A study on the kinetic mechanism of apoenzyme reconstitution from Aerococcus viridans lactate oxidase

The preparation of a reconstitutable apoprotein is widely recognized as an important tool for studying the interactions between protein and coenzyme and also for characterizing the coenzyme-binding site of the protein. Here is described the kinetic analysis of the reconstitution of Aerococcus viridans lactate oxidase apoenzyme with FMN and FAD in the presence of substrate. The reconstitution was followed by measuring the increase in catalytic capacity with time. Lactate oxidase activity was easily removed by obtaining its apoenzyme in an acidic saturated ammonium sulphate solution. When the apoenzyme was reconstituted by the addition of FMN or FAD, a marked lag period was observed, after which the system reached a steady state (linear rate). To explain the binding mechanism of the cofactors to the apoenzyme, a kinetic model is proposed, in which the constants, k3 and k-3, representing the interaction of apoenzyme with cofactor are considered slow and responsible for the lag in the expression of activity. The affinity of apoenzyme was 51-fold higher for FMN than FAD.     

Polyamine oxidase from Zea mays shoots

Polyamine oxidase of maize shoots purified 10-fold had a pH optimum of 6·3 with spermidine as substrate, and K_m of 6 × 10^?4 M. The enzyme was inhibited by the acridine compounds quinacrine, 6,9-diamino-2-ethoxyacridine and acriflavin, but carbonyl reagents, typical thiol inhibitors and copper-binding agents were without effect. Inhibition by quinacrine was reversed by FMN and FAD. Furthermore, about 50 % of the activity of the apoenzyme was restored by the addition of FAD, but not by FMN or riboflavin, indicating that the maize polyamine oxidase is an FAD-dependent flavoprotein.     

Comparative fluorescence studies of native and crosslinked glucose oxidase

The binding characteristics of flavin adenine dinucleotide (FAD) to apoenzyme preparations obtained from native and intramolecularly crosslinked glucose oxidase were determined and compared. The dissociation constants K_diss as well as rates of recombination of FAD with the two apoenzyme preparations, were independently evaluated from fluorescence quenching of either the tryptophans of FAD. The K_diss values thus obtained were <10^?19M for native glucose oxidase and 4 ± 1 × 10^?7M for the crosslinked enzyme. The recombination of apo glucose oxidase with FAD, which is presumably diffusion controlled, is followed by an apparent first order decrease in fluorescence intensity of both the protein tryptophans and FAD, with a rate constant around 0.2 min^?1. This could be related to conformational changes which occur immediately after binding of FAD to the apoenzyme, an interpretation which is supported by the markedly different results obtained in the analogous experiments with the crosslinked enzyme. A model for the conformational characteristics of glucose oxidase, based on this study, is proposed.     

Molecular and Enzymatic Properties of Pyridoxamine (Pyridoxine) 5′-Phosphate Oxidase from Baker’s Yeast

Pyridoxamine (pyridoxine) 5′-phosphate oxidase purified from baker’s yeast was found to have a molecular weight of ca, 55,000 daltons based on polyacrylamide gel electrophoresis. The size of the enzyme subunit was analyzed by gel electrophoresis in the presence of sodium dodecylsulfate. This showed that the enzyme was composed of two nonidentical subunits with a molecular weight of 27,000 and 25,000 daltons. Fluorescence titration of the apoenzyme with FMN suggested that the holoenzyme contained one mol of FMN per mol of the enzyme. The K_m value of FMN for apoenzyme was calculated to be ca. 16 nm on both activities of pyridoxamine 5′-phosphate oxidase and pyridoxine 5′-phosphate oxidase.     

Electrochemical and enzymatic activity of flavin adenine dinucleotide and glucose oxidase immobilized by adsorption on carbon

Flavin adenine dinucleotide (FAD) and glucose oxidase were adsorbed on medium porosity spectroscopic graphite (SG) and on low porosity glassy carbon (GC) with retention of electrochemical activity, as measured by cyclic and differential pulse voltammetry. Adsorption on the SG was very strong, while that on GC was much weaker. Enzyme activity could be partially restored by the addition of the apoenzyme of glucose oxidase to the SG-adsorbed FAD preparation. The holoenzyme of glucose oxidase also was adsorbed on SG with retention of enzyme activity. The mechanism for the reconstitution of active enzyme from adsorbed FAD and soluble apoenzyme is not clear. The data suggest that the reconstituted enzyme stays adsorbed to the SG, but it is not clear whether the FAD or protein portions (or both) are adsorbed after reconstitution. The data also indicate that substrate mass transfer resistance may be important with the reconstituted-adsorbed enzyme.     

Reconstitution of native Escherichia coli pyruvate oxidase from apoenzyme monomers and FAD

Pyruvate oxidase, a tetrameric enzyme consisting of 4 identical subunits, dissociates into apoenzyme monomers and free FAD when treated with acid ammonium sulfate in the presence of high concentrations of potassium bromide. Reconstitution of the native enzymatically active protein can be accomplished by incubating equimolar concentrations of apomonomers and FAD at pH 6.5. The kinetics of the reconstitution reaction have been measured by 1) enzyme activity assays, 2) spectrophotometric assays to measure FAD binding, and 3) high performance liquid chromatography analysis measuring the distribution of monomeric, dimeric, and tetrameric species during reconstitution. The kinetic analysis indicates that the second order reaction of apomonomers with FAD to form an initial monomer-FAD complex is fast. The rate-limiting step for enzymatic reactivation appears to be the folding of the polypeptide chain in the monomer-FAD complex to reconstitute the three-dimensional FAD binding site prior to subunit reassociation. The subsequent formation of native tetramers appears to proceed via an essentially irreversible dimer assembly pathway.     

The recognition of glycolate oxidase apoprotein with flavin analogs in higher plants

The net photosynthetic efficiency in C3 plants (such asrice, wheat and other major crops) can be decreased by30% due to the metabolism of photorespiration [1], inwhich glycolate oxidase (GO) serves as a key enzyme. Itis known that GO, with flavin mononucleotide (FMN) asa cofactor, belongs to flavin oxidase [2]. But it differs fromother flavoproteins in that FMN is loosely bound to itsapoprotein and there exists a dissociation balance betweenthem, which indicates that FMN probably regulate…     

Study of the thermal stability of D-amino acid oxidase from Trigonopsis variabilis reveals enzyme inactivation via multiple steps

The thermal stability of a highly purified preparation of D-amino acid oxidase from Trigonopsis variabilis (TvDAO), which does not show microheterogeneity due to the partial oxidation of Cys-108, was studied based on dependence of temperature (20–60°C) and protein concentration (5–100 µmol L^?1). The time courses of loss of enzyme activity in 100 mmol L^?1 potassium phosphate buffer, pH 8.0, are well described by a formal kinetic mechanism in which two parallel denaturation processes, partial thermal unfolding and dissociation of the FAD cofactor, combine to yield the overall inactivation rate. Estimates from global fitting of the data revealed that the first-order rate constant of the unfolding reaction (k_a) increased 10⁴-fold in response to an increase in temperature from 20 to 60°C. The rate constants of FAD release (k_b) and binding (k_?b) as well as the irreversible aggregation of the apo-enzyme (k_agg) were less sensitive to changes in temperature, their activation energy (E_a) being about 52 kJ mol^?1 in comparison with an E_a value of 185 kJ mol^?1 for k_a. The rate-determining step of TvDAO inactivation switched from FAD dissociation to unfolding at high temperatures. The model adequately described the effect of protein concentration on inactivation kinetics. Its predictions regarding the extent of FAD release and aggregation during thermal denaturation were confirmed by experiments. TvDAO is shown to contain two highly reactive cysteines per protein subunit whose modification with 5,5′-dithio-bis (2-nitrobenzoic acid) was accompanied by inactivation. Dithiothreitol (1 mmol L^?1) enhanced up to 10-fold the recovery of enzyme activity during ion exchange chromatography of technical-grade TvDAO. However, it did not stabilize TvDAO at all temperatures and protein concentrations, suggesting that deactivation of cysteines was not responsible for thermal denaturation.     

Oxydation von reduziertem Nicotinamid-Adenin-Dinucleotid in Rhodospirillum rubrum

Summary An active form of ApoNADH Dehydrogenase [NADH: (acceptor) oxidoreductase, EC 1.6.99.3] from R. rubrum can be obtained by incubating the apoenzyme at 20° C without flavin. Subsequent lowering the temperature to 0° C decreases the activity to the original level. The whole process can be repeated.The addition of flavin stabilizes the active form, so that no decrease is observed after the temperature has been dropped to 0° C. Here FMN is 30–40 times more effective than FAD. The activated apoenzyme, containing FMN, is again the normal functional form of NADH Dehydrogenase, showing its properties.It is assumed, that during the preparation of the apoenzyme the prosthetic group is not removed from the proteinmolecule but is altered in its binding to or in its position at the molecule. This leads to an equilibrium between a form of low and of higher activity of the apoenzyme, which, by changing the temperature, can be shifted to either side.It is possible to remove the prosthetic group still present at the apoenzyme by dialysis or by gelfiltration with Sephadex. The resulting protein can now be activated only in the presence of flavin.

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Oxydation von reduziertem Nicotinamid-Adenin-Dinucleotid in Rhodospirillum rubrum II. Über eine reversible, temperaturabhängige Aktivierung der ApoNADH-Dehydrogenase

Zusammenfassung Das Apoenzym der NADH-Dehydrogenase [NADH: (acceptor) oxidoreductase, EC 1.6.99.3.] aus R. rubrum kann durch Inkubation bei 20° C ohne Flavin in eine aktive Form übergeführt werden. Durch Absenken der Temperatur auf 0° C wird die wenig aktive Form wiederhergestellt. Der gesamte Vorgang kann beliebig wiederholt werden.Flavin stabilisiert die aktive Form, so daß bei Wechsel der Temperatur auf 0° C keine Abnahme der Aktivität mehr eintritt. Dabei hat FMN etwa 30–40fach größere Wirksamkeit als FAD. Das aktivierte, FMN enthaltende Apoenzym ist wieder die normale funktionsfähige Form der NADH-Dehydrogenase und zeigt deren Eigenschaften.Es wird vermutet, daß bei der Herstellung des Apoenzyms die prosthetische Gruppe in ihrer Bindung oder Lage am Enzymmolekül verändert, jedoch nicht abgespalten wird. Daraus resultiert der Zustand eines durch Temperaturwechsel verschiebbaren Gleichgewichtes zwischen einer wenig aktiven und einer aktiven Form des Apoenzyms.Die noch am Apoenzym befindliche prosthetische Gruppe kann durch Dialyse oder Gelfiltration mit Sephadex entfernt werden. Das Apoenzym ist jetzt nur noch bei Zusatz von Flavin aktivierbar.

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Verwendete Abkürzungen NADH reduziertes Nicotinamid-Adenin-Dinucleotid - FMN Flavinmononucleotid - FAD Flavin-adenin-dinucleotid     

Purification and characterization of recombinant FAD synthetase from Neurospora crassa

FAD Synthetase (FADS) [EC 2.7.7.2], the second enzyme in flavin cofactor biosynthetic pathway converts FMN to FAD, plays an important role in many redox reactions. Neurospora crassa FADS (NcFADS) was cloned and overexpressed in E. coli cells. Recombinant NcFADS was purified in high yields of ～8 mg per liter of bacterial culture using a single step glutathione sepharose affinity chromatography. SDS-PAGE and MALDI-MS revealed that NcFADS has a molecular mass of ～31 kDa. Enzyme kinetic analysis monitored by reverse phase HPLC demonstrate a specific activity and k_cat of 1356 nmol/min/mg and 0.69sec^?1 respectively. Steady state kinetic analysis of NcFADS exhibited a K_m of NcFADS for FMN is 2.7 μM and for MgATP^?2 is 88.7 μM. Isothermal titration calorimetry experiments showed that the recombinant protein binds to the substrates with apparent K_d of 20.8 μM for FMN and 16.6 μM for MgATP^?2. Biophysical characterization using intrinsic fluorescence suggests that the enzyme is in folded conformation. Far-UV CD data suggest that the backbone of the enzyme is predominantly in a helical conformation. Differential scanning calorimetry data shows that the T_m is 53 °C ± 1. This is the first report on cloning, purification and characterization of FADS from N. crassa. The specific activity of NcFADS is the highest than any of the reported FADS from any other source. The results obtained in this study is expected to pave way for intensive research aimed to understand the molecular basis for the extraordinarily high turnover rate of NcFADS.     

Flavin specificity and subunit interaction of Vibrio fischeri general NAD(P)H-flavin oxidoreductase FRG/FRase I

Apoenzyme of the major NAD(P)H-utilizing flavin reductase FRG/FRase I from Vibrio fischeri was prepared. The apoenzyme bound one FMN cofactor per enzyme monomer to yield fully active holoenzyme. The FMN cofactor binding resulted in substantial quenching of both the flavin and the protein fluorescence intensities without any significant shifts in the emission peaks. In addition to FMN binding (K(d) 0.5 microM at 23 degrees C), the apoenzyme also bound 2-thioFMN, FAD and riboflavin as a cofactor with K(d) values of 1, 12, and 37 microM, respectively, at 23 degrees C. The 2-thioFMN containing holoenzyme was about 40% active in specific activity as compared to the FMN-containing holoenzyme. The FAD- and riboflavin-reconstituted holoenzymes were also catalytically active but their specific activities were not determined. FRG/FRase I followed a ping-pong kinetic mechanism. It is proposed that the enzyme-bound FMN cofactor shuttles between the oxidized and the reduced form during catalysis. For both the FMN- and 2-thioFMN-containing holoenzymes, 2-thioFMN was about 30% active as compared to FMN as a substrate. FAD and riboflavin were also active substrates. FRG/FRase I was shown by ultracentrifugation at 4 degrees C to undergo a monomer-dimer equilibrium, with K(d) values of 18.0 and 13.4 microM for the apo- and holoenzymes, respectively. All the spectral, ligand equilibrium binding, and kinetic properties described above are most likely associated with the monomeric species of FRG/FRase I. Many aspects of these properties are compared with a structurally and functionally related Vibrio harveyi NADPH-specific flavin reductase FRP.     

Reconstitution of Escherichia coli photolyase with flavins and flavin analogues

Escherichia coli DNA photolyase contains two chromophore cofactors, 1,5-dihydroflavin adenine dinucleotide (FADH2) and (5,10-methenyltetrahydrofolyl)polyglutamate (5,10-MTHF). A procedure was developed for reversible resolution of apophotolyase and its chromophores. To investigate the structures important for the binding of FAD to apophotolyase and of photolyase to DNA, reconstitution experiments with FAD, FMN, riboflavin, 1-deazaFAD, 5-deazaFAD, and F420 were attempted. Only FAD and 5-deazaFAD showed high-affinity binding to apophotolyase. The apoenzyme had no affinity to DNA but did regain its specific binding to thymine dimer containing DNA upon binding stoichiometrically to FAD or 5-deazaFAD. Successful reduction of enzyme-bound FAD with dithionite resulted in complete recovery of photocatalytic activity.     

Cofactors in sarcosine oxidase from Corynebacterium sp. U-96

Sarcosine oxidase from Corynebacterium sp. U-96 is a heterotetrameric enzyme that was reported to contain 1 mol of covalently bound FAD and 1 mol of non-covalently-bound FAD. This work describes the result of reinvestigation of the cofactors in this enzyme. The enzyme was found to contain 1 mol of non-covalently-bound NAD⁺, 1 mol of non-covalently-bound FAD, and 1 mol of covalent FMN. The covalent FMN was identified by the mass and amino acid sequence analyses of the flavin peptide.     

Cloning of FAD synthetase gene from Corynebacterium ammoniagenes and its application to FAD and FMN production

The cloning of a bifunctional FAD synthetase gene, which shows flavokinase and FMN adenylyltransferase activities, from Corynebacterium ammoniagenes was tried by hybridization with synthetic DNAs corresponding to the N-terminal amino acid sequence. The cloned PstI-digested 4.4 × 10³-base (4.4-kb) fragment could not express the FAD synthetase activity in E. coli, but could increase the two activities by the same factor of about 20 in C. amminoagenes. The FAD-synthetase-gene-amplified C. amminoagenes cells were applied to the production of FAD from FMN or riboflavin. The productivity of FAD from FMN was increased four to five times compared with the parent strain, and reached a 90% molar yield. The productivity of FAD from riboflavin was increased about eight times, with a 50% molar yield. The addition of Zn²⁺ to the reaction mixtures for the conversion from riboflavin to FAD brought about the specific inhibition of adenylyltransferase activity and resulted in the accumulation of FMN.     

Stability and reconstitution of pyruvate oxidase from Lactobacillus plantarum: dissection of the stabilizing effects of coenzyme binding and subunit interaction.

Pyruvate oxidase from Lactobacillus plantarum is a homotetrameric flavoprotein with strong binding sites for FAD, TPP, and a divalent cation. Treatment with acid ammonium sulfate in the presence of 1.5 M KBr leads to the release of the cofactors, yielding the stable apoenzyme. In the present study, the effects of FAD, TPP, and Mn2+ on the structural properties of the apoenzyme and the reconstitution of the active holoenzyme from its constituents have been investigated. As shown by circular dichroism and fluorescence emission, as well as by Nile red binding, the secondary and tertiary structures of the apoenzyme and the holoenzyme do not exhibit marked differences. The quaternary structure is stabilized significantly in the presence of the cofactors. Size-exclusion high-performance liquid chromatography and analytical ultracentrifugation demonstrate that the holoenzyme retains its tetrameric state down to 20 micrograms/mL, whereas the apoenzyme shows stepwise tetramer-dimer-monomer dissociation, with the monomer as the major component, at a protein concentration of < 20 micrograms/mL. In the presence of divalent cations, the coenzymes FAD and TPP bind to the apoenzyme, forming the inactive binary FAD or TPP complexes. Both FAD and TPP affect the quaternary structure by shifting the equilibrium of association toward the dimer or tetramer. High FAD concentrations exert significant stabilization against urea and heat denaturation, whereas excess TPP has no effect. Reconstitution of the holoenzyme from its components yields full reactivation. The kinetic analysis reveals a compulsory sequential mechanism of cofactor binding and quaternary structure formation, with TPP binding as the first step. The binary TPP complex (in the presence of 1 mM Mn2+/TPP) is characterized by a dimer-tetramer equilibrium transition with an association constant of Ka = 2 x 10(7) M-1. The apoenzyme TPP complex dimer associates with the tetrameric holoenzyme in the presence of 10 microM FAD. This association step obeys second-order kinetics with an association rate constant k = 7.4 x 10(3) M-1 s-1 at 20 degrees C. FAD binding to the tetrameric binary TPP complex is too fast to be resolved by manual mixing.     

Conformational plasticity surrounding the active site of NADH oxidase from Thermus thermophilus

Biotechnological applications of enzymes can involve the use of these molecules under nonphysiological conditions. Thus, it is of interest to understand how environmental variables affect protein structure and dynamics and how this ultimately modulates enzyme function. NADH oxidase (NOX) from Thermus thermophilus exemplifies how enzyme activity can be tuned by reaction conditions, such as temperature, cofactor substitution, and the addition of cosolutes. This enzyme catalyzes the oxidation of reduced NAD(P)H to NAD(P)⁺ with the concurrent reduction of O₂ to H₂O₂, with relevance to biosensing applications. It is thermophilic, with an optimum temperature of approximately 65°C and sevenfold lower activity at 25°C. Moderate concentrations (≈1M) of urea and other chaotropes increase NOX activity by up to a factor of 2.5 at room temperature. Furthermore, it is a flavoprotein that accepts either FMN or the much larger FAD as cofactor. We have used nuclear magnetic resonance (NMR) titration and ¹⁵N spin relaxation experiments together with isothermal titration calorimetry to study how NOX structure and dynamics are affected by changes in temperature, the addition of urea and the substitution of the FMN cofactor with FAD. The majority of signals from NOX are quite insensitive to changes in temperature, cosolute addition, and cofactor substitution. However, a small cluster of residues surrounding the active site shows significant changes. These residues are implicated in coupling changes in the solution conditions of the enzyme to changes in catalytic activity.     

Metaphosphate-dependent phosphorylation of riboflavin to FMN by Corynebacterium ammoniagenes

Using the bifunctional FAD synthetase from Corynebacterium ammoniagenes, which has the two sequential activities of flavokinase and FMN adenylyl-transferase in FAD biosynthesis, a method of production of the intermediate FMN without any accumulation of FAD was investigated. Various phosphate polymers having no adenylyl moiety were tested for their ability to phosphorylate riboflavin to FMN, using a crude enzyme from C. ammoniagenes/pKH46, which is an FAD-synthetase-gene-dosed strain. Only metaphosphate, other than ATP, could phosphorylate riboflavin to FMN, but FAD did not accumulate at all. The conditions for the conversion of riboflavin to FMN were optimized. The metaphosphate-dependent phosphorylation reaction required Mg²⁺ as the most effective divalent cation. The best concentrations were 10 mM for MgCl₂ and 3mg/ml for metaphosphate. The riboflavin added to the reaction mixture was almost completely converted into FMN after 6 h incubation in the presence of high concentrations of the enzyme preparation.     

Microbial oxidases of acidic -amino acids

Two microbial oxidases of acidic -amino acids have been purified to homogeneity. One is a -aspartate oxidase of the yeast Cryptococcus humicolus UJ1 that was induced markedly with -aspartate and is far more active toward -aspartate than -glutamate. The other is a -glutamate oxidase of Candida boidinii 2201 that preferred -glutamate to -aspartate as a substrate in terms of k_cat/K_m, but was not induced very effectively by -glutamate. The most potent competitive inhibitor of the C. humicolus -aspartate oxidase was malonate, and that of the C. boidinii -glutamate oxidase was -malate. The former enzyme was a homotetramer of 160 kDa consisting of subunits of 40 kDa, each of which contained 1 mol of FAD, while the latter was a monomer of 45 kDa. The N-terminal sequences of both enzymes were similar to those of other FAD enzymes and contained a consensus sequence common to most enzymes binding ADP-containing nucleotides. Peroxisomal localization of the C. humicolus -aspartate oxidase was shown by subcellular fractionation and morphological analysis via electron microscopy of C. humicolus cells, where induction of the enzyme was accompanied by induction of catalase and development of peroxisomes. The apo-form of C. humicolus -aspartate oxidase, prepared by removal of FAD was a monomeric protein of 40 kDa, and its binding with FAD proceeded in two stages. The K_d for the apoprotein-FAD complex was very low (8.2×10⁻¹² M) consistent with the observed tight binding. The C. humicolus -aspartate oxidase was essentially similar to other flavoprotein oxidases of acidic and neutral -amino acids with respect to its spectral properties and sensitivity to specific modifying reagents for arginyl and histidyl residues.     

Ricerche sul meristema apicale della radice II. II contenuto di coenzimi riboflavinici

Abstract

RESEARCHES ON ROOT APEX MERISTEM. II. RIBOFLAVINE COENZYMES. — FMN and FAD contents have been measured (by the method of BURCH et al.) in the roots of etiolated pea seedlings. It has been found that FMN and FAD content is higher, when referred to fresh weight, in the 2 mm. apical segment than in the next 2–4 mm segment.

On a protein basis, FMN is more abundant in the 2–4 mm segment than in the 0–2 mm apical segment.

The opposite is true for FAD (see table). The apical (meristematic) segment is therefore characterized by a higher FAD/FMN ratio (its value is 1) than the next 2–4 mm segment, where the ratio FAD/FMN is 0.29.     

Nicotinamide adenine dinucleotide-dependent formate dehydrogenase from Rhodopseudomonas palustris

Summary Formate dehydrogenase in extracts of the facultative phototroph, Rhodopseudomonas palustris was shown to be soluble and NAD-linked. The flavin nucleotides, FMN and FAD, stimulated the rate of NAD reduction about fourfold. Reduction of artificial electron acceptors such as DCPIP and cytochrome c was also stimulated by FMN and FAD. The pH optimum for the reduction of NAD was pH 8.0, in contrast to pH 6.8 for cytochrome c and DCPIP reduction. The apparent K _m for formate as measured by NAD reduction was 2.6×10^-4 M. Although the addition of thiosulfate or yeast extract to the formate medium increased both the growth rate and yield of Rhps. palustris, they had little effect on the activity of formate dehydrogenase.