A transient intermediate in the reaction catalyzed by (S)-mandelate dehydrogenase from Pseudomonas putida

(S)-Mandelate dehydrogenase from Pseudomonas putida is a member of a FMN-dependent enzyme family that oxidizes (S)-alpha-hydroxyacids to alpha-ketoacids. The reductive half-reaction consists of the steps involved in substrate oxidation and FMN reduction. In this study, we investigated the mechanism of this half-reaction in detail. At low temperatures, a transient intermediate was formed in the course of the FMN reduction reaction. This intermediate is characteristic of a charge-transfer complex of oxidized FMN and an electron-rich donor and is formed prior to full reduction of the flavin. The intermediate was not due to binding of anionic substrates or inhibitors. It was only observed with efficient substrates that have high k(cat) values. At higher temperatures, it was formed within the dead time of the stopped-flow instrument. The rate of formation of the intermediate was 3-4-fold faster than its rate of disappearance; the former had a larger isotope effect. This suggests that the charge-transfer donor is an electron-rich carbanion/enolate intermediate that is generated by the base-catalyzed abstraction of the substrate alpha-proton. This is consistent with the observation that the intermediate was not observed with the R277K and R277G mutants, which have been shown to destabilize the carbanion intermediate (Lehoux, I. E., and Mitra, B. (2000) Biochemistry 39, 10055-10065). Thus, the MDH reaction has two rate-limiting steps of similar activation energies: the formation and breakdown of a distinct intermediate, with the latter step being slightly more rate limiting. We also show that MDH is capable of catalyzing the reverse reaction, the reoxidation of reduced MDH by the product ketoacid, benzoylformate. The transient intermediate was observed during the reverse reaction as well, confirming that it is indeed a true intermediate in the MDH reaction pathway.     

Arginine 165/arginine 277 pair in (S)-mandelate dehydrogenase from Pseudomonas putida: role in catalysis and substrate binding

(S)-Mandelate dehydrogenase from Pseudomonas putida belongs to a FMN-dependent enzyme family that oxidizes (S)-alpha-hydroxyacids. Active site structures of three homologous enzymes, including MDH, show the presence of two conserved arginine residues in close juxtaposition (Arg165 and Arg277 in MDH). Arg277 has an important catalytic role; it stabilizes both the ground and transition states through its positive charge as well as a hydrogen bond [Lehoux, I. E., and Mitra, B. (2000) Biochemistry 39, 10055-10065]. In this study, we examined the role of Arg165 and the overall importance of the Arg165/Arg277 pair. Single mutants at Arg165 as well as double mutants at Arg165 and Arg277 were characterized. Our results show that Arg165 has a role similar to, but less critical than, that of Arg277. It stabilizes the transition state through its positive charge and the ground state through a charge-independent interaction, most likely, a hydrogen bond. Though the k(cat)s for the charge-conserved mutants, R165K and R277K, were only 3-5-fold lower than those of wild-type MDH (wtMDH), the k(cat) for R165K/R277K was approximately 350-fold lower. Thus, at least one arginine residue is required for the optimal substrate orientation and catalysis. Stopped-flow studies show that the FMN reduction step is completely rate-limiting for both wtMDH and the arginine mutants, with the possible exception of R165E. Substrate isotope effects indicate that the carbon-hydrogen bond-breaking step is only partially rate-limiting for wtMDH but fully rate-limiting for the mutants. pH profiles of R165M conclusively show that the pK(a) of 9.3 in free wtMDH does not belong to Arg165.     

(S)-Mandelate dehydrogenase from Pseudomonas putida: mechanistic studies with alternate substrates and pH and kinetic isotope effects

(S)-Mandelate dehydrogenase from Pseudomonas putida, a member of the flavin mononucleotide-dependent alpha-hydroxy acid oxidase/dehydrogenase family, oxidizes (S)-mandelate to benzoylformate. The enzyme was purified with a carboxy-terminal histidine tag. Steady-state kinetic parameters indicate that it preferentially binds large substrates. A good correlation was obtained between the kcat, the substrate kinetic isotope effect (KIE), and the pKa of the substrate alpha-proton. The kcat decreased and the KIE increased for substrates whose alpha-protons have pKas higher than that of mandelate. These results support a mechanism involving a carbanion intermediate but are difficult to reconcile with one involving a direct hydride transfer. pH effects on steady-state parameters were determined with (S)-mandelate and a slow substrate, (R,S)-3-phenyllactate. The kcat/Km pH profile shows that two groups with apparent pKas of 5.5 and 8.9 in the free enzyme are important for activity. These pKas are shifted to 5.1 and 9.6 on binding (S)-mandelate, as shown in the kcat pH profile. The pH dependence of the KIEs suggests that the residues with these pKas are involved in the alpha-carbon-hydrogen bond-breaking step. pH dependencies of the inhibition constants for competitive inhibitors identified these residues as histidine 274 and arginine 277. We propose that histidine 274 is the base that abstracts the substrate alpha-proton and arginine 277 is important for substrate binding as well as stabilization of the carbanion/enolate intermediate.     

Role of arginine 277 in (S)-mandelate dehydrogenase from Pseudomonas putida in substrate binding and transition state stabilization

(S)-Mandelate dehydrogenase from Pseudomonas putida is an FMN-dependent alpha-hydroxy acid dehydrogenase. Structural studies of two homologous enzymes, glycolate oxidase and flavocytochrome b(2), indicated that a conserved arginine residue (R277 in MDH) interacts with the product carboxylate group [Lindqvist, Y., Branden, C.-I., Mathews, F. S., and Lederer, F. (1991) J. Biol. Chem. 266, 3198-3207]. The catalytic role of R277 was investigated by site-specific mutagenesis together with chemical rescue experiments. The R277K, R277G, R277H, and R277L proteins were generated and purified in active forms. The k(cat) for the charge-conserved mutation, R277K, was only 4-fold lower than wt-MDH, but its K(m) value was 40-fold lower; in contrast, k(cat)s for R277G, R277H, and R277L were 400-1000-fold lower than for wt-MDH and K(m) values were 5-15-fold lower compared to R277K. The K(d)s for negatively charged competitive inhibitors were relatively unaffected in all four R277 mutants. The k(cat) for R277G could be enhanced by the addition of exogenous guanidines or imidazoles; the maximum rescued k(cat) was approximately 70% of the wt-MDH value. Only reagents that were positively charged and could function as hydrogen bond donors were effective rescue agents. Our results indicate that R277 plays a major role in transition state stabilization through its positive charge-consistent with a mechanism involving a carbanion intermediate. The positive charge has a relatively small contribution toward substrate binding. R277 also forms a specific hydrogen bond with both the substrate and the transition state; this interaction contributes significantly to the low K(m) for (S)-mandelate.     

Structure of an active soluble mutant of the membrane-associated (S)-mandelate dehydrogenase

The structure of an active mutant of (S)-mandelate dehydrogenase (MDH-GOX2) from Pseudomonas putida has been determined at 2.15 A resolution. The membrane-associated flavoenzyme (S)-mandelate dehydrogenase (MDH) catalyzes the oxidation of (S)-mandelate to give a flavin hydroquinone intermediate which is subsequently reoxidized by an organic oxidant residing in the membrane. The enzyme was rendered soluble by replacing its 39-residue membrane-binding peptide segment with a corresponding 20-residue segment from its soluble homologue, glycolate oxidase (GOX). Because of their amphipathic nature and peculiar solubilization properties, membrane proteins are notoriously difficult to crystallize, yet represent a large fraction of the proteins encoded by genomes currently being deciphered. Here we present the first report of such a structure in which an internal membrane-binding segment has been replaced, leading to successful crystallization of the fully active enzyme in the absence of detergents. This approach may have general application to other membrane-bound proteins. The overall fold of the molecule is that of a TIM barrel, and it forms a tight tetramer within the crystal lattice that has circular 4-fold symmetry. The structure of MDH-GOX2 reveals how this molecule can interact with a membrane, although it is limited by the absence of a membrane-binding segment. MDH-GOX2 and GOX adopt similar conformations, yet they retain features characteristic of membrane and globular proteins, respectively. MDH-GOX2 has a distinctly electropositive surface capable of interacting with the membrane, while the opposite surface is largely electronegative. GOX shows no such pattern. MDH appears to form a new class of monotopic integral membrane protein that interacts with the membrane through coplanar electrostatic binding surfaces and hydrophobic interactions, thus combining features of both the prostaglandin synthase/squaline-hopine cyclase and the C-2 coagulation factor domain classes of membrane proteins.     

Structural and kinetic analysis of catalysis by a thiamin diphosphate-dependent enzyme,benzoylformate decarboxylase

Benzoylformate decarboxylase is a member of the family of enzymes that are dependent on the cofactor thiamin diphosphate. A structure of this enzyme binding (R)-mandelate, a competitive inhibitor, suggests that at least two hydrogen bonds are formed between the substrate, benzoylformate, and active site side chains. The first is between the carboxylate group of benzoylformate and the hydroxyl group of S26, and the second is between carbonyl group of the substrate and an imidazole nitrogen of H70. Steady-state kinetic studies indicate that the catalytic parameters are strongly affected in three active site mutants, S26A, H70A, and H281A. The K(m) of S26A was increased most dramatically, 25-fold more than that of the wild-type enzyme, while the K(i) of (R)-mandelate was increased 100-fold, suggesting that the serine hydroxyl is important for substrate binding. The k(cat) of H70A is reduced more than 3 orders of magnitude, strongly implicating this residue in catalysis, and H281 showed significant, but smaller magnitude, effects on both K(m) and k(cat). Stopped-flow experiments using an alternative substrate, p-nitrobenzoylformate, lead to kinetic resolution of the fate of key thiamin diphosphate-bound intermediates. Together, the experimental results suggest the following roles for residues in the active site. The residue H70 is important for the protonation of the 2-alpha-mandelyl-ThDP intermediate, thereby assisting in decarboxylation, and for the deprotonation of the 2-alpha-hydroxybenzyl-ThDP intermediate, aiding product release. H281 is involved in protonation of the enamine. Surprisingly, S26 appears to be involved not only in substrate binding but also in other steps of the reaction.     

Spectroscopic and kinetic characterization of the bifunctional chorismate synthase from Neurospora crassa: evidence for a common binding site for 5-enolpyruvylshikimate 3-phosphate and NADPH

Chorismate synthase catalyzes the anti-1,4-elimination of the phosphate group and the C-(6proR) hydrogen from 5-enolpyruvylshikimate 3-phosphate to yield chorismate, a central building block in aromatic amino acid biosynthesis. The enzyme has an absolute requirement for reduced FMN, which in the case of the fungal chorismate synthases is supplied by an intrinsic FMN:NADPH oxidoreductase activity, i.e. these enzymes have an additional catalytic activity. Therefore, these fungal enzymes have been termed "bifunctional." We have cloned chorismate synthase from the common bread mold Neurospora crassa, expressed it heterologously in Escherichia coli, and purified it in a three-step purification procedure to homogeneity. Recombinant N. crassa chorismate synthase has a diaphorase activity, i.e. it catalyzes the reduction of oxidized FMN at the expense of NADPH. Using NADPH as a reductant, a reduced flavin intermediate was observed under single and multiple turnover conditions with spectral features similar to those reported for monofunctional chorismate synthases, thus demonstrating that the intermediate is common to the chorismate synthase-catalyzed reaction. Furthermore, multiple turnover experiments in the presence of oxygen have provided evidence that NADPH binds in or near the substrate (5-enolpyruvylshikimate 3-phosphate) binding site, suggesting that NADPH binding to bifunctional chorismate synthases is embedded in the general protein structure and a special NADPH binding domain is not required to generate the intrinsic oxidoreductase activity.     

13C-NMR studies on the reaction intermediates of porcine kidney D-amino acid oxidase reconstituted with 13C-enriched flavin adenine dinucleotide

The 13C-NMR spectra of the reaction intermediates of D-amino acid oxidase (DAO) were measured with DAO reconstituted with FAD in which the 2-, 4-, 4a-, and 10a-positions of the isoalloxazine moiety were selectively 13C-enriched. The reaction intermediates used include charge-transfer complexes of the oxidized DAO with substrate intermediates and those of the reduced enzyme with substrate intermediates. For the former type of complex, the reaction intermediates with beta-cyano-D-alanine (D-BCNA) and D-proline were used, while for the latter the purple intermediates with D-alanine and D-proline were chosen. The 13C-resonances of 2-13C in the reaction intermediates with D-BCNA and D-proline were downfield-shifted by about 1 ppm relative to the free oxidized DAO. The 4-13C signal for the DAO-D-BCNA intermediate was observed at 1.2 ppm upfield from that of the oxidized DAO, though that for DAO-D-proline intermediate showed no shift. These results suggest modulation of the hydrogen bondings at C(2) = 0 and/or C(4) = 0 in these reaction intermediates. Comparison of the 13C-resonances of reduced DAO with those of free reduced FMN in the neutral and anionic forms indicate that FAD in reduced DAO is in the anionic reduced form. The 4a-13C resonance of reduced DAO is upfield-shifted by about 3 ppm from that of free reduced anionic FMN. Comparison of the 13C-resonances for the purple intermediates with those of reduced FMN and reduced DAO indicate unequivocally that FAD in the purple intermediate is in the anionic reduced state. The 4a-13C resonances for the purple intermediates were substantially upfield-shifted (by 2.4 ppm with D-alanine and 1.9 ppm with D-proline) relative to reduced DAO. This indicates that the electron density, and hence the nucleophilicity, of the 4a-carbon is elevated in the purple intermediate relative to free reduced DAO. This leads to a model in which the oxidative half reaction proceeds via the reaction of molecular oxygen at the 4a-position of the reduced FAD in the purple intermediate. This provides a rational molecular basis for the oxidative half reaction by way of the purple intermediate prior to product release rather than by way of free reduced enzyme after product release.     

Reaction intermediate analogues for mandelate racemase: interaction between Asn 197 and the alpha-hydroxyl of the substrate promotes catalysis

Mandelate racemase (MR) catalyzes the interconversion of the enantiomers of mandelic acid, stabilizing the altered substrate in the transition state by 26 kcal/mol relative to the substrate in the ground state. To understand the origins of this binding discrimination, carboxylate-, phosphonate-, and hydroxamate-containing substrate and intermediate analogues were examined for their ability to inhibit MR. Comparison of the competitive inhibition constants revealed that an alpha-hydroxyl function is required for recognition of the ligand as an intermediate analogue. Two intermediate analogues, alpha-hydroxybenzylphosphonate (alpha-HBP) and benzohydroxamate, were bound with affinities approximately 100-fold greater than that observed for the substrate. Furthermore, MR bound alpha-HBP enantioselectively, displaying a 35-fold higher affinity for the (S)-enantiomer relative to the (R)-enantiomer. In the X-ray structure of mandelate racemase [Landro, J. A., Gerlt, J. A., Kozarich, J. W., Koo, C. W., Shah, V. J., Kenyon, G. L., Neidhart, D. J., Fujita, J., and Petsko, G. A. (1994) Biochemistry 33, 635-643], the alpha-hydroxyl function of the competitive inhibitor (S)-atrolactate is within hydrogen bonding distance of Asn 197. To demonstrate the importance of the alpha-hydroxyl function in intermediate binding, the N197A mutant was constructed. The values of k(cat) for N197A were reduced 30-fold for (R)-mandelate and 179-fold for (S)-mandelate relative to wild-type MR; the values of k(cat)/K(m) were reduced 208-fold for (R)-mandelate and 556-fold for (S)-mandelate. N197A shows only a 3.5-fold reduction in its affinity for the substrate analogue (R)-atrolactate but a 51- and 18-fold reduction in affinity for alpha-HBP and benzohydroxamate, respectively. Thus, interaction between Asn 197 and the substrate's alpha-hydroxyl function provides approximately 3.5 kcal/mol of transition-state stabilization free energy to differentially stabilize the transition state relative to the ground state.     

Role of glycine 81 in (S)-mandelate dehydrogenase from Pseudomonas putida in substrate specificity and oxidase activity

(S)-Mandelate dehydrogenase from Pseudomonas putida belongs to a FMN-dependent enzyme family that oxidizes (S)-alpha-hydroxyacids. Despite a high degree of sequence and structural similarity, this family can be divided into three subgroups based on the different oxidants utilized in the second oxidative half-reaction. Only the oxidases show high reactivity with molecular oxygen. Structural data indicate that the relative position of a peptide loop and the isoalloxazine ring of the FMN is slightly different in the oxidases compared to the dehydrogenases; the last residue on this loop is either an alanine or glycine. We examined the effect of the G81A, G81S, G81V, and G81D mutations in MDH on the overall reaction and especially on the suppression of activity with oxygen. G81A had a higher specificity for small substrates compared to that of wtMDH, though the affinity for (S)-mandelate was relatively unchanged. The rate of the first half-reaction was 20-130-fold slower for G81A and G81S; G81D and G81V had extremely low activity. Redox-potential measurements indicate that the reduction in activity is due to the decrease in electrophilicity of the FMN. The affinity for oxygen increased 10-15-fold for G81A and G81S relative to wtMDH; the rate of oxidation increased 2-fold for G81A. The increased reactivity with molecular oxygen did not correlate with the redox potentials and appears to primarily result from a higher affinity for oxygen. These results suggest that one of the ways the oxidase activity of MDH is controlled is through steric effects because of the relative positions of the FMN and the Gly81 loop.     

Hydrogen-1, carbon-13, and nitrogen-15 NMR spectroscopy of Anabaena 7120 flavodoxin: assignment of beta-sheet and flavin binding site resonances and analysis of protein-flavin interactions

Sequence-specific 1H and 13C NMR assignments have been made for residues that form the five-stranded parallel beta-sheet and the flavin mononucleotide (FMN) binding site of oxidized Anabaena 7120 flavodoxin. Interstrand nuclear Overhauser enhancements (NOEs) indicate that the beta-sheet arrangement is similar to that observed in the crystal structure of the 70% homologous long-chain flavodoxin from Anacystis nidulans [Smith et al. (1983) J. Mol. Biol. 165, 737-755]. A total of 62 NOEs were identified: 8 between protons of bound FMN, 29 between protons of the protein in the flavin binding site, and 25 between protons of bound FMN and protons of the protein. These constraints were used to determine the localized solution structure of the FMN binding site. The electronic environment and conformation of the protein-bound flavin isoalloxazine ring were investigated by determining 13C chemical shifts, one-bond 13C-13C and 15N-1H coupling constants, and three-bond 13C-1H coupling constants. The carbonyl edge of the flavin ring was found to be slightly polarized. The xylene ring was found to be nonplanar. Tyrosine 94, located adjacent to the flavin isoalloxazine ring, was shown to have a hindered aromatic ring flip rate.     

Thermodynamic analysis of the binding of oxidized and reduced FMN cofactor to Vibrio harveyi NADPH-FMN oxidoreductase FRP apoenzyme

The Vibrio harveyi NADPH-specific flavin reductase FRP follows a ping-pong mechanism but switches to a sequential mechanism in the luciferase-coupled reaction. The bound FMN co-isolated with FRP, while acting as a genuine cofactor in the single-enzyme reaction, functions in the luciferase-coupled reaction as a prebound substrate and is directly transferred to luciferase once it is reduced [Lei, B., and Tu, S.-C. (1998) Biochemistry 37, 14623-14629]. With the aim of better understanding the functions of FMN in the FRP holoenzyme, this study was undertaken to quantify and compare the thermodynamic properties of the binding of oxidized and reduced FMN by the FRP apoenzyme. By isothermal titration calorimetry (ITC) measurements in various buffers at pH 7.0 and 15-30 degrees C, the binding of FMN by apo-FRP was found to be noncooperative, exothermic, and primarily enthalpy driven. The binding free energy change (hence, the association constant) was nearly invariant over this temperature range. Significant conformational changes in FRP upon binding of FMN were indicated. Equilibrium bindings of reduced flavins by flavin-dependent proteins have rarely been studied. In this work, the thermodynamic properties of binding of reduced FMN by apo-FRP were found to closely resemble those of FMN binding under three sets of experimental conditions via ITC measurements and, in one case, fluorescence quenching. The kinetically deduced ping-pong mechanism of FRP is now supported by direct measurements of binding affinities of the oxidized and reduced FMN cofactors. These findings are also discussed in relation to the function of FRP as a reduced flavin donor in the FRP-luciferase couple.     

Transient kinetics and intermediates formed during the electron transfer reaction catalyzed by Candida albicans estrogen binding protein

The transient kinetics of the reaction of the estrogen binding protein (EBP1) from Candida albicans in which hydride is transferred from NADPH to trans-2-hexenal (HXL) in two half-reactions were analyzed using UV-visible spectrophotometric and fluorometric stopped-flow techniques. The simplest model of the first half-reaction involves four steps including very rapid, tight binding (K(d) 相似文献   

Perturbing the hydrophobic pocket of mandelate racemase to probe phenyl motion during catalysis

Mandelate racemase (MR, EC 5.1.2.2) from Pseudomonas putida catalyzes the Mg(2+)-dependent 1,1-proton transfer that interconverts the enantiomers of mandelate. Crystal structures of MR reveal that the phenyl group of all ground-state ligands is located within a hydrophobic cavity, remote from the site of proton abstraction. MR forms numerous electrostatic and H-bonding interactions with the alpha-OH and carboxyl groups of the substrate, suggesting that these polar groups may remain relatively fixed in position during catalysis while the phenyl group is free to move between two binding sites [i.e., the R-pocket and the S-pocket for binding the phenyl group of (R)-mandelate and (S)-mandelate, respectively]. We show that MR binds benzilate (K(i) = 0.67 +/- 0.12 mM) and (S)-cyclohexylphenylglycolate (K(i) = 0.50 +/- 0.03 mM) as competitive inhibitors with affinities similar to that which the enzyme exhibits for the substrate. Therefore, the active site can simultaneously accommodate two phenyl groups, consistent with the existence of an R-pocket and an S-pocket. Wild-type MR exhibits a slightly higher affinity for (S)-mandelate [i.e., K(m)(S)(-)(man) < K(m)(R)(-)(man)] but catalyzes the turnover of (R)-mandelate slightly more rapidly (i.e., k(cat)(R)(-->)(S) > k(cat)(S)(-->)(R)). Upon introduction of steric bulk into the S-pocket using site-directed mutagenesis (i.e., the F52W, Y54W, and F52W/Y54W mutants), this catalytic preference is reversed. Although the catalytic efficiency (k(cat)/K(m)) of all the mutants was reduced (11-280-fold), all mutants exhibited a higher affinity for (R)-mandelate than for (S)-mandelate, and higher turnover numbers with (S)-mandelate as the substrate, relative to those with (R)-mandelate. (R)- and (S)-2-hydroxybutyrate are expected to be less sensitive to the additional steric bulk in the S-pocket. Unlike those for mandelate, the relative binding affinities for these substrate analogues are not reversed. These results are consistent with steric obstruction in the S-pocket and support the hypothesis that the phenyl group of the substrate may move between an R-pocket and an S-pocket during racemization. These conclusions were also supported by modeling of the binary complexes of the wild-type and F52W/Y54W enzymes with the substrate analogues (R)- and (S)-atrolactate, and of wild-type MR with bound benzilate using molecular dynamics simulations.     

(S)-Mandelate dehydrogenase from Pseudomonas putida: mutations of the catalytic base histidine-274 and chemical rescue of activity.

(S)-Mandelate dehydrogenase from Pseudomonas putida, an FMN-dependent alpha-hydroxy acid dehydrogenase, oxidizes (S)-mandelate to benzoylformate. The generally accepted catalytic mechanism for this enzyme involves the formation of a carbanion intermediate. Histidine-274 has been proposed to be the active-site base that abstracts the substrate alpha-proton to generate the carbanion. Histidine-274 was altered to glycine, alanine, and asparagine. All three mutants were completely inactive. The mutants were able to form adducts with sulfite, though with much weaker affinity than the wild-type enzyme. Binding of the inhibitor, (R)-mandelate, was not greatly affected by the mutation, unlike that of the substrate, (S)-mandelate, indicating that H274 plays a role in substrate binding. The activity of H274G and, to a lesser extent, H274A could be partially restored by the addition of exogenous imidazoles. The maximum rescued activity for H274G with imidazole was approximately 0.1% of the wild-type value. Saturation kinetics obtained for rescued activity suggest that formation of a ternary complex of imidazole, enzyme, and substrate is required for catalysis. pH-dependence studies confirm that the free base form of imidazole is the rescue agent. An earlier study of pH profiles of the wild-type enzyme indicated that deprotonation of a residue with a pK(a) of 5.5 in the free enzyme was essential for activity (Lehoux, I. E., and Mitra, B. (1999) Biochemistry 38, 5836-5848). Data obtained in this work confirm that the pK(a) of 5.5 belongs to histidine-274.     

Structural requirements for transformation of substrates by the S-adenosyl-L-methionine:delta 24(25)-sterol methyltransferase. Inhibition by analogs of the transition state coordinate.

Microsomes from sunflower seedlings were used to investigate the transition state coordinate for the C-24 methylation reaction that mediates phytosterol biosynthesis. They were then used to study structurally related cationic and uncharged compounds of the natural sterol substrate, which were designed to interfere with the reaction progress. The hypothetical reaction course is described to proceed through an Sn2 formation of an activated complex involving the initial production of a covalent structure with a dative bond (methyl from AdoMet attacks si-face of the 24,25-double bond of the sterol) and the secondary production of a series of high energy intermediates, the stabilization of which determines the final C-24 methylated product. Derivatives of lanosterol and cholesterol with a methyl, hydrogen, oxygen, or bromine atom introduced into the side chain and/or at C-3 in place of the natural nucleophile were studied as inhibitors that interfere with the formation of the hypothetical tertiary isopropylcarbinyl cation intermediate in the conversion of cycloartenal to 24(28)-methylene cycloartanol. The data indicate the most potent inhibitor is a sterol with an aziridine group attached to C-24(25), which mimics the bridged C-24(25) carbenium ion generated in the transition state, and the methyltransferase possesses two strategic sites: one that recognizes the proximal end of the sterol acting as a proton donor and the other that recognizes the distal end that acts as a proton acceptor. The best fit (binding/catalysis) involves a flat sterol (including substrate and inhibitor) with intact unsubstituted nucleophilic centers at C-3 and C-24 and a freely rotating side chain that can assume the pseudocyclic conformation.     

Mechanism of flavin reduction in the alkanesulfonate monooxygenase system

The alkanesulfonate monooxygenase system from Escherichia coli is involved in scavenging sulfur from alkanesulfonates under sulfur starvation. An FMN reductase (SsuE) catalyzes the reduction of FMN by NADPH, and the reduced flavin is transferred to the monooxygenase (SsuD). Rapid reaction kinetic analyses were performed to define the microscopic steps involved in SsuE catalyzed flavin reduction. Results from single-wavelength analyses at 450 and 550 nm showed that reduction of FMN occurs in three distinct phases. Following a possible rapid equilibrium binding of FMN and NADPH to SsuE (MC-1) that occurs before the first detectable step, an initial fast phase (241 s(-1)) corresponds to the interaction of NADPH with FMN (CT-1). The second phase is a slow conversion (11 s(-1)) to form a charge-transfer complex of reduced FMNH(2) with NADP(+) (CT-2), and represents electron transfer from the pyridine nucleotide to the flavin. The third step (19 s(-1)) is the decay of the charge-transfer complex to SsuE with bound products (MC-2) or product release from the CT-2 complex. Results from isotope studies with [(4R)-(2)H]NADPH demonstrates a rate-limiting step in electron transfer from NADPH to FMN, and may imply a partial rate-limiting step from CT-2 to MC-2 or the direct release of products from CT-2. While the utilization of flavin as a substrate by the alkanesulfonate monooxygenase system is novel, the mechanism for flavin reduction follows an analogous reaction path as standard flavoproteins.     

Structure-function correlation of fatty acyl-CoA dehydrogenase and fatty acyl-CoA oxidase

We have employed a new pseudosubstrate, beta-(2-furyl)propionyl coenzyme A (FPCoA), to study the functional properties of two enzymes, fatty acyl-CoA dehydrogenase from porcine liver and fatty acyl-CoA oxidase from Candida tropicalis, involved in the oxidation of fatty acids. Previous studies from our laboratory have shown that the dehydrogenase exhibits oxidase activity at the rate of dissociation of the product charge-transfer complex. This raises the question of the difference in functionality between these two flavoproteins. To investigate these differences, we have compared the pH dependence of product formation, the isotope effects using tetradeuterio-FPCoA, and the spectral properties and chemical reactivity of the product charge-transfer complexes formed with the two enzymes. The pH dependencies of the reaction of FPCoA with electron-transfer flavoprotein (ETF) for the dehydrogenase and of the reaction of FPCoA with O2 for the oxidase are quite similar. Both reactions proceed more rapidly at basic pH values while substrate binds more tightly at acidic pH values. These data for both enzymes are consistent with a mechanism in which enzyme is involved in protonation of the carbonyl group of substrate followed by base-catalyzed removal of the C-2 proton from substrate. The C-2 anion of substrate may then serve as the active species in reduction of enzyme-bound flavin. The deuterium isotope effects for both enzyme systems are primary across the entire pH range, assuring that the chemically important step of substrate oxidation is rate limiting in these steady-state kinetic experiments. The two enzymes differ in the chemical reactivity of their product charge-transfer complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Identification of a magnesium-dependent NAD(P)(H)-binding domain in the nicotinoprotein methanol dehydrogenase from Bacillus methanolicus

The Bacillus methanolicus methanol dehydrogenase (MDH) is a decameric nicotinoprotein alcohol dehydrogenase (family III) with one Zn(2+) ion, one or two Mg(2+) ions, and a tightly bound cofactor NAD(H) per subunit. The Mg(2+) ions are essential for binding of cofactor NAD(H) in MDH. A B. methanolicus activator protein strongly stimulates the relatively low coenzyme NAD(+)-dependent MDH activity, involving hydrolytic removal of the NMN(H) moiety of cofactor NAD(H) (Kloosterman, H., Vrijbloed, J. W., and Dijkhuizen, L. (2002) J. Biol. Chem. 277, 34785-34792). Members of family III of NAD(P)-dependent alcohol dehydrogenases contain three unique, conserved sequence motifs (domains A, B, and C). Domain C is thought to be involved in metal binding, whereas the functions of domains A and B are still unknown. This paper provides evidence that domain A constitutes (part of) a new magnesium-dependent NAD(P)(H)-binding domain. Site-directed mutants D100N and K103R lacked (most of the) bound cofactor NAD(H) and had lost all coenzyme NAD(+)-dependent MDH activity. Also mutants G95A and S97G were both impaired in cofactor NAD(H) binding but retained coenzyme NAD(+)-dependent MDH activity. Mutant G95A displayed a rather low MDH activity, whereas mutant S97G was insensitive to activator protein but displayed "fully activated" MDH reaction rates. The various roles of these amino acid residues in coenzyme and/or cofactor NAD(H) binding in MDH are discussed.     

Disruption of distal interactions of Arg 262 and of substrate binding to Ser 52 affect catalysis of sheep liver cytosolic serine hydroxymethyltransferase

The crystal structure of human liver cytosolic recombinant serine hydroxymethyltransferase (hcSHMT) suggested that Ser53 and Arg 263 could participate in the reaction catalyzed by SHMT. The mutation of Arg262 (corresponding to Arg263 in hcSHMT) to "A" in sheep liver cytosolic SHMT (scSHMT) resulted in a 5-fold increase in Km for L-Ser and a 5-fold decrease in kcat compared to scSHMT. Further, in R262A SHMT-glycine complex, the peak at 343 nm (geminal diamine) was more pronounced, compared to wild-type enzyme. Stopped-flow studies showed that the rate constant for the formation of glycine-geminal diamine for R262A SHMT was also decreased. The rate of reaction, concentration of spectral intermediates, fluorescence excitation maximum of glycine geminal diamine and interaction with methoxyamine were altered in R262A SHMT. Although Arg263 in hcSHMT is located outside the PLP binding pocket, it positions Tyr73 for interaction with PLP, by forked H-bonding with the carbonyl groups of main chain residues, Asn71 and Lys72 of the other subunit of the tight dimer. Mutation of Arg262 to Ala and the consequent alteration in orientation of PLP leads to decreased catalytic efficiency. Ser53 (in hcSHMT) is in hydrogen bonding distance to one of the carboxylate oxygens of the amino acid substrate, which also interacts with Tyr83 and Arg402. Replacement of Ser53 with Cys (using 'O' software program) in the structure of hcSHMT resulted in disruption of these interactions, whereas replacement with Ala (S53A) only weakened the substrate interactions. There was a 10-fold increase in Km and 20-fold decrease in catalytic activity efficiency for S52C SHMT, whereas S52A SHMT retained 20% of the activity without change in Km for serine. These results suggest that S52 affects substrate binding and catalysis.