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Arogenate dehydratase (ADT) is the key limiting enzyme of plant phenylalanine biosynthesis, but some ADTs display a prephenate decarboxylase/dehydratase activity-conferring (PAC) domain. The genome resources of 70 species were employed to identify genes and outline their characteristics, especially the number and type of PAC domain structures. We obtained 522 ADTs, and their size, exon number, amino acid number and putative protein isoelectric point greatly varied from 306 to 2520 bp, 1 to 15, 101 to 839 and 4.37 to 11.18, respectively. We classified the ADTs into Class α (without a PAC domain) (115, 22.0 %), β (with a type I PAC domain) (244, 46.7 %) and γ (with a type II PAC domain) (163, 31.2 %), and their distribution frequencies exhibited large differences among various branches of angiosperms. We found that Class γ members are more conserved than Class β members, although they commonly experienced multiple duplication events and strong purifying selection, which resulted in a small number, and the putative origin order was from Class α to β and then to γ. In addition, the co-occurrence of both Class β and γ members could ensure the survival of angiosperms, while their optimized composition and strategically intertwined regulation may facilitate core eudicot success., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The native Putranjiva roxburghii family 1 glycoside hydrolase enzyme showed β-D-fucosidase activity in addition to β-D-glucosidase and β-D-galactosidase activities reported in our previous study. A single step concanvalin A affinity chromatography for native PRGH1 improved the yield and reduced the purification time. The PRGH1 gene was cloned and overexpressed in E. coli. The full length gene contained an ORF of 1617 bp encoding a polypeptide of 538 amino acids. The amino acid sequence of PRGH1 showed maximum similarities to β-glucosidases and myrosinases. Both native and recombinant protein showed maximum hydrolytic activity for pNP-Fuc followed by pNP-Glc and pNP-Gal. Significant enzyme activity was also observed for cellobiose, however it decreased with increase in chain-length for glycan substrates. The enzyme showed significant resistant to D-glucose concentration up to 500 mM. Mutational studies confirmed the predicted catalytic acid/base Glu173 and nucleophile Glu389 as key residues for its activity. Moreover, Glu446 and Asn172 played essential role in substrate binding by interacting with the -1 subsite of substrates. Bioinformatic analysis suggested the possible reasons for the broad substrate specificity and other properties of the enzyme. PRGH1 had high sequence similarity towards S-glucosidase and may be involved in defence. The broad specificity, catalytic efficiency and thermostability make PRGH1 potentially an important industrial enzyme., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

The early evolution of angiosperms was marked by a number of innovations of the reproductive cycle including an accelerated fertilization process involving faster transport of sperm to the egg via a pollen tube. Fast pollen tube growth rates in angiosperms are accompanied by a hard shank-soft tip pollen tube morphology. A critical actor in that morphology is the wall-embedded enzyme pectin methylesterase (PME), which in type II PMEs is accompanied by a co-transcribed inhibitor, PMEI. PMEs convert the esterified pectic tip wall to a stiffer state in the subapical flank by pectin de-esterification. It is hypothesized that rapid and precise targeting of PME activity was gained with the origin of type II genes, which are derived and have only expanded since the origin of vascular plants. Pollen-active PMEs have yet to be reported in early-divergent angiosperms or gymnosperms. Gene expression studies in Nymphaea odorata found transcripts from four type II VGD1-like and 16 type I AtPPME1-like homologs that were more abundant in pollen and pollen tubes than in vegetative tissues. The near full-length coding sequence of one type II PME (NoPMEII-1) included at least one PMEI domain. The identification of possible VGD1 homologs in an early-diverging angiosperm suggests that the refined control of PMEs that mediate de-esterification of pectins near pollen tube tips is a conserved feature across angiosperms. The recruitment of type II PMEs into a pollen tube elongation role in angiosperms may represent a key evolutionary step in the development of faster growing pollen tubes., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Carotenoids are essential isoprenoid pigments produced by plants, algae, fungi and bacteria. Lycopene cyclase (LYC) commonly cyclize carotenoids, which is an important branching step in the carotenogenesis, at one or both end of the backbone. Plants have two types of LYC (β-LCY and ϵ-LCY). In this study, plant LYCs were analyzed. Based on domain analysis, all LYCs accommodate lycopene cyclase domain (Pf05834). Furthermore, motif analysis indicated that motifs were conserved among the plants. On the basis of phylogenetic analysis, β-LCYs and ϵ-LCYs were classified in β and ϵ groups. Monocot and dicot plants separated from each other in the phylogenetic tree. Subsequently, Oryza sativa Japonica Group and Zea mays of LYCs as monocot plants and Vitis vinifera and Solanum lycopersicum of LYCs as dicot plants were analyzed. According to nucleotide diversity analysis of β-LCY and ϵ-LCY genes, nucleotide diversities were found to be π: 0.30 and π: 0.25, respectively. The result highlighted β-LCY genes showed higher nucleotide diversity than ϵ-LCY genes. LYCs interacting genes and their co-expression partners were also predicted using String server. The obtained data suggested the importance of LYCs in carotenoid metabolism. 3D modeling revealed that depicted structures were similar in O. sativa, Z mays, S. lycopersicum, and V. vinifera β-LCYs and ϵ-LCYs. Likewise, the predicted binding sites were highly similar between O. sativa, Z mays, S. lycopersicum, and V. vinifera LCYs. Most importantly, analysis elucidated the V/IXGXGXXGXXXA motif for both type of LYC (β-LCY and ϵ-LCY). This motif related to Rossmann fold domain and probably provides a flat platform for binding of FAD in O. sativa, Z mays, S. lycopersicum, and V. vinifera β-LCYs and ϵ-LCYs with conserved structure. In addition to lycopene cyclase domain, the V/IXGXGXXGXXXA motif can be used for exploring LYCs proteins and to annotate the function of unknown proteins containing lycopene cyclase domain. Overall results indicated that a high degree of conserved signature were observed in plant LYCs., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

A class III chitinase belonging to the GH18 family from Nepenthes alata (NaCHIT3) was expressed in Escherichia coli. The enzyme exhibited hydrolytic activity toward colloidal chitin, ethylene glycol chitin, and (GlcNAc)(n) (n=5 and 6). The enzyme hydrolyzed the fourth glycosidic linkage from the non-reducing end of (GlcNAc)(6). The anomeric form of the products indicated it was a retaining enzyme. The colloidal chitin hydrolytic reaction displayed high activity between pH 3.9 and 6.9, but the pH optimum of the (GlcNAc)(6) hydrolytic reaction was 3.9 at 37 °C. The optimal temperature for activity was 65 °C in 50 mM sodium acetate buffer (pH 3.9). The pH optima of NaCHIT3 and NaCHIT1 might be related to their roles in chitin degradation in the pitcher fluid., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Most cardenolides used in the therapy of cardiac insufficiency are 5 beta-configured and thus the stereo-specific reduction of the Delta(4,5)-double bond of a steroid precursor is a crucial step in their biosynthesis. This step is thought to be catalysed by progesterone 5 beta-reductases. We report here on the isolation of 11 progesterone 5 beta-reductase (P5 beta R) orthologues from 5 beta-cardenolide-free and 5 beta-cardenolide-producing plant species belonging to five different angiosperm orders (Brassicales, Gentianales, Lamiales, Malvales and Solanales). Amino acid sequences of the P5 beta R described here were highly conserved. They all contain certain motifs qualifying them as members of a class of stereo-selective enone reductases capable of reducing activated C=C double bonds by a 1,4-addition mechanism. Protein modeling revealed seven conserved amino acids in the substrate-binding/catalytic site of these enzymes which are all supposed to exhibit low substrate specificity. Eight P5 beta R genes isolated were expressed in Escherichia coli. Recombinant enzymes reduced progesterone stereo-specifically to 5 beta-pregane-3,20-dione. The progesterone 5 beta-reductases from Digitalis canariensis and Arabidopsis thaliana reduced activated C=C double bonds of molecules much smaller than progesterone. The specific role of progesterone 5 beta-reductases of P5 beta Rs in cardenolide metabolism is challenged because this class of enone reductases is widespread in higher plants, and they accept a wide range of enone substrates., ((c) 2010 Elsevier Ltd. All rights reserved.)

To isolate cDNAs involved in the biosynthesis of acetate-derived naphthoquinones in Drosophyllum lusitanicum, an expressed sequence tag analysis was performed. RNA from callus cultures was used to create a cDNA library from which 2004 expressed sequence tags were generated. One cDNA with similarity to known type III polyketide synthases was isolated as full-length sequence and termed DluHKS. The translated polypeptide sequence of DluHKS showed 51-67% identity with other plant type III PKSs. Recombinant DluHKS expressed in Escherichia coli accepted acetyl-coenzyme A (CoA) as starter and carried out sequential decarboxylative condensations with malonyl-CoA yielding alpha-pyrones from three to six acetate units. However, naphthalenes, the expected products, were not isolated. Since the main compound produced by DluHKS is a hexaketide alpha-pyrone, and the naphthoquinones in D. lusitanicum are composed of six acetate units, we propose that the enzyme provides the backbone of these secondary metabolites. An involvement of accessory proteins in this biosynthetic pathway is discussed.

Natural and anthropogenic activities lead to deposition of leaves into water bodies, releasing allelochemicals that might influence aquatic biota. Time-response experiments were performed with Ceratophyllum demersum using aqueous Quercus robur and Phragmites australis leaf extracts. The aim was to investigate the ability of C. demersum to acclimate to stress imposed by leaf extracts using antioxidative enzymes (GR, POD, GST, GPx) and photosynthesis as endpoints. Highest enzyme activity and photosynthetic reduction was observed after 24 h in both extracts. After 48 h, photosynthesis and enzyme levels were significantly different from the ones noted at 24 h. Enzyme activities, except GR, and oxygen production did not return to control levels after 1 week in Q. robur extracts. In contrast, enzyme and photosynthetic oxygen levels in P. australis extract recovered after 168 h. Results indicate that increase in antioxidative enzyme levels is part of the protection strategy of C. demersum against oxidative stress.

Programmed cell death during senescence in plants is associated with proteolysis that helps in remobilization of nitrogen to other growing tissues. In this paper, we provide one of the few reports for the expression of specific serine proteases during senescence associated proteolysis in Gladiolus grandiflorus flowers. Senescence in tepals, stamens and carpels results in an increase in total protease activity and a decrease in total protein content. Of the total protease activity, serine proteases account for about 67-70% while cysteine proteases account for only 23-25%. In-gel assays using gelatin as a substrate and specific protease inhibitors reveal the enhanced activity of two trypsin-type serine proteases of sizes 75 kDa and 125 kDa during the course of senescence. The activity of the 125 kDa protease increases not only during tepal senescence but also during stamen and carpel senescence indicating that it is responsive to general senescence signals.

A recombinant alpha-farnesene synthase from apple (Malus x domestica), expressed in Escherichia coli, showed features not previously reported. Activity was enhanced 5-fold by K(+) and all four isomers of alpha-farnesene, as well as beta-farnesene, were produced from an isomeric mixture of farnesyl diphosphate (FDP). Monoterpenes, linalool, (Z)- and (E)-beta-ocimene and beta-myrcene, were synthesised from geranyl diphosphate (GDP), but at 18% of the optimised rate for alpha-farnesene synthesis from FDP. Addition of K(+) reduced monoterpene synthase activity. The enzyme also produced alpha-farnesene by a reaction involving coupling of GDP and isoprenyl diphosphate but at <1% of the rate with FDP. Mutagenesis of active site aspartate residues removed sesquiterpene, monoterpene and prenyltransferase activities suggesting catalysis through the same active site. Phylogenetic analysis clusters this enzyme with isoprene synthases rather than with other sesquiterpene synthases, suggesting that it has evolved differently from other plant sesquiterpene synthases. This is the first demonstration of a sesquiterpene synthase possessing prenyltransferase activity.

A cDNA clone encoding a sesquiterpene synthase, (+)-germacrene D synthase, has been isolated from ginger (Zingiber officinale). The full-length cDNA (AY860846) contains a 1650-bp open reading frame coding for 550 amino acids (63.8kDa) with a theoretical pI=5.59. The deduced amino acid sequence is 30-46% identical with sequences of other sesquiterpene synthases from angiosperms. The recombinant enzyme, produced in Escherichia coli, catalyzed the formation of a major product, (+)-germacrene D (50.2% of total sesquiterpenoids produced) and a co-product, germacrene B (17.1%) and a number of minor by-products. The optimal pH for the recombinant enzyme is around 7.5. Substantial (+)-germacrene D synthase activity is observed in the presence of Mg2+, Mn2+, Ni2+ or Co2+, while the enzyme is inactive when Cu2+ or Zn2+ is used. The Km- and kcat-values are 0.88 microM and 3.34 x 10(-3) s(-1), respectively. A reaction mechanism involving a double 1,2-hydride shift has been established using deuterium labeled substrates in combination with GC-MS analysis.

The ability of eight species of plants to assimilate 2,4,6-trinitrotoluene (TNT) was investigated. Glycine max (soybean), in particular, demonstrated rapid assimilation of high concentrations of this explosive. Penetration and localization of [1-(14)C]-TNT in plant root cells and leaves were studied via electron microscopic autoradiography. TNT was shown to be localized primarily on membrane structures involved in the transportation of nicotinamide coenzymes (membranes of endoplasmic reticulum, mitochondria, plastids). [1-(14)C]-TNT in roots was incorporated mainly in low-molecular-weight metabolites; however, in stems and leaves, the radiocarbon was incorporated in biopolymers. Enzymatic transformation of TNT in roots was studied, and it was found that degradation involved mainly nitroreductase acting on the TNT nitro groups. The process was intensified in the presence of electron donors--NADH and NADPH. Nitroreductase activity was revealed in root cell cytosol and expression was strongly induced by plant cultivation on TNT-containing media. Oxidation of [C(3)H(3)]TNT by peroxidase and phenoloxidase was also studied. In contrast to the strongly induced nitroreductase, levels of these enzymes changed very little with TNT addition. This suggests that the main pathway of TNT transformation in plant cells is nitro group reduction. A plant's nitroreductase activity and its ability to incorporate TNT from aqueous solutions were correlated in four plants that were studied. The results suggest that plant nitroreductase activity may serve as a good biochemical indicator of plants that can be used for phytoremediation of soils contaminated with TNT.

beta-Galactosidase (EC. 3.2.1.23) from ripe carambola (Averrhoa carambola L. cv. B10) fruit was fractionated through a combination of ion exchange and gel filtration chromatography into four isoforms, viz. beta-galactosidase I, II, III and IV. This beta-galactosidases had apparent native molecular masses of 84, 77, 58 and 130 kDa, respectively. beta-Galactosidase I, the predominant isoform, was purified to electrophoretic homogeneity; analysis of the protein by SDS-PAGE revealed two subunits with molecular masses of 48 and 36 kDa. N-terminal amino acid sequence of the respective polypeptides shared high similarities albeit at different domains, with the deduced amino acid sequence of certain plant beta-galactosidases, thus, explaining the observed low similarity between the two subunits. beta-Galactosidase I was probably a heterodimer that have glycoprotein properties and a pI value of 7.2, with one of the potential glycosylation sites appeared to reside within the 48-kDa-polypeptide. The purified beta-galactosidase I was substantially active in hydrolyzing (1-->4)beta-linked spruce and a mixture of (1-->3)beta- and (1-->6)beta-linked gum arabic galactans. This isoform also had the capability to solubilize and depolymerize structurally intact pectins as well as to modify alkaline-soluble hemicelluloses, reflecting in part changes that occur during ripening.

Pectin methylesterases (PMEs) are enzymes produced by bacteria, fungi and higher plants. They belong to the carbohydrate esterase family CE-8. This study deals with comparison of 127 amino acid sequences of this family containing the five characteristic sequence segments: 44&#95;GxYxE, 113&#95;QAVAL, 135&#95;QDTL, 157&#95;DFIFG, 223&#95;LGRPW (Daucus carota numbering). Six strictly conserved residues (Gly44, Gly154, Asp157, Gly161, Arg225 and Trp227) and six conservative ones (Ile39, Ser86, Ser137, Ile152, Ile159 and Leu223) were identified. A set of 70 representative PMEs was created. The sequences were aligned and the evolutionary tree based on the alignment was calculated. The tree reflected the taxonomy: the fungal and bacterial PMEs formed their own clusters and the plant enzymes were grouped into eight separate clades. The plant PME from Vitis riparia was placed in a common clade with fungi. Three plant clades (Plant 1, 2 and 3) were relatively homogenous reflecting high degree of mutual sequence identity. The clade Plant 4 contained PMEs from flower parts (mostly form pollen) and was heterogenous, like the clades Plant 1a and 2a, which moreover exhibit an intermediate character. The clades Plant X1 and X2 were situated in the tree close to microbial clades and represented atypical plant PMEs. Taking into account the remaining plant PMEs, an expanded plant alignment and tree (with most Arabidopsis thaliana and Oryza sativa enzymes), were prepared. An exclusive Arabidopsis alignment and tree indicated the existence of a new plant clade X3. In the pre pro region of most plant enzymes a longer conserved segment containing basic dipeptide, R(K)/R(K), that precedes the N-terminal end of PME was revealed. This was not observed in the clade Plant X1 and majority of the clade Plant X2. This study brings further the description of occurrence of potential glycosylation sites in pre pro sequences and in mature enzymes as well as important amino acid residues, such as aspartates, cysteines, histidines and other aromatic residues (Tyr, Phe and Trp), with discussion of their possible function in the activity of PMEs.

Linoleic acid (18:2) is found in a large variety of plant oils but to date there is limited knowledge about the substrate selectivity of acyltransferases required for its incorporation into storage triacylglycerols. We have compared the incorporation of oleoyl (18:1) and linoleoyl (18:2) acyl-CoAs onto lysophosphatidic acid acceptors by sub-cellular fractions prepared from a variety of plant and microbial species. Our assays demonstrated: (1). All lysophosphatidic acid acyltransferase (LPA-AT) enzymes tested incorporated 18:2 acyl groups when presented with an equimolar mix of 18:1 and 18:2 acyl-CoA substrates. The ratio of 18:1 to 18:2 incorporation into phosphatidic acid varied between 0.4 and 1.4, indicating low selectivity between these substrates. (2). The presence of either stearoyl (18:0) or oleoyl (18:1) groups at the sn-1 position of lysophosphatidic acid did not affect the selectivity of incorporation of 18:1 or 18:2 into the sn-2 position of phosphatidic acid. (3). All LPA-AT enzymes tested incorporated the saturated palmitoyl (16:0) acyl group from equimolar mixtures of 16:0- and 18:1-CoA. The ratios of 18:1 to 16:0 incorporation are generally much higher than those of 18:1 to 18:2 incorporation, varying between 2.1 and 8.6. (4). The LPA-AT from oil palm kernel is an exception as 18:1 and 16:0 are utilised at comparable rates. These results show that, in the majority of species examined, there is no correlation between the final sn-2 composition of oil or membrane lipids and the ability of an LPA-AT to use 18:2 as a substrate in in vitro assays.

This study investigated the effects of Na(2)SO(3), which releases SO(2) in apoplastic water, on the growth of geranium seedlings and on the activities of various antioxidant enzymes including peroxidase. Sodium sulfite (Na(2)SO(3)) addition both inhibited primary root growth and stimulated lateral root growth of the seedlings respectively. In addition, the contents of chlorophyll and Rubisco protein of the seedlings were greatly reduced with Na(2)SO(3) treatment. Total peroxidase activities of the seedlings also increased proportionally with the amount of Na(2)SO(3), this presumably correlating with oxidative stress levels. Notably, about an 8-fold enhancement of total peroxidase activity occurred in seedlings treated with 60 nM Na(2)SO(3) at pH 4.0. This enhancement of total peroxidase activity was mainly due to the increase of a strong cationic isoperoxidase, strong anionic isoperoxidase and neutral isoperoxidase activities. The strong cationic isoperoxidase from geranium seedlings was found to be the same enzyme as PC3 from geranium callus in terms of its physicochemical and catalytic properties. Moreover, the activities of superoxide dismutase and glutathione reductase were greatly enhanced with Na(2)SO(3) treatments at pH 4.0 without significant alteration of catalase activity. These results suggest that Na(2)SO(3) exposure, activities the plants defense mechanism against the reactive oxygen species generated.

This study assesses whether the phylogenetic relationships between SODs from different organisms could assist in elucidating the functional relationships among these enzymes from evolutionarily distinct species. Phylogenetic trees and intron positions were compared to determine the relationships among these enzymes. Alignment of Cu/ZnSOD amino acid sequences indicates high homology among plant sequences, with some features that distinguish chloroplastic from cytosolic Cu/ZnSODs. Among eukaryotes, the plant SODs group together. Alignment of the Mn and FeSOD amino acid sequences indicates a higher degree of homology within the group of MnSODs (>70%) than within FeSODs (approximately 60%). Tree topologies are similar and reflect the taxonomic classification of the corresponding species. Intron number and position in the Cu/Zn Sod genes are highly conserved in plants. Genes encoding cytosolic SODs have seven introns and genes encoding chloroplastic SODs have eight introns, except the chloroplastic maize Sod1, which has seven. In Mn Sod genes the number and position of introns are highly conserved among plant species, but not among nonplant species. The link between the phylogenetic relationships and SOD functions remains unclear. Our findings suggest that the 5' region of these genes played a pivotal role in the evolution of function of these enzymes. Nevertheless, the system of SODs is highly structured and it is critical to understand the physiological differences between the SODs in response to different stresses in order to compare their functions and evolutionary history.

A strongly cationic isoperoxidase named PC3 was purified to homogeneity from scented-geranium (Pelargonium graveolens) callus by using DEAE-Sephacel chromatography, CM-cellulose chromatography and Sephacryl S-200 gel filtration, respectively. The enzyme was a glycoprotein with M(r) of ca. 58 kDa estimated by SDS-PAGE and Sephadex G-150 gel filtration. The pI value of the enzyme was 9.1. Kinetic studies revealed that PC3 had a very low K(m) value for scopoletin of 0.01 mM and could use ascorbate as a substrate. Interestingly, PC3 could not oxidize ferulic acid as a substrate. Chemical modification of the enzyme showed that PC3 was rapidly inactivated by His, Cys, Trp and Lys-specific reagents. The second order rate constants and reaction orders with respect to these inactivations were determined. Notably, ca. 4-7-fold activity boosting of PC3 occurred by adenine and imidazole when anilino substrates, such as o-dianisidine and o-phenylenediamine were oxidized, whereas this activity boosting did not occur when several phenolic substrates were used.

Three groups of carambola fruits (Averrhoa carambola L.) were stored at 2 and 10 degrees C (85-90% relative humidity). The major physicochemical, physiological, and enzymatic responses of fruit were measured in each group over a 30-day period: chilling injury index (CII), decay (%), intracuticular waxes, cuticle permeability, pulp firmness, weight loss, sucrose, fructose and glucose contents, ion electrolyte leakage in pulp (%), ethylene and carbon dioxide production rates, and the activities of peroxidase (POD), polyphenol oxidase (PPO), and phenylalanine ammonia-lyase (PAL) enzymes. CII values were statistically different at 2 and 10 degrees C, showing high significance with respect to sucrose content and weight loss (P < 0.05). Chilling injury included darkened ribs and skin desiccation. According to the CI symptom development, a possible relationship of POD and PPO activities was found at 2 degrees C. A significant sucrose content increase was observed at 10 degrees C. CI symptoms were associated with POD and PAL activities., (Copyright 2001 Academic Press.)

In Crassulacean acid metabolism (CAM) plants, phosphoenolpyruvate carboxylase (PEPC) is subject to day-night regulatory phosphorylation of a conserved serine residue in the plant enzyme's N-terminal domain. The dark increase in PEPC-kinase (PEPC-k) activity is under control of a circadian oscillator, via the enhanced expression of the corresponding gene (1). The signaling cascade leading to PEPC-k up-regulation was investigated in leaves and mesophyll cell protoplasts of the facultative, salt-inducible CAM species, Mesembryanthemum crystallinum. Mesophyll cell protoplasts had the same PEPC-k activity as leaves from which they were prepared (i.e., high at night, low during the day). However, unlike C(4) protoplasts (2), CAM protoplasts did not show marked PEPC-k up-regulation when isolated during the day and treated with a weak base such as NH(4)Cl. Investigations using various pharmacological reagents established the operation, in the darkened CAM leaf, of a PEPC-k cascade including the following components: a phosphoinositide-dependent phospholipase C (PI-PLC), inositol 1,4,5 P (IP(3))-gated tonoplast calcium channels, and a putative Ca(2+)/calmodulin protein kinase. These results suggest that a similar signaling machinery is involved in both C(4) (2, 3) and CAM plants to regulate PEPC-k activity, the phosphorylation state of PEPC, and, thus, carbon flux through this enzyme during CAM photosynthesis., (Copyright 2001 Academic Press.)

The activity of 7-deoxyloganin 7-hydroxylase, an enzyme catalyzing the conversion of 7-deoxyloganin into loganin, was detected in a microsomal preparation from the cell suspension cultures of Lonicera japonica. It was dependent on NADPH and molecular oxygen. The enzymatic reaction was inhibited by carbon monoxide as well as by several cytochrome P450 inhibitors, especially ketoconazole, indicating that the reaction was mediated by cytochrome P450. The enzyme showed substrate specificity for 7-deoxyloganin. The K(m) values for 7-deoxyloganin and NADPH were estimated as 170 and 18 microM, respectively, from Lineweaver-Burk plots.

Betaine aldehyde dehydrogenase was purified to homogeneity from wild-type amaranth plants subjected to water deficit. The enzyme has a native molecular mass of 125 kDa; it is formed by two subunits, one of the subunits with a molecular mass of 63 kDa and the second one of 70 kDa as determined by SDS-PAGE and double dimension electrophoresis. IEF studies showed two bands with pI values of 4.93 and 4.85, respectively. Possible glycosilation of the 63- and 70-kDa subunits were tested with negative results. Both subunits cross-reacted strongly with polyclonal antibody raised against porcine kidney BADH. Also antiserum rose against HSP70 cross-reacted strongly with the wild amaranth BADH 70-kDa subunit. The enzyme was stable to extreme pH's and temperatures, and high KCl concentrations. Product inhibition of BADH was not observed., (Copyright 2001 Academic Press.)

The regiospecificity for the gene product of fad2,(1) the microsomal oleoyl-PC desaturase from higher plants, differs from some previous suggestions. Rather than only referencing the carboxyl group (a Delta(12) desaturase) or the methyl terminus (an omega-6 desaturase), this desaturase locates the second double bond in its substrates by first referencing the existing double bond. This specificity was demonstrated for the oleoyl-PC desaturase cDNA from the developing seeds of peanut (Arachis hypogaea L) expressed in yeast (Saccharomyces cerevisae). The expressed enzyme was capable of desaturating monounsaturated fatty acyl groups in membrane lipids. Endogenous palmitoleate was desaturated to cis, cis 9,12 hexadecadienoate (9(Z)12(Z)C16:2), endogenous oleate to linoleate (9(Z)12(Z) octadecadienoate), and cis 10-nonadecenoate (provided as a supplement in the growth medium) to 10(Z)13(Z)C19:2. The rule, Delta(x+3) where x=9 is the double bond location in the substrate, best describes the consistent placement of the second double bond in the above monounsaturated substrates for the oleoyl-PC desaturase of higher plants.

The similarities between delta12- and delta5-fatty acyl desaturase sequences were used to construct degenerate primers for PCR experiments with cDNA transcribed from mRNA of developing borage seeds. Screening of a borage seed cDNA library with an amplified DNA fragment resulted in the isolation of a full-length cDNA corresponding to a deduced open-reading frame of 446 amino acids. The protein showed high similarity to plant delta8-sphingolipid desaturases as well as to the delta6-fatty acyl desaturase from Borago officinalis. The sequence is characterized by the presence of a N-terminal cytochrome b5 domain. Expression of this open-reading frame in Saccharomyces cerevisiae resulted in the formation of delta8-trans/cis-phytosphingenines not present in wild-type cells, as shown by HPLC analysis of sphingoid bases as their dinitrophenyl derivatives. GLC-MS analysis of the methylated di-O-trimethylsilyl ether derivatives confirmed the presence of delta8-stereoisomers of C18- and C20-phytosphingenine. Furthermore, Northern blotting showed that the gene encoding a stereo-unselective delta8-sphingolipid desaturase is primarily expressed in young borage leaves.

The kinetic mechanism of betaine aldehyde dehydrogenase from leaves of the plant Amaranthus hypochondriacus is ordered with NAD(+) adding first. NADH is a noncompetitive inhibitor against NAD(+), which was interpreted before as evidence of an iso mechanism, in which NAD(+) and NADH binds to different forms of free enzyme. With the aim of testing the proposed kinetic mechanism, we have now investigated the ability of NADH to form different complexes with the enzyme. By initial velocity and equilibrium binding studies, we found that the steady-state levels of E.glycine betaine are negligible, ruling out binding of NADH to this complex. However, NADH readily bind to E.betaine aldehyde, whose levels most likely are kinetically significant given its low dissociation constant. Also, NADH combined with E.NADH and E.NAD(+). Finally, NADH was not able to revert the hydride transfer step, what suggest that there is no acyl-enzyme intermediate, i.e. the release of the reduced dinucleotide takes place after the deacylation step. Although formation of the complex E.NAD(+).NADH would produce an uncompetitive effect in the inhibition of NADH against NAD(+), the iso mechanism cannot be conclusively discarded.

Combinatorial chemistry is common place today in chemical synthesis. Virtually thousands of derivatives of a molecule can be achieved by automated systems. The use of biological systems to exploit combinatorial chemistry (combinatorial biochemistry) now has multiple examples in the polyketide field. The modular functional domain structure of polyketide synthases have been recombined through genetic engineering into unnatural constellations in heterologous hosts in order to produce polyketide structures not yet discovered in nature. We present herein an example for a potential type of combinatorial biochemistry in alkaloidal systems using various combinations of Thalictrum tuberosum (meadow rue) O-methyltransferase subunits that result in heterodimeric enzymes with substrate specificities that differ from those of the homodimeric native enzymes.

We have investigated distinguishing features in cells of the abscission zone of a monocotyledon fruit, the oil palm Elaeis guineensis. The cell walls of the abscission zone and the subtending mesocarp and pedicel have been analysed by light and transmission electron microscopy, by chemical methods and by solid state 13C CP/MAS NMR spectroscopy. Results show that these abscission zone cells have specific characteristics which include high levels of unmethylated pectin in the walls and an inducible (x35) polygalacturonase enzyme expression. Together these findings help to explain the localised precision of cell separation events.

The Borago officinalis Delta6 fatty acid desaturase (Boofd6) shares 58% identity in its amino acid sequence with Boofd8, a Delta8 sphingolipid desaturase from the same plant species. In order to localise the distinct catalytic properties of Boofd6 and Boofd8 to individual regions within them, a set of chimeras of these two enzymes were constructed and expressed in yeast. Chimera 2 is different from the other chimeras and Boofd6 in that it did not have any detectable desaturase activity on 18 carbon fatty acids. However, it desaturated C16 palmitoleic and C14 myristoleic acid, and the conversion rate for the later one was more than three times higher than that of Boofd6. These results suggest that the predicted membrane helices 1 and 2 of Boofd6 are involved in forming the substrate-binding site. This site appears to place constraints on the chain length of fatty acid substrates, which is similar to hydrophobic substrate binding pockets.

Substrate specificity of recombinant chalcone synthase (CHS) from Scutellaria baicalensis (Labiatae) was investigated using chemically synthesized aromatic and aliphatic CoA esters. It was demonstrated for the first time that CHS converted benzoyl-CoA to phlorobenzophenone (2,4,6-trihydroxybenzophenone) along with pyrone by-products. On the other hand, phenylacetyl-CoA was enzymatically converted to an unnatural aromatic polyketide, phlorobenzylketone (2, 4,6-trihydroxyphenylbenzylketone), whose structure was finally confirmed by chemical synthesis. Furthermore, in agreement with earlier reports, S. baicalensis CHS also accepted aliphatic CoA esters, isovaleryl-CoA and isobutyryl-CoA, to produce phloroacylphenones. In contrast, hexanoyl-CoA only afforded pyrone derivatives without formation of a new aromatic ring. It was noteworthy that both aromatic and aliphatic CoA esters were accepted in the active site of the enzyme as a starter substrate for the complex condensation reaction. The low substrate specificity of CHS thus provided further insight into the structure and function of the enzyme., (Copyright 2000 Academic Press.)

Upon illumination, the cotyledons of Catharanthus roseus seedlings readily synthesise vindoline from late biosynthetic intermediates, which accumulate in etiolated seedlings. The cellular localisation of tryptophan decarboxylase (TDC) and desacetoxyvindoline 4-hydroxylase (D4H), which catalyse the first and penultimate reactions of vindoline biosynthesis, was identified by immunocytochemistry in developing seedlings. The expression of TDC was restricted to the upper epidermis of cotyledons, whereas that of D4H was confined to laticifer cells. Light exposure of etiolated seedlings significantly induced D4H enzyme activity without changing the steady-state levels of D4H immunoreactive protein or modifying the cellular distribution of D4H expression in dark-grown seedlings. These results suggest that the early and late stages of vindoline biosynthesis occupy different cellular compartments, even in the early phases of etiolated seedling development. The role of light in activating the late stages of vindoline biosynthesis does not, therefore, seem to be related to the formation of the laticifer and idioblast cell types. It is concluded that light is not required for formation of these cell types, whereas regulatory factors, restricted to idioblasts and laticifers, may respond to light to activate localised expression of the late stages of vindoline biosynthesis.

We report the isolation and expression analysis of two cDNAs encoding 3-ketoacyl-acyl carrier protein synthases (KAS) that are involved in the de novo synthesis of fatty acids in plastids of perilla (Perilla frutescens L.). The cDNAs, designated PfFAB1 and PfFAB24, encoded polypeptides with high sequence identities to those of KAS I and KAS II/IV, respectively, of various plants. Genomic Southern blots revealed that there was a single PfFAB1 gene but two PfFAB24 genes in the perilla genome. Of interest is that the expression of both genes was developmentally regulated in seeds. Their mRNA expression patterns in seeds were also discussed in comparison with the profile of fatty acid accumulation.

A 2-oxoglutarate-dependent dioxygenase [EC 1.14.11-] that catalyzes the 6-hydroxylation of partially methylated flavonols has been purified to near homogeneity from Chrysosplenium americanum. Enzyme purification was achieved by fast protein liquid chromatography on Superose 12 and Mono Q columns as well as by affinity chromatography on 2-oxoglutarate-Sepharose and immunoaffinity columns. The specific activity of the 6-hydroxylase eluted from Mono Q (97.1 pkat/mg) was enriched 538-fold, with a 0.63% recovery. Both affinity chromatography steps resulted in the elimination of most contaminating proteins, but not without loss of enzyme activity and stability. The molecular mass of both the native and denatured enzyme was found to be 42 and 45 kDa, respectively, suggesting a monomeric protein. The enzyme exhibits strict specificity for position 6 of partially methylated flavonols possessing a 7-methoxyl group, indicating its involvement in the biosynthesis of polymethylated flavonols in this plant. The cofactor dependence of the enzyme is similar to that of other plant dioxygenases, particularly its dependence on ferrous ions for catalytic activity and reactivation. Internal amino acid sequence information indicated its relatedness to other plant flavonoid dioxygenases. The results of substrate interaction kinetics and product inhibition studies suggest an ordered, sequential reaction mechanism (TerTer), where 2-oxoglutarate is the first substrate to bind, followed by O2 and the flavonol substrate. Product release occurs in the reverse order where the hydroxylated flavonol is the first to be released, followed by CO2 and succinate. To our knowledge, this is the first reported 2-oxoglutarate-dependent dioxygenase that catalyzes the aromatic hydroxylation of a flavonoid compound.

The solution structure of diadenosine 5',5'''-P1,P4-tetraphosphate hydrolase from Lupinus angustifolius L., an enzyme of the Nudix family, has been determined by heteronuclear NMR, using a torsion angle dynamics/simulated annealing protocol based on approximately 12 interresidue NOEs per residue. The structure represents the first Ap4A hydrolase to be determined, and sequence homology suggests that other members will have the same fold. The family of structures shows a well-defined fold comprised of a central four-stranded mixed beta-sheet, a two-stranded antiparallel beta-sheet and three helices (alphaI, alphaIII, alphaIV). The root-mean-squared deviation for the backbone (C',O,N,Calpha) of the rigid parts (residues 9 to 75, 97 to 115, 125 to 160) of the protein is 0.32 A. Several regions, however, show lower definition, particularly an isolated helix (alphaII) that connects two strands of the central sheet. This poor definition is mainly due to a lack of long-range NOEs between alphaII and other parts of the protein. Mapping conserved residues outside of the Nudix signature and those sensitive to an Ap4A analogue suggests that the adenosine-ribose moiety of the substrate binds into a large cleft above the four-stranded beta-sheet. Four conserved glutamate residues (Glu55, Glu58, Glu59 and Glu125) form a cluster that most likely ligates an essential magnesium ion, however, Gly41 also an expected magnesium ligand, is distant from this cluster.

The sensitivity of an important aquatic macrophyte, duckweed (Lemna major), to UV-B radiation was studied under experimental conditions at three different doses designated as no, mild, and severe injury dose by observing visible injury symptoms and estimating levels of chlorophyll, pheophytin, carotenoids, protein, starch, free sugar, and peroxidase activity. Laboratory-grown duckweed plants were exposed to UV-B radiation at 0.4 mW/cm(2) intensity for different time periods. Mild and severe injury were developed at 6.48 and 8.64 J, respectively. Peroxidase activity increased at all the exposure levels. Dose-dependent decrease in chlorophyll and starch with drastic depletion in protein and free sugar content were observed. Pheophytin and carotenoids content increased at no injury level, but decreased at higher exposure level. The results indicate that ambient UV-B radiation at the indicated level acts as a physiological stress in Lemna major., (Copyright 2000 Academic Press.)

Deacetylipecoside synthase (DIS), the enzyme catalyzing the condensation of dopamine and secologanin to form the (R)-epimer of deacetylipecoside, has been purified 570-fold from the leaves of Alangium lamarckii and partially characterized. The isolated enzyme is a single polypeptide with Mr 30,000, and has a pH optimum at 7.5 and a temperature optimum at 45 degrees C. The apparent Km values for dopamine and secologanin are 0.7 and 0.9 mM, respectively. DIS exhibits high substrate specificity toward dopamine, whereas neither tyramine nor tryptamine are utilized. The enzyme activity is not inhibited by its substrate dopamine, but is inhibited by alangimakine and dehydroalangimakine with similar I50 values of 10 microM. DIS presumably provides (R)-deacetylipecoside for the formation of tetrahydroisoquinoline monoterpene glucosides that also possess an (R)-configuration at the same chiral center.

Monoterpenoid indole alkaloids are a vast and structurally complex group of plant secondary compounds. In contrast to other groups of plant products which produce many glycosides, indole alkaloids rarely occur as glucosides. Plants of Rauvolfia serpentina accumulate ajmaline as a major alkaloid, whereas cell suspension cultures of Rauvolfia mainly accumulate the glucoalkaloid raucaffricine at levels of 1.6 g/l. Cell cultures do contain a specific glucosidase. known as raucaffricine-O-beta-D-glucosidase (RG), which catalyzes the in vitro formation of vomilenine, a direct intermediate in ajmaline biosynthesis. Here, we describe the molecular cloning and functional expression of this enzyme in Escherichia coli. RG shows up to 60% amino acid identity with other glucosidases of plant origin and it shares several sequence motifs with family 1 glucosidases which have been characterized. The best substrate specificity for recombinant RG was raucaffricine (KM 1.3 mM, Vmax 0.5 nkat/microg protein) and only a few closely related structural derivatives were also hydrolyzed. Moreover, an early intermediate of ajmaline biosynthesis, strictosidine, is a substrate for recombinant RG (KM 1.8 mM, Vmax 2.6 pkat/microg protein) which was not observed for the low amounts of enzyme isolated from Rauvolfia cells.

In a reaction between (-)-epicatechin (EC) and 2-mercaptoethanol (2ME), catalyzed by partially purified polyphenol oxidase (PPO) extracted from the style of Rhododendron mucronatum, 2'-(2-hydroxyethylthio)-(-)-epicatechin (2'-HETEC), 5'-(2-hydroxyethylthio)-(-)-epicatechin (5'-HETEC), and 2',5'-bis(2-hydroxyethylthio)-(-)-epicatechin (2',5'-HETEC) were formed. The rate of formation of 2',5'-HETEC from 5'-HETEC was faster than that from 2'-HETEC. In the absence of 2ME, the concentration of EC decreased rapidly and the reaction mixture turned brown; 2'-, 5'-, and 2',5'-HETEC, especially 2'-substituted HETECs. reacted more slowly. These data indicate that 2ME acts both as an inhibitor of the polymerization of O-quinone, presumably by binding to it and as a reductant involved in the conversion of O-quinone to O-dihydroxyphenol, Inhibition of enzymatic browning by other thiol compounds such as cysteine and dithiothreitol was also investigated.

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.

Stimulation of the lipoxygenase pathway in olive fruit initiates a cascade of reactions that begins with the regio- and stereospecific di-oxygenation of polyunsaturated fatty acids containing a cis, cis-1,4 pentadiene moiety. Later products of the pathway include volatiles that influence the organoleptic properties of harvested olive oil. In this study, we have investigated lipoxygenase activity in olive callus cultures, and found that there is evidence of several isoforms of the enzyme with different pH optima and substrate specificities. Endogenous lipoxygenase activity was detected throughout the growth cycle of olive callus, particularly during the log phase of growth, suggesting that olive lipoxygenases are intimately involved in growth. The most prominent lipoxygenase activity in tissue cultures was found to be soluble but significant activities were detected in the plastid fraction. In addition, hydroperoxide lyase (HPL) activity was measured in the calli; both 13- and 9-HPL activities were found which were particulate.

Secologanin synthase, an enzyme catalyzing the oxidative cleavage of the cyclopentane ring in loganin to form secologanin, was detected in microsomal preparations from cell suspension cultures of Lonicera japonica. The reaction required NADPH and molecular oxygen, and was blocked by carbon monoxide as well as by several other cytochrome P450 inhibitors, indicating that the reaction was mediated by cytochrome P450. Of the substrates examined, only specificity for loganin was demonstrated. A possible reaction mechanism is described.

Among the large number of plant O-methyltransferases that are involved in secondary metabolism, only a few have been enzymatically characterized, and little information is available on the structure of their substrate binding site and the mechanism which determines their substrate specificity and methylation regiospecificity. We have previously reported the isolation of two O-methyltransferases, S-adenosyl-l-methionine:(iso)eugenol O-methyltransferase (IEMT) and S-adenosyl-l-methionine:caffeic acid O-methyltransferase (COMT) from Clarkia breweri, an annual plant from California. While IEMT and COMT (which methylate eugenol/isoeugenol and caffeic acid/5-hydroxyferulic acid, respectively) share 83% identity at the amino acid level, they have distinct substrate specificity and methylation regiospecificity. We report here that seven amino acids play a critical role in discriminating between eugenol/isoeugenol and caffeic acid/5-hydroxyferulic acid. When these amino acids in IEMT were replaced by the corresponding residues of COMT, the hybrid protein showed activity only with caffeic acid/5-hydroxyferulic acid. Conversely, when these amino acids in COMT were replaced by corresponding IEMT residues, the hybrid protein had activity only with eugenol/isoeugenol. These results provide strong evidence that O-methyltransferase substrate preference could be determined by a few amino acid residues and that new OMTs with different substrate specificity could begin to evolve from an existing OMT by mutation of a few amino acids. Phylogenetic analysis confirms that C. breweri IEMT evolved recently from COMT., (Copyright 1999 Academic Press.)

Recently, and for the first time, a diisopropylphosphorofluoridate (DFP)-hydrolyzing enzyme, i.e. an organophosphorus acid anhydrolase (OPAA), has been reported in a plant-source. Based on this and other suggestive evidence, the ability of three plant sources and a protist to hydrolyze DFP and 1,2,2-trimethylpropyl methylphosphonofluoridate (Soman) were tested, and the effects of Mn2+ and ethylenediamine tetraacetate (EDTA) on this activity. The plants are duckweed (Lemna minor), giant duckweed (Spirodela oligorhiza), and germinated mung bean (Vigna radiata); the protist is a slime mold (Dictyostelium discoidium). The tests are based on a crude classification of OPAAs as 'squid type' (DFP hydrolyzed more rapidly than Soman) and all of the others termed by us, with questionable justification, as 'Mazur type' (Soman hydrolyzed more rapidly than DFP). Of the two duckweeds, Spirodela oligorhiza hydrolyzes Soman but not DFP, and Lemna minor does not hydrolyze either substrate. In contrast to the report of Yu and Sakurai, mung bean does not hydrolyze DFP and hydrolyzes Soman with a 5-fold stimulation by Mn2+ and a marked inhibition by EDTA. The slime mold hydrolyzes Soman more rapidly than DFP (but does hydrolyze DFP) and the hydrolysis is Mn2+ stimulated. The failure of these plant sources to hydrolyze DFP is similar to the behavior of OPAA from Bacillus stearothermophilus.

A microplate assay for mevalonate and 5-phosphomevalonate kinase activities has been developed using an enzyme-coupled system of pyruvate kinase and lactate dehydrogenase. Mevalonate and 5-phosphomevalonate kinase activities were measured in crude and partially purified enzyme preparations from Catharanthus roseus suspension-cultured plant cells. The assay was validated with respect to protein and substrate concentration. Mevalonate and 5-phosphomevalonate kinase showed Michaelis-Menten kinetics with respect to ATP and their specific substrates; the apparent Km values of mevalonate kinase for ATP and mevalonate were 210 and 65 microM, respectively, and of 5-phosphomevalonate kinase for ATP and 5-phosphomevalonate were 0.41 and 0.4 mM, respectively. Considering mevalonate kinase, the relative standard deviation of enzyme activity within a determination (n = 3) is always less than 2.5% and in between determinations (n = 9) is less than 2%. The method can be used in a continuous assay as well as in a discontinuous assay., (Copyright 1999 Academic Press.)

Increased binding of ruthenium red to pectin as the number of methyl esters attached to the pectin decreases was used as the basis for a gel diffusion assay for pectin methylesterase (PME, EC 3.1.1.11) activity. The stained zone diameters resulting from the hydrolysis of 0.1% (w/v) 90% esterified pectin in an agarose gel by diffused, commercial PME were log-linear over 4 orders of magnitude, with a minimum detection limit of 3.6 pkatals. Pectin deesterification as the cause for a stained zone after PME incubation was confirmed when only 1 N NaOH, which will chemically deesterify the pectin, and not methanol or acid, the two products formed when PME acts on a methyl ester, resulted in the characteristic stained zone. The stained zone diameters decreased with increasing percentage of substrate esterification, were independent of pH, and were insensitive to simultaneous incubation with two forms of pectin lyase (EC 4.2.2.10), polygalacturonase (EC 3.2.1.15), or all combinations. PME extracted from tomato seeds, cotton fibers, and melon fruit showed pH optima of 6, 6, and 8, respectively. Using individual tomato seed parts, the assay was adapted to quantify diffusate activity and to localize activity in tissue prints. The sensitivity, specificity, and simplicity of this PME assay are superior to all others.

This report describes the activation of the alternative oxidase (AOX) of higher plant mitochondria by a high reduction level of the ubiquinone pool in the presence of pyruvate. In mitochondria from both thermogenic (Arum italicum spadices) and nonthermogenic (Glycine max cotyledons) tissues AOXis activated when the Q-pool becomes highly reduced in the presence of pyruvate. Pyruvate is essential for this activation. The enzyme is not activated when pyruvate is added after a transient high reduction level of the Q-pool, but is when pyruvate is added before the transient reduction. Pyruvate also protects the enzyme against inhibition during catalytic turnover. Although this activation is not accompanied by a reduction of the covalent disulfide bond, the same activation can be achieved with dithiothreitol (DTT). It is suggested that a part of the activation by DTT is not the result of reducing the covalent disulfide bond, and the relation between these types of activation is discussed. The importance of this activation for the in vivo regulation and its relation to previously reported activators is discussed. A mechanism is proposed in which it is suggested that AOX is inactivated by its product (oxidized ubiquinone) during catalysis and that this inhibition is prevented in the presence of pyruvate. The inhibition can be reversed by a reductive process, achieved by high levels of reduction of the Q-pool or by DTT, but not by pyruvate. This restoration of activity is not related to the redox process involved in reducing the covalent disulfide bond., (Copyright 1998 Academic Press.)

Peroxidase active preparations from three Ancistrocladus species and from Triphyophyllum peltatum have been partially purified. They catalyze the oxidative dimerization of korupensamines A and B to michellamines A and C, respectively, as well as the mixed coupling to michellamines A, B, and C. All of these enzymes consist of single polypeptides of approximately 65 kDa with peroxidase activity as monomers. They were characterized as soluble proteins predominantly localized in the leaf phloem of all species examined. Comparative studies with various naphthol precursors revealed an unexpected substrate specificity. Only korupensamines were dimerized by the enzymes, while other monomers, even if closely related, were not accepted as substrates. The coupling reactions described here represent the first direct synthesis of michellamines from korupensamines without previous protection of these precursors.

The three pinene synthases (cyclases) from common sage (Salvia officinalis) catalyze the conversion of geranyl pyrophosphate to the bicyclic olefins (+)-alpha-pinene and (+)-camphene (cyclase I), (-)-alpha-pinene, (-)-beta-pinene, and (-)-camphene (cyclase II), and (+)-alpha-pinene and (+)-beta-pinene (cyclase III), in addition to smaller amounts of monocyclic and acyclic monoterpene olefins. [1-3H,4-2H2]- and [10-2H2]-geranyl pyrophosphates were prepared and used in conjunction with 1-3H- and 1-3H,10-2H3-labeled geranyl precursors to examine the isotope effects attending the C4- and C10-deprotonation steps in the enzymatic synthesis of the pinenes. The observation of isotopically sensitive branching within each set of stereochemically related bicyclic olefins confirmed that each product set was synthesized by the respective pinene synthase by partitioning of common carbocationic intermediates along different reaction channels at the active site. The changes in product distribution resulting from deuterium substitution at C4 and C10 of the substrate were used to determine kinetic isotope effects (KIEs) for the terminating deprotonations; these observed KIEs represent the lower limits of the intrinsic isotope effects. The intramolecular isotope effects for the methyl-methylene elimination in beta-pinene formation by cyclases II and III were also evaluated with [10-2H2]geranyl pyrophosphate as substrate and by MS analysis of the olefin products. The intramolecular KIEs (kH/kD = 3.0 and 3.5) were significantly higher than the observed KIEs determined from product ratios (kH/kD = 1.7 and 2.6) since the former involves considerably less masking of the intrinsic isotope effects.

The three pinene synthases (cyclases) from common sage (Salvia officinalis) catalyze the conversion of geranyl pyrophosphate to the bicyclic olefins (+)-alpha-pinene and (+)-camphene (cyclase I), (-)-alpha-pinene, (-)-beta-pinene, and (-)-camphene (cyclase II), and (+)-alpha-pinene and (+)-beta-pinene (cyclase III), in addition to smaller amounts of monocyclic and acyclic monoterpene olefins. (1R)-4-2H1- and (1S)-4-2H1-labeled geranyl pyrophosphates were prepared and used to examine the stereochemistry of the C3-proton elimination from the pinyl cation intermediates in the formation of the alpha-pinene enantiomers. Mass spectrometric analysis of the biosynthetic products derived from the chirally deuterated substrates revealed that cyclase I and cyclase III removed the C4-proR-hydrogen of the substrate (C3 proton trans to the dimethyl bridge of the pinyl nucleus) with a stereoselectivity exceeding 94% in the formation of (+)-alpha-pinene. Similarly, cyclase II removed the C4-proS-hydrogen of the substrate (C3-trans proton of the corresponding pinyl cation) with a stereoselectivity exceeding 78% in the formation of (-)-alpha-pinene. The stereoselectivity of these C3-axial hydrogen eliminations is rationalized on the basis of a stereochemical model for the electrophilic isomerization-cyclization reaction sequence catalyzed by the pinene cyclases. The changes in the overall rates of olefin biosynthesis by these enzymes and in the product ratios resulting from deuterium substitution also permitted confirmation of isotopically sensitive branching in pinene biosynthesis and allowed the observation of primary kinetic isotope effects in isolation.

Monoterpene cyclases catalyze the divalent metal ion-dependent conversion of geranyl pyrophosphate, the ubiquitous C10 intermediate of isoprenoid biosynthesis, to a variety of monoterpene skeletons, and the pyrophosphoryl moiety is a primary determinant for substrate binding by these enzymes. To determine what specific features of this functional group are critical for enzymatic recognition, inorganic pyrophosphate and a series of structurally related analogs were examined as inhibitors of geranyl pyrophosphate:(+)-alpha-pinene cyclase and geranyl pyrophosphate:(+)-bornyl pyrophosphate cyclase from sage (Salvia officinalis). Analysis of trends in the magnitude of inhibition by the analogs relative to inorganic pyrophosphate indicated that the combination of ionization state (formal charge) at the enzymatic pH optimum, ability to chelate divalent metal ions, and intramolecular flexibility is required for effective interaction with both cyclases. Only when all of these criteria are met is inhibition of cyclization comparable to that observed with inorganic pyrophosphate.