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The flavoprotein dihydroorotate dehydrogenase (DHOD) catalyzes the oxidation of dihydroorotate to orotate. Dihydrooxonate is an analogue of dihydroorotate in which the C5 carbon is substituted by a nitrogen atom. We have investigated dihydrooxonate as a substrate of three DHODs, each representing a distinct evolutionary class of the enzyme, namely the two family 1 enzymes from Lactococcus lactis, DHODA and DHODB, and the enzyme from Escherichia coli, which, like the human enzyme, belongs to family 2. Dihydrooxonate was accepted as a substrate although much less efficiently than dihydroorotate. The first half-reaction was rate limiting according to pre-steady-state and steady-state kinetics with different electron acceptors. Cysteine and serine have been implicated as active site base residues, which promote substrate oxidation in family 1 and family 2 DHODs, respectively. Mutants of DHODA (C130A) and E. coli DHOD (S175A) have extremely low activity in standard assays with dihydroorotate as substrate, but with dihydrooxonate the mutants display considerable and increasing activity above pH 8.0. Thus, the absence of the active site base residue in the enzymes seems to be compensated for by a lower pKa of the 5-position in the substrate. Oxonate, the oxidation product of dihydrooxonate, was a competitive inhibitor versus dihydroorotate, and DHODA was the most sensitive of the three enzymes. DHODA was reinvestigated with respect to product inhibition by orotate. The results suggest a classical one-site ping-pong mechanism with fumarate as electron acceptor, while the kinetics with ferricyanide is highly dependent on the detailed reaction conditions.

Mutants of Magnaporthe griseaharboring a defective gene for 1,3,8-trihydroxynaphthalene reductase retain the capability to produce scytalone, thus suggesting the existence of a second naphthol reductase that can catalyze the reduction of 1,3,6,8-tetrahydroxynaphthalene to scytalone within the fungal melanin biosynthetic pathway. The second naphthol reductase gene was cloned from M. griseaby identification of cDNA fragments with weak homology to the cDNA of trihydroxynaphthalene reductase. The amino acid sequence for the second naphthol reductase is 46% identical to that of trihydroxynaphthalene reductase. The second naphthol reductase was produced in Esherichia coliand purified to homogeneity. Substrate competition experiments indicate that the second reductase prefers tetrahydroxynaphthalene over trihydroxynaphthalene by a factor of 310; trihydroxynaphthalene reductase prefers trihydroxynaphthalene over tetrahydroxynaphthalene by a factor of 4.2. On the basis of the 1300-fold difference in substrate specificities between the two reductases, the second reductase is designated tetrahydroxynaphthalene reductase. Tetrahydroxynaphthalene reductase has a 200-fold larger Kifor the fungicide tricyclazole than that of trihydroxynaphthalene reductase, and this accounts for the latter enzyme being the primary physiological target of the fungicide. M. griseamutants lacking activities for both trihydroxynaphthalene and tetrahydroxynaphthalene reductases do not produce scytalone, indicating that there are no other metabolic routes to scytalone.

We report here the first three-dimensional structure of the D1 C-terminal processing protease (D1P), which is encoded by the ctpA gene. This enzyme removes the C-terminal extension of the D1 polypeptide of photosystem II of oxygenic photosynthesis. Proteolytic processing is necessary to allow the light driven assembly of the tetranuclear manganese cluster, which is responsible for photosynthetic water oxidation. The X-ray structure of the Scenedesmus obliquus enzyme has been determined at 1.8 Å resolution using the multiwavelength anomalous dispersion method. The enzyme is monomeric and is composed of three folding domains. The middle domain is topologically homologous to known PDZ motifs and is proposed to be the site at which the substrate C-terminus binds. The remainder of the substrate likely extends across the face of the enzyme, interacting at its scissile bond with the enzyme active site Ser 372 / Lys 397 catalytic dyad, which lies at the center of the protein at the interface of the three domains.

Yeast dihydroorotate dehydrogenase (DHOD) was purified 2800-fold to homogeneity from its natural source. Its sequence is 70% identical to that of the Lactococcus lactis DHOD (family IA) and the two active sites are nearly the same. Incubations of the yeast DHOD with dideuterodihydroorotate (deuterated in the positions eliminated in the dehydrogenation) as the donor and [14C]orotate as the acceptor revealed that the C5 deuteron exchanged with H2O solvent at a rate equal to the 14C exchange rate, whereas the C6 deuteron was infrequently exchanged with H2O solvent, thus indicating that the C6 deuteron of the dihydroorotate is sticky on the flavin cofactor. The pH dependencies of the steady-state parameters (kcat and kcat/Km) are similar, indicating that kcat/Km reports the productive binding of substrate, and the parameters are dependent on the donor–acceptor pair. The lower pKa values for kcat and kcat/Km observed for substrate dihydroorotate (around 6) in comparison to the values determined for dihydrooxonate (around 8) suggest that the C5 pro S hydrogen atom of dihydroorotate (but not the analogous hydrogen of dihydrooxonate), which is removed in the dehydrogenation, assists in lowering the pKa of the active site base (Cys133). The pH dependencies of the kinetic isotope effects on steady-state parameters observed for the dideuterated dihydroorotate are consistent with the dehydrogenation of substrate being rate limiting at low pH values, with a pKa value approximating that assigned to Cys133. Electron acceptors with dihydroorotate as donor were preferred in the following order: ferricyanide (1), DCPIP (0.54), Qo (0.28), fumarate (0.15), and O2 (0.035). Orotate inhibition profiles versus varied concentrations of dihydroorotate with ferricyanide or O2 as acceptors suggest that both orotate and dihydroorotate have significant affinities for the reduced and oxidized forms of the enzyme.

Famoxadone is a newly commercialized fungicide and potent Qo-site inhibitor of cytochrome bc1. The S-(−)-enantiomer of famoxadone (the active component) was synthesized by two routes and was analyzed computationally and by X-ray crystallography. The molecule displays an extended conformation with flexibility in the structure imparted by the two terminal phenyl groups. In the crystal lattice, intermolecular hydrogen bonds occur between the NH and the oxygen atoms of the heterocycle.

A new fungicide lead has been identified by enzyme screening of a focused combinatorial library. The lead compound 4Figure 1Inhibitors of scytalone dehydratase (SD)., a potent inhibitor of scytalone dehydratase (SD), exhibits fungicidal activity upon foliar application but does not show systemic activity. The X-ray crystal structure of the enzyme–inhibitor complex and an appreciation for the relationship between physical properties and systemic activity enabled us to rapidly improve upon this initial lead. The geminal halogen–methyl group combination was found to be optimal for interaction with the bounding serine and asparagine side-chain residues. Replacement of CF3with methyl was a key discovery, giving inhibitors with slightly diminished enzyme inhibition potency while significantly increasing systemic activity. Amides prepared from amines with 2,4-dichloro substitution on the phenyl ring gave the most potent enzyme inhibitors. Two compounds from this series showed systemic activity comparable to the commercial standard and were selected for outdoor testing in flooded plots which simulate rice paddies.

Lumazine synthase has a slow rate of catalysis: steady-state kcatvalues for the Escherichia coli, Magnaporthe grisea, and spinach enzymes are 0.024, 0.052, and 0.023 s−1, respectively, at pH 7.5 and 25°C. Following the formation of an imine connecting the two substrates 3,4-dihydroxy-2-butanone 4-phosphate and 4-ribitylamino-5-amino-2,6-dihydroxypyrimidine, there is a chemically difficult isomerization. Calculated estimates of the free energy barrier for the isomerization are equal to or greater than 15 kcal/mol at 25°C. Free energies calculated from the steady-state kcatvalues at 25°C for the E. coli, M. grisea, and spinach enzymes are 19.7, 19.2, and 19.7 kcal/mol, respectively. The single-turnover rate (presteady state) at pH 7.5 and 25°C for the M. griseaenzyme is 140-fold greater than the steady-state rate and it has a free energy barrier of 16.3 kcal/mol. In the pre-steady state the M. griseaenzyme has a pKaof 5.8, plausibly reporting the proposed general base of catalysis (His127). The M. griseaenzyme has an off rate of 0.37 s−1for its product, 6,7-dimethyl-8-ribityllumazine, approximately 7-fold higher than kcatand 20-fold lower than the single-turnover rate. The off rate for the product orthophosphate is about 1 s−1. Thus, for the M. griseaenzyme at pH 7.5 and 25°C, product dissociation is significantly rate limiting to the steady-state rate of catalysis, whereas the isomerization step limits the single turnover rate. The spinach and E. colienzymes display a significant lag in pre-steady state, suggesting that substrate association is significantly rate limiting for these catalysts. Temperature studies on the enzyme-catalyzed rates for the three enzymes indicate a dominating enthalpic term.

Two water molecules reside between inhibitors and active site residues of scytalone dehydratase. A molecular dynamics study is consistent with one water molecule binding less tightly than the other. Inhibitor binding studies with site-directed mutants indicate that the hydrogen bonding network around the less mobile water molecule contributes much greater binding energy than that around the more mobile one.

A method for the determination of inhibition constants for catalytically-debilitated mutant enzymes is described. The inhibitor is partitioned between the mutant and wild-type enzymes. Catalytic rates of the wild-type enzyme are used as the signal of inhibitor binding to the mutant enzyme. The method is validated with scytalone dehydratase, the Y50F mutant, and a potent inhibitor. The Kivalue for Y50F determined by this method is 0.49 ± 0.10 nM. The Kivalue determined using the Y50F catalytic report for inhibitor binding in the absence of wild-type enzyme is 0.20 ± 0.030 nM. The wild-type enzyme binds the inhibitor ten-fold less tightly, thus indicating that the hydrogen-bonding interaction between the Y50 hydroxyl group and the inhibitor (suggested by X-ray crystallography) is weak. The method is most useful when the catalytic activity of the wild-type enzyme is the most sensitive report of inhibitor binding and the mutant enzyme is greatly crippled in catalytic activity.

The catalytic mechanism of scytalone dehydratase was examined by studying alternative substrates and site-directed mutations of active-site residues. Searches for an enol intermediate by looking for a half-reaction with authentic scytalone and 3,4-dihydro-6,8-dihydroxy-1-(2H)-2-[¹³C]naphthalenone were negative. An alternative substrate, 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one (DDBO), was nearly equal to scytalone as substrate for the enzyme, and DDBO's anomeric effect in stabilizing a partial carbocation center at C3 does not substantially contribute to the mechanism. Kinetic analysis of site-directed mutations of active-site amino acid side chains within the enzyme's active site provided an account for the role of these residues in the enzyme-catalyzed dehydration reactions. A concerted E2 elimination for the catalytic mechanism is proposed. © 1999 Society of Chemical Industry

Famoxadone is a preventative and curative fungicide recently developed for plant disease control. The molecule and its oxazolidinone analogs (OADs) are potent inhibitors of mitochondrial electron transport, specifically inhibiting the function of the enzyme ubiquinol:cytochrome c oxidoreductase (cytochrome bc1). Visible absorbance spectral studies on the purified enzyme suggested that famoxadone bound close to the low potential heme of cytochrome b. This binding mode was confirmed in competitive binding experiments by studying the displacement of a radiolabelled OAD from submitochondria. EPR studies on the binding of famoxadone to submitochondria and purified bc1 suggested its binding mode was more like that of myxothiazol than that of stigmatellin (ligands known to bind near the low potential heme). Zoospores of Phytophthora infestans, when given low concentrations of famoxadone and other OADs, were observed to cease oxygen consumption and motility within seconds and later the cells disintegrated, releasing the cellular contents. Famoxadone was a potent inhibitor of the growth of Saccharomyces cerevisiae when grown on non-fermentable carbon sources and it was an approximately 50-fold less potent inhibitor of growth when the yeast was grown on a fermentable carbon source, glucose. Such physiological observations are consistent with the loss of mitochondrial function imposed by famoxadone and OADs. Single amino acid changes in the apocytochrome b of baker's yeast cytochrome b located near the low potential heme altered the inhibition constants for the inhibitors famoxadone, myxothiazol, azoxystrobin and kresoxim-methyl differentially, thus strongly suggesting different binding interactions of the protein with the inhibitors. © 1999 Society of Chemical Industry

Lumazine synthase, which catalyzes the penultimate step of riboflavin biosynthesis, has been cloned from three higher plants (spinach, tobacco, and arabidopsis) through functional complementation of an Escherichia coliauxotroph. Whereas the three plant proteins exhibit some structural similarities to known microbial homologs, they uniquely possess N-terminal polypeptide extensions that resemble typical chloroplast transit peptides. In vitroprotein import assays with intact chloroplasts and immunolocalization experiments verify that higher plant lumazine synthase is synthesized in the cytosol as a larger molecular weight precursor protein, which is post-translationally imported into chloroplasts where it is proteolytically cleaved to its mature size. The authentic spinach enzyme is estimated to constitute <0.02% of the total chloroplast protein. Recombinant “mature” spinach lumazine synthase is expressed in E. coliat levels exceeding 30% of the total soluble protein and is readily purified to homogeneity using a simple two-step procedure. ApparentVmaxand Kmvalues obtained with the purified plant protein are similar to those reported for microbial lumazine synthases. Electron microscopy and hydrodynamic studies reveal that native plant lumazine synthase is a hollow capsid-like structure comprised of 60 identical 16.5-kDa subunits, resembling its icosahedral counterparts in E. coliand Bacillus subtilis.

Scytalone dehydratase is a molecular target of inhibitor design efforts aimed at preventing the fungal disease caused by Magnaporthe grisea. A method for cocrystallization of enzyme with inhibitors at neutral pH has produced several crystal structures of enzyme–inhibitor complexes at resolutions ranging from 1.5 to 2.2 Å. Four high resolution structures of different enzyme–inhibitor complexes are described. In contrast to the original X‐ray structure of the enzyme, the four new structures have well‐defined electron density for the loop region comprising residues 115–119 and a different conformation between residues 154 and 160. The structure of the enzyme complex with an aminoquinazoline inhibitor showed that the inhibitor is in a position to form a hydrogen bond with the amide of the Asn131 side chain and with two water molecules in a fashion similar to the salicylamide inhibitor in the original structure, thus confirming design principles. The aminoquinazoline structure also allows for a more confident assignment of donors and acceptors in the hydrogen bonding network. The structures of the enzyme complexes with two dichlorocyclopropane carboxamide inhibitors showed the two chlorine atoms nearly in plane with the amide side chain of Asn131. The positions of Phe53 and Phe158 are significantly altered in the new structures in comparison to the two structures obtained from crystals grown at acidic pH. The multiple structures help define the mobility of active site amino acids critical for catalysis and inhibitor binding. Proteins 1999;35:425–439. © 1999 Wiley‐Liss, Inc.

Scytalone dehydratase is a molecular target of inhibitor design efforts aimed at preventing the fungal disease caused by Magnaporthe grisea. A method for cocrystallization of enzyme with inhibitors at neutral pH has produced several crystal structures of enzyme–inhibitor complexes at resolutions ranging from 1.5 to 2.2 Å. Four high resolution structures of different enzyme–inhibitor complexes are described. In contrast to the original X-ray structure of the enzyme, the four new structures have well-defined electron density for the loop region comprising residues 115–119 and a different conformation between residues 154 and 160. The structure of the enzyme complex with an aminoquinazoline inhibitor showed that the inhibitor is in a position to form a hydrogen bond with the amide of the Asn131 side chain and with two water molecules in a fashion similar to the salicylamide inhibitor in the original structure, thus confirming design principles. The aminoquinazoline structure also allows for a more confident assignment of donors and acceptors in the hydrogen bonding network. The structures of the enzyme complexes with two dichlorocyclopropane carboxamide inhibitors showed the two chlorine atoms nearly in plane with the amide side chain of Asn131. The positions of Phe53 and Phe158 are significantly altered in the new structures in comparison to the two structures obtained from crystals grown at acidic pH. The multiple structures help define the mobility of active site amino acids critical for catalysis and inhibitor binding. Proteins 1999;35:425–439. © 1999 Wiley-Liss, Inc.

A new fungicide lead has been identified by in vitro screening of a focused combinatorial library. Amides (768) were synthesized in pools of four and assayed as inhibitors of scytalone dehydratase. Deconvolution of one of the most active pools led to the discovery of a potent inhibitor of the enzyme 3b(Ki= 26 pM), which has fungicidal properties.

We have synthesized an alternate substrate for trihydroxynaphthalene reductase (3HNR) and scytalone dehydratase (SD), two enzymes in the fungal melanin biosynthetic pathway. The oxidation of 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one (DDBO) to 4,5-dihydroxy-2H-benzopyran-2-one (DBO) with concomitant reduction of NADP+is catalyzed by 3HNR. DDBO is dehydrated by SD to 5-hydroxy-4H-1-benzopyran-4-one (HBO). These reactions can be monitored using continuous spectrophotometric assays. DDBO racemizes rapidly, so chiral synthesis to mimic the natural substrate is not required. DDBO, DBO, and HBO are stable in aerated aqueous solution, in contrast to the rapidly autooxidizing trihydroxynaphthalene, a physiological substrate for 3HNR and product of SD. Unlike the natural substrates, DDBO, DBO, and HBO do not change protonation state between pH's 4 and 9. Oxidation of DDBO is effectively irreversible at pH 7, as DBO deprotonates with a pKaof 2.5. At pH 7.0 and 25°C, thekcatfor 3HNR catalyzed DDBO oxidation is 14 s−1and theKmis 5 μM; thekcatfor SD catalyzed DDBO dehydration is 400 s−1and theKmis 15 μM. Based on these kinetic constants, DDBO is a better substrate than the natural substrate scytalone for both 3HNR and SD at neutral pH. An explanation for the preference of DDBO over scytalone in the oxidation and dehydration reactions is offered.

We describe a multiwavelength method for measuring an enzyme's discrimination of one substrate over another by continuously monitoring the reactions of the two substrates simultaneously. This method is generally applicable to ultraviolet–visible diode array or rapid-scanning spectrophotometers and the measurement requires a single incubation of enzyme with two substrates. Rates at each of the wavelengths may be fit globally by using a nonlinear least-squares fitting procedure which provides adequate statistical analysis. The specificity of trypsin forN-α-benzoyl-l-argininep-nitroanilide (BRpNA) overN-t-butyloxycarbonyl-l-alanine-p-nitrophenylester (BocApNP) was 2.1 as measured by the multiwavelength partition method and 2.3 by comparing the individualkcat/Km's for the two substrates. Multiwavelength analysis was applied to two enzymes in the biosynthetic pathway for fungal melanin: scytalone dehydratase and trihydroxynaphthalene reductase fromMagnaporthea grisea.The specificity of trihydroxynaphthalene reductase for 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one compared toscytalone, a natural substrate for the enzyme, was 95. Scytalone dehydratase was eight-fold more specific for 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one than it was for scytalone. Multiwavelength analysis was also used to measure an equilibrium constant of 0.040 for the reaction {dihydroorotate + oxonic acid ↔ orotate + dihydrooxonic acid} catalyzed by dihydroorotate dehydrogenase. Advantages, limitations, and further applications of this steady-state method, which directly measures relative substrate specificities, are delineated. All studies described in this paper were at pH 7.0 and 25°C.

The substrate specificity factor, VcKo/VoKc, of spinach (Spinacia oleracea L.) ribulose 1,5-bisphosphate carboxylase/oxygenase was determined at ribulosebisphosphate concentrations between 0.63 and 200 µM, at pH values between 7.4 and 8.9, and at temperatures in the range of 5° C to 40° C. The CO2/O2 specificity was the same at all ribulosebisphosphate concentrations and largely independent of pH. With increasing temperature, the specificity decreased from values of about 160 at 5° C to about 50 at 40° C. The primary effects of temperature were on Kc [Km(CO2)] and Vc [Vmax (CO2)], which increased by factors of about 10 and 20, respectively, over the temperature range examined. In contrast, Ko [Ki (O2)] was unchanged and Vo [Vmax (O2)] increased by a factor of 5 over these temperatures. The CO2 compensation concentrations (G) were calculated from specificity values obtained at temperatures between 5° C and 40° C, and were compared with literature values of G. Quantitative agreement was found for the calculated and measured G values. The observations reported here indicate that the temperature response of ribulose 1,5-bisphosphate carboxylase/oxygenase kinetic parameters accounts for two-thirds of the temperature dependence of the photorespiration/photosynthesis ratio in C3 plants, with the remaining one-third the consequence of differential temperature effects on the solubilities of CO2 and O2.

Polypeptide D1 of the photosystem II reaction center of oxygenic photosynthesis is expressed in precursor form (pre-D1), and it must be proteolytically processed at its C terminus to enable assembly of the manganese cluster responsible for photosynthetic water oxidation. A rapid and highly sensitive enzyme-linked immunosorbent assay-based microtiter plate method is described for assaying this D1 C-terminal processing protease. A protocol is described for the isolation and purification to homogeneity of the enzyme from the green alga, Scenedesmus obliquus. Amino acid sequence information on the purified protease was used to clone the corresponding gene, the translated sequence of which is presented. A comparison of the gene product with homologous proteases points to a region of conserved residues that likely corresponds to the active site of a new class of serine protease. The LF-1 mutant strain ofScenedesmus(isolated by Dr. Norman Bishop) is incapable of processing pre-D1. We show here that the C-terminal processing protease gene in this strain contains a single base deletion that causes a frame shift and a premature stop of translation within the likely active site of the enzyme. A suppressor strain, LF-1-RVT-1, which is photoautotrophic and capable of processing pre-D1 has a nearby single base insertion that restores the expression of active enzyme. These observations provide the first definitive proof that the enzyme isolated is responsible for in vivoproteolytic processing of pre-D1 and that no other protease can compensate for its loss.

Famoxadone is a preventative and curative fungicide recently commercialized for plant-disease control. The molecule and its oxazolidinone analogs are potent inhibitors of mitochondrial ubiquinol : cytochrome c oxidoreductase (cytochrome bc1) and they bind in the Q0 site of the enzyme near the low potential heme of cytochrome b. Inhibitor binding constants for five mutant cytochrome bc1 enzymes from Saccharomyces cerevisiae having single amino acid changes in their apocytochrome b located near the low potential heme were compared with their two parental wild-type enzymes. The five individual amino acid changes altered the inhibition constants for the inhibitors famoxadone, myxothiazol, azoxystrobin, and kresoxim-methyl in dissimilar fashion. The log scale differences in binding constants relative to those of their parentals provide fingerprints for the effects of the amino acid changes on binding of the individual inhibitors, thus reflecting the structural diversity of the inhibitors. ©1999 Society of Chemical Industry

The balance between photosynthesis and photorespiration in many species, including most crop plants, is determined by the kinetic properties of ribulose-l,5-bisphosphate (RuBP) carboxylase/oxygenase1,2. Photosynthesis is initiated by the carboxylase activity3while the oxygenase activity catalyses the first reaction in the photorespiratory pathway1,2,4. In these reactions, CO2and O2are competitive substrates2. Because O2inhibits carboxylation, and photorespiration oxidizes reduced carbon to CO2with no known benefit to the plant, it has been suggested that photosynthetic efficiency and thus productivity might be increased by chemical or genetic alterations of the enzyme which increase carboxylation or decrease oxygenation5–7. On the other hand, it has been argued that RuBP carboxylase/oxygenase cannot completely discriminate between CO2and O2, so that photorespiration is unavoidable8. From analyses of RuBP carboxylase/oxygenase enzymes purified from several different species, we report here large differences in specificity towards the substrates CO2and O2. Evolutionary pressures seem to have directed the enzyme towards more efficient utilization of CO2.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.