Old Yellow Enzyme Research Articles

Crystal Structure of Bacterial Morphinone Reductase and Properties of the C191A Mutant Enzyme

The crystal structure of the NADH-dependent bacterial flavoenzyme morphinone reductase (MR) has been determined at 2.2-A resolution in complex with the oxidizing substrate codeinone. The structure reveals a dimeric enzyme comprising two 8-fold beta/alpha barrel domains, each bound to FMN, and a subunit folding topology and mode of flavin-binding similar to that found in Old Yellow Enzyme (OYE) and pentaerythritol tetranitrate (PETN) reductase. The subunit interface of MR is formed by interactions from an N-terminal beta strand and helices 2 and 8 of the barrel domain and is different to that seen in OYE. The active site structures of MR, OYE, and PETN reductase are highly conserved reflecting the ability of these enzymes to catalyze "generic" reactions such as the reduction of 2-cyclohexenone. A region of polypeptide presumed to define the reducing coenzyme specificity is identified by comparison of the MR structure (NADH-dependent) with that of PETN reductase (NADPH-dependent). The active site acid identified in OYE (Tyr-196) and conserved in PETN reductase (Tyr-186) is replaced by Cys-191 in MR. Mutagenesis studies have established that Cys-191 does not act as a crucial acid in the mechanism of reduction of the olefinic bond found in 2-cyclohexenone and codeinone.

X-Ray Structure of 12-Oxophytodienoate Reductase 1 Provides Structural Insight into Substrate Binding and Specificity within the Family of OYE

Background: 12-Oxophytodienoate reductase (OPR) is a flavin mononucleotide (FMN)-dependent oxidoreductase in plants that belongs to the family of Old Yellow Enzyme (OYE). It was initially characterized as an enzyme involved in the biosynthesis of the plant hormone jasmonic acid, where it catalyzes the reduction of the cyclic fatty acid derivative 9 S,13 S-12-oxophytodienoate (9 S,13 S-OPDA) to 1 S,2 S-3-oxo-2(2′[ Z]-pentenyl)-cyclopentane-1-octanoate. Several isozymes of OPR are now known that show different stereoselectivities with regard to the four stereoisomers of OPDA. Results: Here, we report the high-resolution crystal structure of OPR1 from Lycopersicon esculentum and its complex structures with the substrate 9 R,13 R-OPDA and with polyethylene glycol 400. OPR1 crystallizes as a monomer and folds into a (βα) 8 barrel with an overall structure similar to OYE. The cyclopentenone ring of 9 R,13 R-OPDA is stacked above the flavin and activated by two hydrogen bonds to His187 and His190. The olefinic bond is properly positioned for hydride transfer from the FMN N(5) and proton transfer from Tyr192 to Cβ and Cα, respectively. Comparison of the OPR1 and OYE structures reveals striking differences in the loops responsible for binding 9 R,13 R-OPDA in OPR1. Conclusions: Despite extensive biochemical characterization, the physiological function of OYE still remains unknown. The similar catalytic cavity structures and the substrate binding mode in OPR1 strongly support the assumption that α,β-unsaturated carbonyl compounds are physiological substrates of the OYE family. The specific binding of 9 R,13 R-OPDA by OPR1 explains the experimentally observed stereoselectivity and argues in favor of 9 R,13 R-OPDA or a structurally related oxylipin as natural substrate of OPR1.

Computational studies of electron-transfer processes in old yellow enzyme

Old Yellow Enzyme (OYE) is a flavoenzyme that was first isolated from brewer's bottom yeast. Homologues have been identified in other strains of yeast, bacteria, and plants. In plants, the OYE homologue functions enzymatically in the synthesis of plant hormones, but the biological function of OYE in yeast is still unknown. Flavin mononucleotide (FMN) is the cofactor that is noncovalently bound in the enzyme. OYE binds several phenolic ligands that serve as models for reactive biological substrates. These complexes have broad long-wavelength absorption bands, which have been ascribed to charge-transfer interactions, with the phenolate anion acting as the electron donor and the FMN as the acceptor [Abramovitz, A. S.; Massey, V. J Bio Chem 1976, 251, 5327–5336]. The computational characterization of these electronic transitions in the active site will help in understanding the biological processes in the enzyme. It was found that at several levels of computational methods, and through computationally mutating relevant amino acids, a charge-transfer process is occurring. This result agrees with previous experimental work and is consistent with all ultraviolet–visible spectrophotometric data. The preliminary results for the computational studies of these electron-transfer processes will be presented. © 2001 John Wiley & Sons, Inc. Int J Quantum Chem, 2001

A novel stress-inducible 12-oxophytodienoate reductase from Arabidopsis thaliana provides a potential link between Brassinosteroid-action and Jasmonic-acid synthesis

To isolate brassinosteroid (BR) inducible genes, a subtractive cDNA-cloning strategy was applied. One of the isolated genes encodes a plant homologue to yeast old yellow enzymes (OYE) with strong sequence similarity to two cloned 12-oxo-phytodienoic acid reductases ( OPR1 and OPR2 ) from A. thaliana and was termed 12-oxophytodienoate reductase 3 ( OPR3 ; accession number: AJ238149). The expression of the OPR3 gene is induced by brassinosteroids, jasmonic acid (JA), and by a variety of stimuli like UV-light, touch, wind, wounding, and application of a detergent. Recombinant OPR3 protein converts 12-oxophytodienoate (OPDA) into 12-oxo phytoenoic acid (OPC8: 0), indicating the participation of OPR3 in the biosynthesis of JA from linolenic acid via the Vick-Zimmerman-pathway. In plants, OPC8: 0 is inevitably metabolized to JA by three cycles of β-oxidation. Both OPDA and JA are signal molecules involved in developmental processes and stress responses. Depending on environmental or developmental conditions, OPR potentially regulates the ratio between these two signal molecules. The yeast old yellow enzymes act on various enones and phenols including steroids, catalyzing reduction and disproportionation reactions. Thus, in addition to OPDA to OPC8: 0 conversion, OPR3 might be involved in further biosynthetic or degradative pathways in plants. As OPR3 expression is increased through treatment with brassinosteroids, it provides a potential link between brassinosteroid action and JA synthesis. BRs may thus influence the stress responses of plants through stimulation of JA synthesis.

An Arabidopsis gene induced by wounding functionally homologous to flavoprotein oxidoreductases.

The regulation of genes in response to wounding is mediated in part by the octadecanoids 12-oxo-phytodienoic acid (OPDA), jasmonic acid (JA) and its methyl ester methyl jasmonate (MeJA). We identified, by differential display, an Arabidopsis gene (OPR3) induced after wounding. OPR3 is homologous to members of the flavin mononucleotide (FMN) binding proteins, including the old yellow enzyme (OYE) from yeast and 12-oxophytodienoate-10,11-reductase (OPR) from Arabidopsis. Transcripts of OPR3 rapidly accumulated in leaves after wounding and MeJA treatment, but they were detected in various tissues of unwounded plants at relatively low levels. Expression of the OPR3 gene was significantly reduced in wounded leaves of the coil mutant, indicating partial dependence on jasmonate perception for full induction of the gene. The recombinant protein of OPR3 cross-reacted with an antiserum raised against the OYE protein, and showed oxidation of beta-NADPH when OPDA or 15-deoxy-delta(12,14) prostaglandin J2 (PGJ2), an analogue of OPDA, was used as substrate. Beta-NADPH oxidation was not observed when MeJA, which lacks the double bond in the ketone ring, was used as substrate. The recombinant OPR3 protein also showed beta-NADPH oxidation activity in the presence of cyclohexenone, but not cyclohexanone, suggesting that the enzyme has specificity to cleavage of olefinic bonds in cyclic enones. The results show that the OPR3 gene product represents a new OPR of Arabidopsis induced after wounding.

A Homolog of Old Yellow Enzyme in Tomato: SPECTRAL PROPERTIES AND SUBSTRATE SPECIFICITY OF THE RECOMBINANT PROTEIN

A cDNA was isolated and characterized from a tomato shoot cDNA library, the deduced amino acid sequence of which exhibited similarity with yeast Old Yellow Enzymes (OYEs) and related enzymes of bacterial and plant origin. Sequence identity was particularly high with 12-oxophytodienoate 10,11-reductase (OPR) from Arabidopsis thaliana. The cDNA-encoded protein was expressed as a glutathione S-transferase fusion protein in Escherichia coli and was purified from bacterial extracts. The protein was found to be a flavoprotein catalyzing the NADPH-dependent reduction of the olefinic bond of alpha,beta-unsaturated carbonyl compounds, including 12-oxophytodienoic acid. Thus, the tomato enzyme was termed LeOPR. The catalytic efficiency of LeOPR was highest with N-ethylmaleimide followed by 12-oxophytodienoic acid and maleic acid as substrates. Photoreduction of the LeOPR-bound FMN resulted in the formation of a red, anionic semiquinone prior to the formation of the fully reduced flavin dihydroquinone. Spectroscopic characterization of LeOPR revealed the formation of charge transfer complexes upon titration with para-substituted phenolic compounds, a distinctive feature of the enzymes of the OYE family. The ligand binding properties were compared between LeOPR and OYE, and the findings are discussed with respect to structural differences between the active sites of OYE and LeOPR.

The role of threonine 37 in flavin reactivity of the old yellow enzyme.

Threonine 37 is conserved among all the members of the old yellow enzyme (OYE) family. The hydroxyl group of this residue forms a hydrogen bond with the C-4 oxygen atom of the FMN reaction center of the enzyme [Fox, K. M. & Karplus, P. A. (1994) Structure 2, 1089-1105]. The position of Thr-37 and its interaction with flavin allow for speculations about its role in enzyme activity. This residue was mutated to alanine and the mutant enzyme was studied and compared with the wild-type OYE1 to evaluate its mechanistic function. The mutation has different effects on the two separate half-reactions of the enzyme. The mutant enzyme has enhanced activity in the oxidative half-reaction but the reductive half-reaction is slowed down by more than one order of magnitude. The peaks of the absorption spectra for enzyme bound with phenolic compounds are shifted toward shorter wavelengths than those of wild-type OYE1, consistent with its lower redox potential. It is suggested that Thr-37 in the wild-type OYE1 increases the redox potential of the enzyme by stabilizing the negative charge of the reduced flavin through hydrogen bonding with it.

The Oxidative Half-reaction of Old Yellow Enzyme: THE ROLE OF TYROSINE 196

Tyrosine 196 in Old Yellow Enzyme (OYE) was mutated to phenylalanine, and the resulting mutant enzyme was characterized to evaluate the mechanistic role of the residue. The residue demonstrates little effect on ligand binding and the reductive half-reaction, but a dramatic slowing by nearly 6 orders of magnitude of its oxidative half-reaction with 2-cyclohexenone. Observation of the oxidative half-reaction with a series of substrates allows us to propose a model describing the mechanism of the oxidative half-reaction. In addition, the curtailed reactivity with enones allows for characterization of the manner in which reduced enzyme primes the substrate for the redox reaction by observation of the Michaelis complex with reduced enzyme bound to substrate.

On the Active Site of Old Yellow Enzyme: ROLE OF HISTIDINE 191 AND ASPARAGINE 194

Old Yellow Enzyme (OYE) binds phenolic ligands forming long wavelength (500-800 nm) charge-transfer complexes. The enzyme is reduced by NADPH, and oxygen, quinones, and alpha,beta-unsaturated aldehydes and ketones can act as electron acceptors to complete catalytic turnover. Solution of the crystal structure of OYE1 from brewer's bottom yeast (Fox, K. M., and Karplus, P. A. (1994) Structure 2, 1089-1105) made it possible to identify histidine 191 and asparagine 194 as amino acid residues that hydrogen-bond with the phenolic ligands, stabilizing the anionic form involved in charge-transfer interaction with the FMN prosthetic group. His-191 and Asn-194 are also predicted to interact with the nicotinamide ring of NADPH in the active site. Mutations of His-191 to Asn, Asn-194 to His, and a double mutation, H191N/N194H, were made of OYE1. It was not possible to isolate the N191H mutant enzyme, but the other two mutant forms had the expected effect on phenolic ligand binding, i.e. decreased binding affinity and decreased charge-transfer absorbance. Reduction of the H191N mutant enzyme by NADPH was similar to that of OYE1, but the reduction rate constant for NADH was greatly decreased. The double mutant enzyme had an increased rate constant for reduction by NADPH, but the reduction rate constant with NADH was lower by a factor of 15. The reactivity of OYE1 and the mutant enzymes with oxygen was similar, but the reactivity of 2-cyclohexenone was greatly decreased by the mutations. The crystal structures of the two mutant forms showed only minor changes from that of the wild type enzyme.

Binding and Reactivity of Candida albicans Estrogen Binding Protein with Steroid and Other Substrates

In this report recombinant estrogen binding protein (EBP1), isolated originally from Candida albicans as a result of its high affinity for 17beta-estradiol, has been purified extensively using a modified affinity purification scheme originally developed for a homolog of EBP1, old yellow enzyme (OYE). It is shown that like OYE, the protein binds a variety of compounds with a phenolic structure, including 17beta-estradiol, and compounds with an alpha, beta-unsaturated keto or aldehyde structure. In addition, EBP1 exhibits an NADPH oxidoreductase activity, transferring electrons from NADPH to all alpha,beta-unsaturated ketones and aldehydes tested via the tightly bound FMN cofactor. Analysis of the steady-state kinetics of these reactions indicate a tetra uni ping-pong mechanism. Inhibition of the steady-state reaction by 17beta-estradiol gives a Ki = 10 +/- 2 nM, and indicates exclusive binding of this steroid to the enzyme in its oxidized state. In contrast, 19-nortestosterone binds to both oxidized and reduced forms of the enzyme with dissociation constants of 600 +/- 100 and 650 +/- 90 nM, respectively. EBP1 also catalyzes a disproportionation reaction with certain compounds, in which two molecules of a cylic alpha,beta-unsaturated ketone, including the steroid 19-nortestosterone, are individually aromatized and reduced to the corresponding saturated ketone. Despite the extensive similarity in sequence and enzymic activity, notable differences between EBP1 and the OYE family of proteins exist with regard to the binding behavior and reactivity with the two steroids tested here, estradiol and 19-nortestosterone.

Synthesis and Properties of 8-CN-flavin Nucleotide Analogs and Studies with Flavoproteins

A high potential analog of riboflavin with a cyano function at the 8-position was synthesized by employing novel reaction conditions, starting from 8-amino-riboflavin. This was converted to the FAD level with FAD synthetase. The reduced 8-CN-riboflavin, unlike normal reduced flavin, has a distinctive absorption spectrum with two distinctive peaks in the near ultraviolet region. The oxidation-reduction potential of the new flavin was determined to be -50 mV, approximately 160 mV more positive than that of normal riboflavin. The 8-CN-riboflavin and 8-CN-FMN were found to be photoreactive and need to be protected from exposure to light. However such complications were not encountered with protein-bound flavins. The apoproteins of flavodoxin and Old Yellow Enzyme (OYE) were reconstituted with the 8-CN-FMN and apoDAAO was reconstituted with 8-CN-FAD. Spectral properties of the enzyme-bound neutral and anionic semiquinones were determined from these reconstituted proteins. In the case of 8-CN-FMN-OYE I, it was shown that the comproportionation reaction of a mixture of reduced and oxidized enzyme bound flavin is very rapid, compared with the same reaction with native protein, resulting in approximately 100% thermodynamically stable anionic semiquinone. In the case of 8-CN-OYE I, it was shown that the rate of reduction of the enzyme bound flavin by NADPH is approximately 40 times faster, and the rate of reoxidation of reduced enzyme bound flavin by oxygen is an order of magnitude slower than with the normal FMN enzyme. This is in accord with the high oxidation-reduction potential of the flavin, which thermodynamically stabilizes the reduced enzyme.

Molecular Cloning and Characterization of 12-Oxophytodienoate Reductase, an Enzyme of the Octadecanoid Signaling Pathway fromArabidopsis thaliana: STRUCTURAL AND FUNCTIONAL RELATIONSHIP TO YEAST OLD YELLOW ENZYME

Using partial amino acid sequence information for 12-oxophytodienoate-10,11-reductase obtained from Corydalis sempervirens we have cloned the homologous enzyme from Arabidopsis thaliana. The open reading frame of the cDNA encodes a polypeptide of 372 amino acids (Mr = 41,165) with significant similarity to the sequence of Old Yellow Enzyme from Saccharomyces carlsbergensis (Saito, K., Thiele, D. J., Davio, M., Lockridge, O., and Massey, V. (1991) J. Biol. Chem. 266, 20720-20724), a flavin (FMN)-protein catalyzing the NADPH-dependent reduction of the olefinic bond of alpha,beta-unsaturated carbonyls. Specifically, all residues required for binding of FMN in Old Yellow Enzyme are conserved in the A. thaliana sequence, as are all residues associated with catalytic activity. The enzyme was functionally expressed from its cDNA in Escherichia coli and thus proven to encode OPDA reductase. Further similarities of OPDA reductase and yeast Old Yellow Enzyme include their binding to and elution by reductant from N-(4-hydroxybenzoyl)aminohexyl-Sepharose, the immunoreactivity of yeast Old Yellow Enzyme with an antiserum raised against plant OPDA reductase and the demonstration that Old Yellow Enzyme is an active OPDA reductase. It is thus conceivable that the physiological role of Old Yellow Enzymes now known from bacteria, yeasts, and higher plants, is in oxylipin metabolism.

Flavoprotein structure and mechanism. 8. Structure-function relations for old yellow enzyme.

The past 5 years have seen tremendous progress in our knowledge of old yellow enzyme (OYE) as a number of OYEs have been cloned and expressed, a high-resolution crystal structure has been determined for one of these, and new substrates have been found that can be turned over by the enzyme. Together these studies do not yet define the physiological role of OYE, but they lead to significant new insights into the enzymatic properties and structure-function relations of OYE.

A New Old Yellow Enzyme of Saccharomyces cerevisiae

In 1993, the first gene of Old Yellow Enzyme (OYE) of Saccharomyces cerevisiae was cloned (Stott, K., Saito, K., Thiele, D. J., and Massey, V. (1993) J. Biol. Chem. 268, 6097-6106) and named OYE2 to distinguish it from the first OYE gene cloned from Saccharomyces carlsbergenesis (Saito, K., Thiele, D. J., Davio, M., Lockridge, O., and Massey, V. (1991) J. Biol. Chem. 266, 20720-20724). The analysis of an OYE2 deletion mutant suggested that S. cerevisiae had at least two OYE genes. In the present study, we cloned a new OYE species named OYE3 and analyzed the OYE3 protein expressed in Escherichia coli. OYE3 consists of 400 amino acid residues and its molecular mass calculated by electrospray mass spectrometry is 44,788 daltons, in good agreement with the value of 44,920 daltons predicted from the amino acid sequence derived from the DNA sequence. In the downstream region of the OYE3 gene, the cytochrome oxidase (COX10) gene exists with a 426-base pair intermediate sequence. Some of the physicochemical and kinetic properties of OYE2 and OYE3 have been determined. Although the two enzymes are clearly closely related, they show differences in ligand binding properties and in their catalytic activities with oxygen and cyclohexen-2-one as acceptors.

Old yellow enzyme at 2 Å resolution: overall structure, ligand binding, and comparison with related flavoproteins

Old yellow enzyme (OYE) was the first flavoenzyme purified, but its function is still unknown. Nevertheless, the NADPH oxidase activity, the flavin mononucleotide environment and the ligand-binding properties of OYE have been extensively studied by biochemical and spectroscopic approaches. Full interpretation of these data requires structural information. The crystal structures of oxidized and reduced OYE at 2 A resolution reveal an alpha/beta-barrel topology clearly related to trimethylamine dehydrogenase. Complexes of OYE with p-hydroxybenzaldehyde, beta-estradiol, and an NADPH analog show all three binding at a common site, stacked on the flavin. The putative NADPH binding mode is novel as it involves primary recognition of the nicotinamide mononucleotide portion. This work shows that the striking spectral changes seen upon phenol binding are due to close physical association of the flavin and phenolate. It also identifies the structural class of OYE and suggests that if NADPH is its true substrate, then OYE has adopted NADPH dependence during evolution.

Crystallization of Old Yellow Enzyme Illustrates an Effective Strategy for Increasing Protein Crystal Size

Old Yellow Enzyme (OYE) was the first flavoenzyme discovered, and has been widely used as a model flavoenzyme. Three crystal forms have been grown from natural protease-nicked OYE and one has been grown from intact recombinant OYE. The recombinant OYE crystals are best suited for structure determination and grow from a solution containing polyethylene glycol and MgCl 2. They belong to space group P4 32 12 with unit cell dimensions a= b=142·88 Å, c=43·01 Å and have a single OYE chain per asymmetric unit. In order to optimize the production of large single crystals, we have carried out a systematic study of crystal size versus drop volume. Crystallographic lore holds that the volume of the medium in which the crystals are grown should have a significant effect on the final size of the crystals produced. However, a systematic investigation of the degree to which the volume of the crystallization sample affects the final size of the crystals has not been reported. In order to investigate this phenomenon, drops were set up in both hanging and sandwich geometry at a variety of drop volumes, but otherwise identical crystallization conditions. A 60-fold increase in drop volume produced a 730-fold increase in crystal volume, and a simultaneous increase in the effective diffraction limit of the crystals from near 2·5 Å to well beyond 2·0 Å resolution. The dramatic crystal volume increase appears to be due both to the increased amount of protein and the slower equilibration rates associated with larger drops. Our results also emphasize that the quality of diffraction data may often be limited by crystal size rather than intrinsic crystal order even in the case of reasonably sized (200 to 400 μm) crystals.

Studies on the Flavin-Binding Region of Old Yellow Enzyme with an Active Site Probe, 8-Fluoro-8-Demethyl FMN<xref ref-type="fn" rid="fn1"><sup>1</sup></xref>

The brewer's yeast old yellow enzyme (OYE) was reconstituted with 8-fluoro-8-demethyl FMN (8F-FMN). The reconstituted enzyme exhibited absorption maxima at 355 and 450 nm in the visible region. This reconstituted enzyme underwent no further spectral changes, showing no evidence of modification in the flavin moiety. However, when the reconstituted enzyme was subjected to specific limited proteolysis with bovine alpha-chymotrypsin, gradual spectral changes were observed with disappearance of the 355- and 450-nm bands accompanied by the appearance of a new band at 496 nm. Identical spectral changes were observed when the proteolytically cleaved OYE (nicked OYE) was reconstituted with 8F-FMN. The process associated with these spectral changes was found to be unimolecular by kinetic analysis. Reverse-phase HPLC analysis revealed that these spectral changes resulted from covalent bond formation between 8F-FMN and the protein moiety after the proteolytic cleavage of the protein into 14K and 34K fragments. The reverse-phase HPLC monitored at 490 nm showed that the chromophore with 496 nm absorption maximum was covalently attached to the 14K fragment. The amino acid sequence analysis of the flavinylated 14K fragment together with that of the 14K fragment of native OYE indicated that the N-terminal leucine of the 14K fragment is the site of flavinylation. These findings imply that the amino group of the N-terminal leucine of the 14K fragment became available as the result of proteolysis and that this amino group nucleophilically attacked the 8-position of 8F-FMN, forming a covalent bond between the flavin moiety and the 14K fragment.(ABSTRACT TRUNCATED AT 250 WORDS)

The heterogeneity of brewer's yeast old yellow enzyme.

Heterogeneity of brewer's yeast old yellow enzyme (OYE) was found by anion-exchange high-performance liquid chromatography (HPLC) as well as by 13C-NMR spectroscopy of [4a-13C]FMN reconstituted into apo OYE. Though the OYE sample prepared according to the conventional procedure gave a single protein band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the OYE sample was found to consist of five species on anion-exchange HPLC. The 13C-NMR spectrum of the [4a-13C]FMN-reconstituted OYE gave multiple peaks corresponding to 4a-13C. This multiplicity indicates that this OYE preparation possesses heterogeneity in the environment surrounding FMN, i.e., the active site of OYE. The different species of OYE were separately obtained by preparative HPLC on an anion-exchange column. These species as well as the unresolved sample showed identical mobility on SDS-PAGE and similar but slightly different NADPH oxidase activities. This heterogeneity was shown not to have resulted from proteolytic modification during the conventional purification procedure, which includes autolysis of the yeast cells, since the enzyme extracted by mechanical destruction of the yeast cells in the presence of various protease inhibitors exhibited identical heterogeneity. The pure OYE forms obtained by preparative anion-exchange HPLC are homogeneous in the flavin environment as revealed by a single 13C-NMR signal for the [4a-13C]FMN-reconstituted species.

31P- and 13C-NMR studies on the flavin-protein and flavin-ligand interactions in brewer's yeast old yellow enzyme.

The 31P- and 13C-NMR spectra of old yellow enzyme (OYE) were measured. The 31P-NMR signal of FMN bound to apo OYE-I, one of the pure forms of OYE, was observed at a substantially lower field compared to that of free FMN. While the 31P-signal of free FMN is pH-titratable with a pK value of about 6.5, which corresponds to the monoanion-dianion transition of the phosphate group, the 31P-signal of FMN bound to OYE-I shows no pH-dependence at pH 5-9, indicating that the phosphate group of FMN bound to OYE-I is fixed in the dianionic form in the pH region of 5-9. Apo OYE(0), i.e., the OYE preparation obtained by the conventional method, was reconstituted with [2-13C]FMN or [4,10a-13C2]FMN, while apo OYE-I was reconstituted with [4a-13C]FMN. The 13C-NMR spectra of these reconstituted OYE species were measured in the absence and presence of phenolic compounds which form complexes with OYE. Each 13C-signal of the 13C-labeled FMN became broader in the bound state compared to the free state, indicating restriction of flavin mobility in the bound form. Complex formation of the reconstituted OYE species with p-bromophenol did not shift the 10a-13C signal but shifted the 2- and 4-13C signals slightly upfield, whereas the 4a-13C signal was shifted significantly upfield in the complexed form. This complex-induced upfield shift of the 4a-13C signal was measured with various p-substituted phenols.(ABSTRACT TRUNCATED AT 250 WORDS)

Reactivity of old yellow enzyme with alpha-NADPH and other pyridine nucleotide derivatives.

The reaction of Old Yellow Enzyme (OYE) with pyridine nucleotides has been examined using steady state kinetics, rapid reaction kinetics, and equilibrium binding. alpha-NADPH, beta-NADPH, and the acid breakdown products of NADPH all bind to oxidized OYE with dissociation constants below 1 microM. These complexes produce characteristic red shifts in the absorption spectrum of OYE. A similar red shift which occurs after multiple turnovers of OYE with NADPH has been found to be due to an impurity in the NADPH preparation, possibly an acid breakdown product. Anions such as chloride, acetate, azide, and phenolates compete with the pyridine nucleotides for binding to a common site in oxidized OYE. Anaerobic reduction of OYE by NADPH proceeds via two intermediates to establish a readily reversible equilibrium. In contrast to most other NADPH-dependent enzymes, both alpha- and beta-NADPH are capable of reducing OYE, and alpha-NADPH is more effective. Using beta-[4(R)-2H]NADPH, a primary deuterium isotope effect was observed in the reduction reaction. Results from rapid reaction and steady state studies showed that reduction of OYE was rate limiting in turnover. Consistent with this, the turnover number with alpha-NADPH was significantly higher than that with beta-NADPH.