Crystal structure at 1.1 Angstroms resolution of an insect myrosinase from Brevicoryne brassicae shows its close relationship to beta-glucosidases

The aphid Brevicoryne brassicae is a specialist feeding on Brassicaceae plants. The insect has an intricate defence system involving a beta-D-thioglucosidase (myrosinase) that hydrolyses glucosinolates sequestered from the host plant into volatile isothiocyanates. These isothiocyanates act synergistically with the pheromone E-beta-farnesene to form an alarm system when the aphid is predated. In order to investigate the enzymatic characteristics of the aphid myrosinase and its three-dimensional structure, milligram amounts of pure recombinant aphid myrosinase were obtained from Echerichia coli. The recombinant enzyme had similar physiochemical properties to the native enzyme. The global structure is very similar to Sinapis alba myrosinase and plant beta-O-glucosidases. Aphid myrosinase has two catalytic glutamic acid residues positioned as in plant beta-O-glucosidases, and it is not obvious why this unusual enzyme hydrolyses glucosinolates, the common substrates of plant myrosinases which are normally not hydrolyzed by plant beta-O-glucosidases. The only residue specific for aphid myrosinase in proximity of the glycosidic linkage is Tyr180 which may have a catalytic role. The aglycon binding site differs strongly from plant myrosinase, whereas due to the presence of Trp424 in the glucose binding site, this part of the active site is more similar to plant beta-O-glucosidases, as plant myrosinases carry a phenylalanine residue at this position.     

Functional expression and characterization of the myrosinase MYR1 from Brassica napus in Saccharomyces cerevisiae

Myrosinases are thioglucosidases that hydrolyze the natural plant products glucosinolates. We have expressed the myrosinase MYR1 from Brassica napus in Saccharomyces cerevisiae. The recombinant myrosinase was enzymatically active which shows that the MYR1, which in the plant is complex bound with myrosinase-binding proteins and myrosinase-associated proteins, is functional in its free form. Characterization of the recombinant MYR1 with respect to pH optimum, substrate specificity, activation by ascorbic acid, and inhibitors showed similar characteristics as previously observed for other plant myrosinases. The indolizidine alkaloid castanospermine, an inhibitor of O-glycosidases, inhibited the hydrolysis of p-hydroxybenzylglucosinolate with a K(i) value of 0.3 microM and 2-deoxy-2-fluoroglucotropaeolin, a specific inhibitor of thioglucosidases, inhibited the enzyme with a K(i) value of 1 mM. The expression of the myrosinase in yeast was transient and the growth of the yeast cells was significantly reduced during the period of expression of the myrosinase. Immunoblot analysis showed that the highest level of expression of MYR1 was obtained 24 h after induction with galactose. The amount of myrosinase protein correlated with the level of enzyme activity. The transient expression of myrosinase indicates that myrosinase is toxic to the cells. This is the first report on successful heterologous expression of a myrosinase and provides an important tool for, e.g., further characterization of myrosinase by site-directed mutagenesis and for studying the interaction between myrosinase and myrosinase-binding proteins, myrosinase-associated proteins, and epithiospecifier proteins.     

Revised determination of free and complexed myrosinase activities in plant extracts.

The enzyme myrosinase (thioglucoside glucohydrolase, EC 3.2.1.147, formerly EC 3.2.3.1) catalyzes the hydrolysis of glucosinolates after tissue damage in plants of the order Brassicales. The various myrosinase isoforms occur either as free soluble dimers or as insoluble complexes. We propose a reliable method for determination of both soluble and insoluble myrosinase activity concentrations in partially purified plant extracts. The procedure requires the removal of endogenous glucosinolates through ion-exchange columns previous to enzyme measurements. Myrosinase activity was assayed in continuous mode by photometric quantification of the released glucose using glucose-oxidase with peroxidase and colorimetric indicators. The measurement of the colored product at 492nm has a favorable signal to noise ratio both in clear extract solutions (free dimers) and in turbid pellet suspensions (insoluble complexes). No interferences by ascorbic acid were found in continuous analyses. With the recommended sample preparation methods and assay conditions potential activities in damaged plant tissues can be characterized which are involved in plant defense mechanisms.     

Role of divalent metal cations in ATP hydrolysis catalyzed by the hepatitis C virus NS3 helicase: magnesium provides a bridge for ATP to fuel unwinding

This study investigates the role of magnesium ions in coupling ATP hydrolysis to the nucleic acid unwinding catalyzed by the NS3 protein encoded by the hepatitis C virus (HCV). Analyses of steady-state ATP hydrolysis rates at various RNA and magnesium concentrations were used to determine values for the 15 dissociation constants describing the formation of a productive enzyme-metal-ATP-RNA complex and the four rate constants describing hydrolysis of ATP by the possible enzyme-ATP complexes. These values coupled with direct binding studies, specificity studies and analyses of site-directed mutants reveal only one ATP binding site on HCV helicase centered on the catalytic base Glu291. An adjacent residue, Asp290, binds a magnesium ion that forms a bridge to ATP, reorienting the nucleotide in the active site. RNA stimulates hydrolysis while decreasing the affinity of the enzyme for ATP, magnesium, and MgATP. The binding scheme described here explains the unusual regulation of the enzyme by ATP that has been reported previously. Binding of either free magnesium or free ATP to HCV helicase competes with MgATP, the true fuel for helicase movements, and leads to slower hydrolysis and nucleic acid unwinding.     

Studies on Myrosinase in Mustard Seed

Various sugars and glucosides inhibited both ascorbate-activated and non-activated plant myrosinases at much higher concentrations than those of the substrate. Among these, glucose and salicin were confirmed to be competitive inhibitors. The resemblance of myrosinase to β-glucosidase was pointed out from these results. The Ki values for them remained unchanged after the activation of the enzyme by l-ascorbate, whereas the Km value for the substrate increased. Glucose had no influence on the binding of ascorbate to the enzyme. Pigman’s model for β-glucosidase can be satisfactorily applied to yield an explanation of these experimental results.

β-Glucosidase activity of mustard myrosinase was confirmed using p-nitrophenyl β-glucoside ( p-NPG) as the substrate. p-NPG hydrolysis was not accelerated by 10^?3 m ascorbate but was inhibited competitively by its higher concentrations. This result forms a distinct contrast with the activating effect of ascorbate in enzymatic sinigrin hydrolysis. The previously noticed feature of the ascorbate effect on the enzyme that the Km value for sinigrin hydrolysis increased after activation is explainable by the fact that ascorbate was a competitive inhibitor as well as a specific activator. A schematic model interpreting the interaction of ascorbate with the enzyme is proposed.     

The structural basis of oligosaccharide binding by rice BGlu1 beta-glucosidase

Rice BGlu1 β-glucosidase is an oligosaccharide exoglucosidase that binds to six β-(1→4)-linked glucosyl residues in its active site cleft. Here, we demonstrate that a BGlu1 E176Q active site mutant can be effectively rescued by small nucleophiles, such as acetate, azide and ascorbate, for hydrolysis of aryl glycosides in a pH-independent manner above pH 5, consistent with the role of E176 as the catalytic acid–base. Cellotriose, cellotetraose, cellopentaose, cellohexaose and laminaribiose are not hydrolyzed by the mutant and instead exhibit competitive inhibition. The structures of the BGlu1 E176Q, its complexes with cellotetraose, cellopentaose and laminaribiose, and its covalent intermediate with 2-deoxy-2-fluoroglucoside were determined at 1.65, 1.95, 1.80, 2.80, and 1.90 Å resolution, respectively. The Q176 Nε was found to hydrogen bond to the glycosidic oxygen of the scissile bond, thereby explaining its high activity. The enzyme interacts with cellooligosaccharides through direct hydrogen bonds to the nonreducing terminal glucosyl residue. However, interaction with the other glucosyl residues is predominantly mediated through water molecules, with the exception of a direct hydrogen bond from N245 to glucosyl residue 3, consistent with the apparent high binding energy at this residue. Hydrophobic interactions with the aromatic sidechain of W358 appear to orient glucosyl residues 2 and 3, while Y341 orients glucosyl residues 4 and 5. In contrast, laminaribiose has its second glucosyl residue positioned to allow direct hydrogen bonding between its O2 and Q176 Oε and O1 and N245. These are the first GH1 glycoside hydrolase family structures to show oligosaccharide binding in the hydrolytic configuration.     

Myrosinase: insights on structural,catalytic, regulatory,and environmental interactions

Glucosinolate–myrosinase is a substrate-enzyme defense mechanism present in Brassica crops. This binary system provides the plant with an efficient system against herbivores and pathogens. For humans, it is well known for its anti-carcinogenic, anti-inflammatory, immunomodulatory, anti-bacterial, cardio-protective, and central nervous system protective activities. Glucosinolate and myrosinase are spatially present in different cells that upon tissue disruption come together and result in the formation of a variety of hydrolysis products with diverse physicochemical and biological properties. The myrosinase-catalyzed reaction starts with cleavage of the thioglucosidic linkage resulting in release of a D-glucose and an unstable thiohydroximate-O-sulfate. The outcome of this thiohydroximate-O-sulfate has been shown to depend on the structure of the glucosinolate side chain, the presence of supplementary proteins known as specifier proteins and/or on the physiochemical condition. Myrosinase was first reported in mustard seed during 1939 as a protein responsible for release of essential oil. Until this date, myrosinases have been characterized from more than 20 species of Brassica, cabbage aphid, and many bacteria residing in the human intestine. All the plant myrosinases are reported to be activated by ascorbic acid while aphid and bacterial myrosinases are found to be either neutral or inhibited. Myrosinase catalyzes hydrolysis of the S-glycosyl bond, O-β glycosyl bond, and O-glycosyl bond. This review summarizes information on myrosinase, an essential component of this binary system, including its structural and molecular properties, mechanism of action, and its regulation and will be beneficial for the research going on the understanding and betterment of the glucosinolate–myrosinase system from an ecological and nutraceutical perspective.     

A novel approach for reliable activity determination of ascorbic acid depending myrosinases

Up to now, a wide array of methods for the determination of myrosinase activity has been described. These vary from the simple photometric estimation to highly sophisticated assays using radioactively labelled substrates. However, ascorbic acid--an effective activator of myrosinases--interferes with most of these enzyme tests. Unfortunately, in the past, such interferences were disregarded in many scientific examinations of myrosinases. Whereas such failings have less effects when the activation of myrosinases is not very distinctive, they are quite relevant in all cases where myrosinases are completely inactive in the absence of ascorbic acid. In this paper, the current methods for myrosinase determination are reviewed critically with special emphasis on putative interferences with ascorbic acid. Thereafter, an alternative and interference-free HPLC-based quantification method of the enzymatically produced glucose is presented. Due to the benzoylation of glucose, it becomes possible to quantify even those exiguous glucose concentrations, which are indispensable for correct determination of kinetic enzyme data in the presence of ascorbic acid. Using this new method, the activity of Tropaeolum majus myrosinase towards glucotropaeolin was analyzed. This enzyme shows a distinctive activation by ascorbic acid with maximal activation at a concentration of about 2 mM.     

Monitoring the reaction of carbachol with acetylcholinesterase by thioflavin T fluorescence and acetylthiocholine hydrolysis

Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. Carbamates are very poor substrates that, like other AChE substrates, form an initial enzyme-substrate complex and proceed to an acylated enzyme intermediate which is then hydrolyzed. However, the hydrolysis of the carbamoylated enzyme is slow enough to resolve the acylation and deacylation steps on the catalytic pathway. Here we show that the reaction of carbachol (carbamoylcholine) with AChE can be monitored both with acetylthiocholine as a reporter substrate and with thioflavin T as a fluorescent reporter group. The fluorescence of thioflavin T is strongly enhanced when it binds to the P-site of AChE, and this fluorescence is partially quenched when a second ligand binds to the A-site to form a ternary complex. These fluorescence changes allow not only the monitoring of the course of the carbamoylation reaction but also the determination of carbachol affinities for the A- and P-sites.     

Steady-state kinetic mechanism of recombinant avocado ACC oxidase: initial velocity and inhibitor studies

The gaseous plant hormone ethylene modulates a wide range of biological processes, including fruit ripening. It is synthesized by the ascorbate-dependent oxidation of 1-aminocyclopropyl-1-carboxylate (ACC), a reaction catalyzed by ACC oxidase. Recombinant avocado (Persea americana) ACC oxidase was expressed in Escherichia coli and purified in milligram quantities, resulting in high levels of ACC oxidase protein and enzyme activity. An optimized assay for the purified enzyme was developed that takes into account the inherent complexities of the assay system. Fe(II) and ascorbic acid form a binary complex that is not the true substrate for the reaction and enhances the degree of ascorbic acid substrate inhibition. The K(d) value for Fe(II) (40 nM, free species) and the K(m)'s for ascorbic acid (2.1 mM), ACC (62 microM), and O(2) (4 microM) were determined. Fe(II) and ACC exhibit substrate inhibition, and a second metal binding site is suggested. Initial velocity measurements and inhibitor studies were used to resolve the kinetic mechanism through the final substrate binding step. Fe(II) binding is followed by either ascorbate or ACC binding, with ascorbate being preferred. This is followed by the ordered addition of molecular oxygen and the last substrate, leading to the formation of the catalytically competent complex. Both Fe(II) and O(2) are in thermodynamic equilibrium with their enzyme forms. The binding of a second molecule of ascorbic acid or ACC leads to significant substrate inhibition. ACC and ascorbate analogues were used to confirm the kinetic mechanism and to identify important determinants of substrate binding.     

Purification and characterization of myrosinase from the cabbage aphid (Brevicoryne brassicae), a brassica herbivore.

Aphids are among the most serious insect pests of agricultural crops in the world. They often have specific hosts, and the cabbage aphid (Brevicoryne brassicae) is a specialist on Cruciferae. It has previously been described that certain insects contain the enzyme myrosinase (EC 3.2.3.1), which is considered an important defence enzyme of crucifers. Myrosinase was purified to homogeneity from cabbage aphid soluble extracts using anion-exchange and phenyl-Sepharose chromatography. The protein has an apparent subunit molecular mass of 57-58 kDa and is a dimer. The isoelectric point is 4.9 and the enzyme has a temperature optimum around 40 degrees C. The enzyme was active towards the glucosinolates tested, sinigrin and glucotropaeolin, but was inhibited by ascorbate at concentrations that normally activate plant myrosinases. Using sinigrin as the substrate Km was determined as 0.41 mM, and the kcat as 36 s(-1). With glucotropaeolin the Km and kcat values were determined as 0.52 mM and 22.8 s(-1), respectively. The enzyme was stable upon storage at 4 degrees C for many months, but lost some activity upon freezing. The insect myrosinase did not cross-react with antibodies raised to plant myrosinase. Peptide sequencing of a tryptic digest of the protein showed homology to beta-glucosidases. The presence of myrosinase in an insect pest specialist may be an example of a coevolution process that facilitates host specialization.     

Determination of myrosinase (thioglucoside glucohydrolase) activity by a spectrophotometric coupled enzyme assay

The hexokinase/glucose-6-phosphate dehydrogenase coupled enzyme system was used to assay for plant thioglucoside glucohydrolase (myrosinase, EC 3.2.3.1) by measuring the rate of glucose released during hydrolysis of glucosinolates. This coupled assay was compared with two other assays for myrosinase: a pH-stat assay that measures the rate of acid released during glucosinolate hydrolysis, and a spectrophotometric assay in which the decrease in the absorbance at 227.5 nm is used to measure the disappearance of the substrate, 2-propenylglucosinolate (DSA assay). The coupled and pH-stat assays were found to give comparable activities and were linear with enzyme concentration over the range 0 to 30 micrograms. The DSA assay gave lower myrosinase activity in comparison to the coupled and pH-stat assays. This is due to the lower concentrations of substrate and activator (ascorbate) which must be used in the assay. The DSA assay was found to give a nonlinear relationship with enzyme concentration over the range 2 to 30 micrograms. For these reasons this assay was found to be unsatisfactory. The coupled assay was found to be more sensitive and more widely applicable than the pH-stat assay as a routine continuous assay for myrosinase activity.     

Characterisation of aphid myrosinase and degradation studies of glucosinolates

Myrosinase from Brevicoryne brassicae was purified by ammonium sulfate fractionation, dialysis, and chromatography on a DEAE column. The chromatography yielded a single peak and a 115.6-fold purification. Further FPLC gel filtration gave a single peak at 120 kDa. Denaturing SDS/PAGE of the protein revealed a single band at 60 kDa, indicating that the native B. brassicae myrosinase is a dimer. Kinetic parameters towards 8 glucosinolates were calculated. Strong differences of V(max) and K(m) were observed depending on the substrate. Degradation products of each glucosinolate were identified and quantified by GC-MS and GLC-FID, respectively. Using both crude aphid homogenates and purified myrosinase, two unique hydroxyglucosinolates, 3-butenyl- and benzyl-isothiocyanates were identified from progoitrin ((2S)-2-hydroxybut-3-enyl-glucosinolate) and sinalbin (4-hydroxybenzyl-glucosinolate) degradation respectively. Addition of ascorbic acid to the reaction mixtures containing sinalbin and progoitrin caused the production of hydroxylated degradation products usually associated with plant myrosinase metabolisation. The occurrence of the myrosinase system in B. brassicae is discussed in terms of similar allelochemical adaptation between the herbivore and its host plant.     

Unmasking tandem site interaction in human acetylcholinesterase. Substrate activation with a cationic acetanilide substrate

Acetylcholinesterase (AChE) contains a narrow and deep active site gorge with two sites of ligand binding, an acylation site (or A-site) at the base of the gorge, and a peripheral site (or P-site) near the gorge entrance. The P-site contributes to catalytic efficiency by transiently binding substrates on their way to the acylation site, where a short-lived acyl enzyme intermediate is produced. A conformational interaction between the A- and P-sites has recently been found to modulate ligand affinities. We now demonstrate that this interaction is of functional importance by showing that the acetylation rate constant of a substrate bound to the A-site is increased by a factor a when a second molecule of substrate binds to the P-site. This demonstration became feasible through the introduction of a new acetanilide substrate analogue of acetylcholine, 3-(acetamido)-N,N,N-trimethylanilinium (ATMA), for which a = 4. This substrate has a low acetylation rate constant and equilibrates with the catalytic site, allowing a tractable algebraic solution to the rate equation for substrate hydrolysis. ATMA affinities for the A- and P-sites deduced from the kinetic analysis were confirmed by fluorescence titration with thioflavin T as a reporter ligand. Values of a >1 give rise to a hydrolysis profile called substrate activation, and the AChE site-specific mutant W86F, and to a lesser extent wild-type human AChE itself, showed substrate activation with acetylthiocholine as the substrate. Substrate activation was incorporated into a previous catalytic scheme for AChE in which a bound P-site ligand can also block product dissociation from the A-site, and two additional features of the AChE catalytic pathway were revealed. First, the ability of a bound P-site ligand to increase the substrate acetylation rate constant varied with the structure of the ligand: thioflavin T accelerated ATMA acetylation by a factor a(2) of 1.3, while propidium failed to accelerate. Second, catalytic rate constants in the initial intermediate formed during acylation (EAP, where EA is the acyl enzyme and P is the alcohol leaving group cleaved from the ester substrate) may be constrained such that the leaving group P must dissociate before hydrolytic deacylation can occur.     

Oxygen exchange between acetate and the catalytic glutamate residue in glutaconate CoA-transferase from Acidaminococcus fermentans. Implications for the mechanism of CoA-ester hydrolysis.

The exchange of oxygen atoms between acetate, glutaryl-CoA, and the catalytic glutamate residue in glutaconate CoA-transferase from Acidaminococcus fermentans was analyzed using [(18)O(2)]acetate together with matrix-assisted laser desorption/ionization time of flight mass spectrometry of an appropriate undecapeptide. The exchange reaction was shown to be site-specific, reversible, and required both glutaryl-CoA and [(18)O(2)]acetate. The observed exchange is in agreement with the formation of a mixed anhydride intermediate between the enzyme and acetate. In contrast, with a mutant enzyme, which was converted to a thiol ester hydrolyase by replacement of the catalytic glutamate residue by aspartate, no (18)O uptake from H(2)(18)O into the carboxylate was detectable. This result is in accord with a mechanism in which the carboxylate of aspartate acts as a general base in activating a water molecule for hydrolysis of the thiol ester intermediate. This mechanism is further supported by the finding of a significant hydrolyase activity of the wild-type enzyme using acetyl-CoA as substrate, whereas glutaryl-CoA is not hydrolyzed. The small acetate molecule in the substrate binding pocket may activate a water molecule for hydrolysis of the nearby enzyme-CoA thiol ester.     

The crystal structure of the formiminotransferase domain of formiminotransferase-cyclodeaminase: implications for substrate channeling in a bifunctional enzyme

BACKGROUND: The bifunctional enzyme formiminotransferase-cyclodeaminase (FTCD) contains two active sites at different positions on the protein structure. The enzyme binds a gamma-linked polyglutamylated form of the tetrahydrofolate substrate and channels the product of the transferase reaction from the transferase active site to the cyclodeaminase active site. Structural studies of this bifunctional enzyme and its monofunctional domains will provide insight into the mechanism of substrate channeling and the two catalytic reactions. RESULTS: The crystal structure of the formiminotransferase (FT) domain of FTCD has been determined in the presence of a product analog, folinic acid. The overall structure shows that the FT domain comprises two subdomains that adopt a novel alpha/beta fold. Inspection of the folinic acid binding site reveals an electrostatic tunnel traversing the width of the molecule. The distribution of charged residues in the tunnel provides insight into the possible mode of substrate binding and channeling. The electron density reveals that the non-natural stereoisomer, (6R)-folinic acid, binds to the protein; this observation suggests a mechanism for product release. In addition, a single molecule of glycerol is bound to the enzyme and indicates a putative binding site for formiminoglutamate. CONCLUSIONS: The structure of the FT domain in the presence of folinic acid reveals a possible novel mechanism for substrate channeling. The position of the folinic acid and a bound glycerol molecule near to the sidechain of His82 suggests that this residue may act as the catalytic base required for the formiminotransferase mechanism.     

Development and characteristics of myrosinase in Brassica napus during early seedling growth

Myrosinase (β-thioglucoside glucohydroase, E. C. 3.2.3.1) proteins with different physical, but similar kinetic characteristics exist in oilseed rape ( Brassica napus L. cv. Bienvenu) seedlings. Two protein fractions have been described which are immunologically, and therefore likely to be structurally, related. Myrosinase I, a dimeric 156 kDa glycosylated protein was purified to apparent homogeneity, and polyclonal antibodies were raised against this protein. Myrosinase II, in comparison, was significantly less glycosylated. The native protein had a molecular weight of approximately 188 kDa, with subunit M_r's of mainly 62 kDa and also 68 kDa. Total 'potential'enzyme activity (assayed in the presence of ascorbic acid activator) increased during early seedling growth. Immunoblot analysis of seedling proteins showed that this is directly related to an increase in the amount of myrosinase protein itself , predominantly myrosinase II proteins, which are not present in the dry seed. Myrosinase II protein is located exclusively in the cotyledons of 5-day-old seedlings, whilst myrosinase I is distributed throughout the seedling.     

Mechanism of ascorbic acid oxidation by cytochrome b(561)

The 1 equiv reaction between ascorbic acid and cytochrome b(561) is a good model for redox reactions between metalloproteins (electron carriers) and specific organic substrates (hydrogen-atom carriers). Diethyl pyrocarbonate inhibits the reaction of cytochrome b(561) with ascorbate by modifying a histidine residue in the ascorbate-binding site. Ferri/ferrocyanide can mediate reduction of DEPC-treated cytochrome b(561) by ascorbic acid, indicating that DEPC-inhibited cytochrome b(561) cannot accept electrons from a hydrogen-atom donor like ascorbate but can still accept electrons from an electron donor like ferrocyanide. Ascorbic acid reduces cytochrome b(561) with a K(m) of 1.0 +/- 0.2 mM and a V(max) of 4.1 +/- 0.8 s(-1) at pH 7.0. V(max)/K(m) decreases at low pH but is approximately constant at pH >7. The rate constant for oxidation of cytochrome b(561) by semidehydroascorbate decreases at high pH but is approximately constant at pH <7. This suggests that the active site must be unprotonated to react with ascorbate and protonated to react with semidehydroascorbate. Molecular modeling calculations show that hydrogen bonding between the 2-hydroxyl of ascorbate and imidazole stabilizes the ascorbate radical relative to the monoanion. These results are consistent with the following mechanism for ascorbate oxidation. (1) The ascorbate monoanion binds to an unprotonated site (histidine) on cytochrome b(561). (2) This complex donates an electron to reduce the heme. (3) The semidehydroascorbate anion dissociates from the cytochrome, leaving a proton associated with the binding site. (4) The binding site is deprotonated to complete the cycle. In this mechanism, an essential role of the cytochrome is to bind the ascorbate monoanion, which does not react by outer-sphere electron transfer in solution, and complex it in such a way that the complex acts as an electron donor. Thermodynamic considerations show that no steps in this process involve large changes in free energy, so the mechanism is reversible and capable of fulfilling the cytochrome's function of equilibrating ascorbate and semidehydroascorbate.     

Reaction mechanism of the Co2+-activated multifunctional bromoperoxidase-esterase from Pseudomonas putida IF-3.

The reaction mechanism of the Co2+-activated bromoperoxidase-esterase of Pseudomonas putida IF-3 was studied. Site-directed mutagenesis suggested that the serine residue of the catalytic triad conserved in serine hydrolases participates in the bromination and ester hydrolysis reactions. The enzyme released a trace amount of free peracetic acid depending on the concentration of H2O2, which had been considered the intermediate in the reaction of nonmetal haloperoxidases to oxidize halide ions to hypohalous acid. However, the formation of free peracetic acid could not explain the enzyme activation effect by Co2+ ions which completely depleted the free peracetic acid. In addition, the kcat value of the enzymatic bromination was 900-fold higher than the rate constant of free peracetic acid-mediated bromination. Those results strongly suggested that the peracetic acid-like intermediate formed at the catalytic site is the true intermediate and that the formation of free peracetic acid is only a minor reaction involving the enzyme. We propose the possible reaction mechanism of this multifunctional enzyme based on these findings.     

Cell Specific,Cross-Species Expression of Myrosinases in Brassica Napus,Arabidopsis Thaliana and Nicotiana Tabacum

A prototypical characteristic of the Brassicaceae is the presence of the myrosinase-glucosinolate system. Myrosinase, the only known S-glycosidase in plants, degrades glucosinolates, thereby initiating the formation of isothiocyanates, nitriles and other reactive products with biological activities. We have used myrosinase gene promoters from Brassica napus and Arabidopsis thaliana fused to the beta -glucuronidase (GUS) reporter gene and introduced into Arabidopsis thaliana, Brassica napus and/or Nicotiana tabacum plants to compare and determine the cell types expressing the myrosinase genes and the GUS expression regulated by these promoters. The A. thaliana TGG1 promoter directs expression to guard cells and phloem myrosin cell idioblasts of transgenic A. thaliana plants. Expression from the same promoter construct in transgenic tobacco plants lacking the myrosinase enzyme system also directs expression to guard cells. The B. napus Myr1.Bn1 promoter directs a cell specific expression to idioblast myrosin cells of immature and mature seeds and myrosin cells of phloem of B. napus. In A. thaliana the B. napus promoter directs expression to guard cells similar to the expression pattern of TGG1. The Myr1.Bn1 signal peptide targets the gene product to the reticular myrosin grains of myrosin cells. Our results indicate that myrosinase gene promoters from Brassicaceae direct cell, organ and developmental specific expression in B. napus, A. thaliana and N. tabacum.