M.Cecilia Rojas

Diferulate and lignin formation is related tobiochemical differences of wall-bound peroxidases

Abstract Purified cell walls from oat coleoptiles contain ionically and covalently bound peroxidaseactivity which correspond to 06% of the total peroxidase activity in the coleoptile Ionicallywall-bound peroxidases showed a 2–3-fold higher efficacy than peroxidases in the covalentfraction in the use of H 2 O 2 and phenolic substrates that are precursors of diferulate bridges andlignin The NADH oxidase activity in both fractions was effectively enhanced by p -coumaric acidand the ionic fraction showed a higher efficacy over the covalent one for NADH utilization in thepresence of this phenol Moreover the isoelectrofocusing pattern revealed marked differences inisoform composition for ionically and covalently bound wall peroxidases A cationic group ofisoperoxidases (pI∼96) was present only in the ionic fraction while the covalent fraction wasenriched with anionic forms (pI∼40–65) In excised coleoptiles incubated for 24 h the ionicallywall-bound peroxidase activity increased by 50% over covalently bound activity for 4 h ofincubation The increase of peroxidase activity preceded the accumulation of diferulic acid andlignin in oat cell walls Thus the evidence here reported suggest a possible functional differenceof peroxidase wall fractions studied related to diferulate and lignin synthesis in oat coleoptiles ©1998 Elsevier Science Ltd All rights reserved

Role of wall peroxidases in oat growth inhibition by DIMBOA

Abstract 2,4-Dihydroxy-7-methoxy-1,4-benzoxazin-3-one (DIMBOA), an effective growth-inhibitor of oat coleoptiles, enhanced 1524 and 246 times the rate of NADH oxidation by ionically and covalently bound oat cell wall peroxidases, a reaction that generates H 2 O 2 . This effect was similar to that of p -coumaric acid, while ferulic acid was a poor activator of this reaction. Coniferyl alcohol did not activate NADH oxidation. DIMBOA was poorly utilized by ionic or covalent oat wall peroxidases in the presence of H 2 O 2 . With horseradish peroxidase, DIMBOA efficiently promoted compound III breakdown with a decomposition rate constant of 4.88×10 −2 s −1 . This effect was 14 times higher than that obtained with p -coumaric acid that gave a decomposition rate constant of 0.35×10 −2 s −1 . The diferulate and lignin levels in the cell walls from oat coleoptiles increased by 56 and 30%, respectively, upon DIMBOA treatment. The effect of DIMBOA on oat wall peroxidases would thus contribute to its growth inhibitory effect by promoting H 2 O 2 synthesis and phenol coupling at the cell wall level.

Gibberellin biosynthesis and gibberellin oxidase activities in Fusarium sacchari, Fusarium konzum and Fusarium subglutinans strains

Several isolates of three Fusarium species associated with the Gibberella fujikuroi species complex were characterized for their ability to synthesize gibberellins (GAs): Fusarium sacchari (mating population B), Fusarium konzum (mating population I) and Fusarium subglutinans (mating population E). Of these, F. sacchari is phylogenetically related to Fusarium fujikuroi and is grouped in the Asian clade of the complex, while F. konzum and F. subglutinans are only distantly related to Fusarium fujikuroi and belong to the American clade. Variability was found between the different F. sacchari strains tested. Five isolates (B-12756; B-1732, B-7610, B-1721 and B-1797) were active in GA biosynthesis and accumulated GA(3) in the culture fluid (2.76-28.4 microg/mL), while two others (B-3828 and B-1725) were inactive. GA(3) levels in strain B-12756 increased by 2.9 times upon complementation with ggs2 and cps-ks genes from F. fujikuroi. Of six F. konzum isolates tested, three (I-10653; I-11616; I-11893) synthesized GAs, mainly GA(1), at a low level (less than 0.1 microg/mL). Non-producing F. konzum strains contained no GA oxidase activities as found for the two F. subglutinans strains tested. These results indicate that the ability to produce GAs is present in other species of the G. fujikuroi complex beside F. fujikuroi, but might differ significantly in different isolates of the same species.

Complexes of bivalent cations with neryl and geranyl pyrophosphate: Their role in terpene biosynthesis

Abstract Kinetic analysis of the nonenzymic solvolysis of neryl and geranyl pyrophosphate (NPP and GPP, respectively) showed that the dissociation constants of the bis-metallic complexes with Mg 2+ and Mn 2+ were larger for NPP than for GPP by approximately one order of magnitude. Rate constants for reaction of the bis-metallic complexes were larger for NPP than for GPP. Qualitatively similar behavior was observed with complexes of Co 2+ . Extents of elimination and cyclization were increased by metal ions. Carbocyclase-catalyzed formation of cyclic monoterpene hydrocarbons in the presence of Mg 2+ involved bis-metallic complexes as the “true” substrates.

Monooxygenases involved in GA12 and GA14 synthesis in Gibberella fujikuroi.

A microsomal preparation from mycelia of the gibberellin (GA)-producing fungus Gibberella fujikuroi catalyzed the first two steps in the conversion of the biosynthetic intermediate GA12-aldehyde to gibberellic acid (GA3). [14C]GA12-Aldehyde was converted to radiolabelled GA14, the major product, together with smaller amounts of non-hydroxylated GA12. The microsomal activities required reduced pyridine nucleotides and molecular oxygen. However, GA12 and GA14 synthesis differed markedly in the preferred electron source. Formation of GA12 required NADH or NADPH, while GA14 synthesis from GA12-aldehyde occurred only with NADPH. Marked differences were also found in the activating effect of FAD. When NADPH was the reductant, the rate of GA14 synthesis was enhanced 3.5 times by 5 microM FAD while this flavin nucleotide did not alter the synthesis of GA12. In contrast, GA12 synthesis was activated 3.8 times by 50 microM FAD in the presence of NADH. Both activities were inhibited by carbon monoxide and cytochrome c. These properties suggest that the 3beta-hydroxylation of GA12-aldehyde and further oxidation of carbon 7 are catalyzed by cytochrome P-450 monooxygenases in Gibberella fujikuroi.

Comparative steady-state fluorescence studies of cytosolic rat liver (GTP), Saccharomyces cerevisiae (ATP) and Escherichia coli (ATP) phosphoenolpyruvate carboxykinases

Two members of the ATP-dependent class of phospho enol pyruvate (PEP) carboxykinases (Saccharomyces cerevisiae and Escherichia coli PEP carboxykinase), and one member of the GTP-dependent class (the cytosolic rat liver enzyme) have been comparatively analyzed by taking advantage of their intrinsic fluorescence. The S. cerevisiae and the rat liver enzymes show intrinsic fluorescence with a maximum emission characteristic of moderately buried tryptophan residues, while the E. coli carboxykinase shows somewhat more average exposure for these fluorophores. The fluorescence of the three proteins was similarly quenched by the polar compound acrylamide, but differences were observed for the ionic quencher iodide. For the ATP-dependent enzymes, these last experiments indicate more exposure to the aqueous media of the tryptophan population of the E. coli than of the S. cerevisiae enzyme. The effect of nucleotides on the emission intensities and quenching efficiencies revealed substrate-induced conformational changes in the E. coli and cytosolic rat liver PEP carboxykinases. The addition of Mn2+ or of the adenosine nucleotides in the presence of Mg2+ induced an enhancement in the fluorescence of the E. coli enzyme. The addition of guanosine or inosine nucleotides to the rat liver enzyme quenched its fluorescence. From the ligand-induced fluorescence changes, dissociation constants of 40 +/- 6 microM, 10 +/- 0.8 microM, and 15 +/- 1 microM were obtained for Mn2+, MgATP and MgADP binding to the E. coli enzyme, respectively. For the cytosolic rat liver PEP carboxykinase, the respective values for GDP, IDP and ITP binding are 6 +/- 0.5 microM, 6.7 +/- 0.4 microM and 10.1 +/- 1.7 microM. A comparison of the dissociation constants obtained in this work with those reported for other PEP carboxykinases is presented.

Synthesis of monoterpene hydrocarbons from [1-3H]linalyl pyrophosphate by carbocyclase from Citrus limonum☆

Abstract A partially purified enzyme preparation from the flavedo of Citrus limonum utilized [1- 3 H]linalyl pyrophosphate as a substrate for cyclic terpene hydrocarbon formation more efficiently than the pyrophosphates of nerol and geraniol. The products formed from all three substrates are α-pinene, β-pinene, limonene, and γ-terpinene. Neryl and geranyl pyrophosphate inhibit the formation of these products from linalyl pyrophosphate. No free linalyl pyrophosphate could be detected during the enzymatic formation of cyclic terpene hydrocarbons from geranyl pyrophosphate. Mn 2+ catalyzes the nonenzymatic solvolysis of linalyl pyrophosphate, forming myrcene and ocymenes and no bicyclic hydrocarbons. Linalyl pyrophosphate is a sterically plausible precursor of cyclic hydrocarbons, but the present data support only its role as an alternative substrate and not as an obligatory free intermediate in terpene biosynthesis.

Substrate and metal specificity in the enzymic synthesis of cyclic monoterpenes from geranyl and neryl pyrophosphate

A partially purified enzyme (carbocyclase) from the flavedo of Citrus limonum formed alpha-pinene, beta-pinene, limonene, and gamma-terpinene from geranyl pyrophosphate (GPP) and neryl pyrophosphate. The maximum specific activities obtained were 7.0 and 3.6 nmol/min/mg, respectively. Cross-inhibition by the two substrates were observed and the ability to utilize neryl pyrophosphate was almost completely lost with aging. Citronellyl pyrophosphate and dimethylallyl pyrophosphate were the most effective inhibitors of carbocyclase. Isopentenyl pyrophosphate, the monophosphate esters of nerol and geraniol, as well as inorganic pyrophosphate were much less effective inhibitors. The enzyme had an absolute requirement for Mn2+. It could be replaced with about 2% effectiveness by Mg2+ and Co2+. Kinetic studies showed that the observed reaction rate correlates with the calculated concentration of the GPP (Mn2+)2 species. Previous evidence with nonenzymatic reactions and the results presented support the view that the mechanism of carbocyclase may be the intramolecular analog of prenyltransferase.

Fluorescent labeling of the nucleotide site in cytosolic rat liver phosphoenolpyruvate carboxykinase

Reaction of rat liver phosphoenolpyruvate carboxykinase (GTP: oxaloacetate carboxy-lyase (transphosphorylating), EC 4.1.1.32) with the alkylating fluorescent probe N-(iodoacetylaminoethyl)-5-naphthylamine-1-sulfonic acid (1,5-I-AEDANS), results in complete loss of enzymatic activity. One mole of the fluorescent reagent is incorporated per mole of the inactivated enzyme. When the modification is carried out in the presence of GDPMn, the enzyme retains 97% of its activity with almost no incorporation of label. The specificity of the reaction is further supported by the detection of a unique fluorescent peptide from the trypsin-treated modified enzyme. Fluorescence emission of enzyme-bound AEDANS shows a broad band centered at 470 nm and presents a monoexponential decay with a lifetime of 19 ns. These data indicate that the probe-binding site is considerably less polar than water and similar in polarity to ethanol. Anisotropy determinations give evidence for restricted rotational freedom for AEDANS bound to the rat carboxykinase, while acrylamide quenching studies reveal limited accessibility to the probe site. The results are consistent with specific labeling of rat liver phosphoenolpyruvate carboxykinase at or near the GDP site. The characteristics of the nucleotide-binding sites of rat liver and yeast (ATP) phosphoenolpyruvate carboxykinase are compared.

Hydrolysis of bismetallic complexes of linalyldiphosphate and their participation in the biosynthesis of cyclic monoterpenes

The allylic diphosphate ester linalyldiphosphate forms mono- and bis-metallic complexes with Mg2+ and Mn2+. Complexes of the trianion of the ligand are 50 to 100 times more stable than those of the dianion. Mg2+ and Mn2+ are bound in the monometallic complex 103 and 104 times more strongly than in the bismetallic species.The rate constant of the uncatalysed hydrolysis of linalyldiphosphate at pH 7.0 is 1.2 × 104s–1, which is higher than reported values for the primary diphosphates. Mg2+ and Mn2+ affect its rate of hydrolysis and it is shown that the rate enhancement observed is a function of the concentration of the bismetallic complexes.The rate of cyclic terpene hydrocarbon biosynthesis catalysed by carbocyclase from Citrus limonum also correlates with the concentration of the bismetallic complexes.Linalyl monophosphate, which is not a substrate for carbocyclase hydrolyses at a rate proportional to the concentration of the monometallic complex of Mn2+. The neutral charge of this complex, as opposed to the positively charged bismetallic complex of linalyl diphosphate may be an explanation of the absence of utilization of this complex as a substrate.It appears from the data presented and from previous evidence that bismetallic complexes of allylic diphosphates are the most reactive species in their hydrolysis and the only reactive species in the enzymic cyclization of these precursors of monoterpenes.