA transcripts and reacted neither at other sites for psi in such transcripts nor with transcripts of 16S or 23S ribosomal RNA or subfragments. The enzyme did not require either a renatured RNA structure or Mg2+, and prior formation of m5U was not required. Stoichiometric formation of psi occurred with no requirement for an external source of energy, indicating that psi synthesis is thermodynamically favored.