Spectroscopic studies of pyruvate oxidase flavoprotein from Escherichia coli trapped in the lipid-activated form by cross-linking

Pyruvate oxidase is a flavoprotein dehydrogenase isolated from Escherichia coli which catalyzes the oxidative decarboxylation of pyruvate to acetate plus CO2. The maximal turnover of the enzyme, measured using a ferricyanide reductase assay, is increased 20-to 30-fold by either of two methods. Proteolysis in the presence of the substrate (pyruvate) and cofactor (Mg2+-thiamin pyrophosphate) results in cleavage at a single locus near the carboxyl terminus and concomitant activation. Phospholipids and detergents can bind to the enzyme and result in a similar activation, which is presumed to be physiologically relevant, since the enzyme functions as a peripheral membrane enzyme. Previous studies showed that proteolytic activation of pyruvate oxidase results in substantial changes in the absorption spectrum of the oxidized form of the bound flavin. Up to this time, similar studies of the lipid-activated form of the enzyme have not been feasible, since it is necessary to reduce the flavoprotein in order to induce binding to the lipids. In this paper, glutaraldehyde cross-linking of the lipid-activated enzyme is used to trap the enzyme in this form. Spectroscopic studies show alterations of the flavin spectrum similar to those observed upon proteolytic activation. This alteration in the flavin binding site is consistent with kinetic studies which suggest that activation results from an acceleration in the rates of electron transfer both into and out of the bound flavin, which appears to be more "accessible" in the activated forms of the enzyme.     

Nucleotide sequence and deduced amino acid sequence of Escherichia coli pyruvate oxidase, a lipid-activated flavoprotein.

The entire nucleotide sequence of the poxB (pyruvate oxidase) gene of Escherichia coli K-12 has been determined by the dideoxynucleotide (Sanger) sequencing of fragments of the gene cloned into a phage M13 vector. The gene is 1716 nucleotides in length and has an open reading frame which encodes a protein of Mr 62,018. This open reading frame was shown to encode pyruvate oxidase by alignment of the amino acid sequences deduced for the amino and carboxy termini and several internal segments of the mature protein with sequences obtained by amino acid sequence analysis. The deduced amino acid sequence of the oxidase was not unusually rich in hydrophobic sequences despite the peripheral membrane location and lipid binding properties of the protein. The codon usage of the oxidase gene was typical of a moderately expressed protein. The deduced amino acid sequence shares homology with the large subunits of the acetohydroxy acid synthase isozymes I, II, and III, encoded by the ilvB, ilvG, and ilvI genes of E. coli.     

Conformational studies of Escherichia coli pyruvate oxidase

In this study the effects of experimental modifications of plasma membrane lipid lateral mobility on the electrical membrane properties and cation transport of mouse neuroblastoma cells, clone Neuro-2A, have been studied. Short-term supplementation of a chemically defined growth medium with oleic acid or linoleic acid resulted in an increase in the lateral mobility of lipids as inferred from fluorescence recovery after photobleaching of the lipid probe 3,3'-dioctadecylindocarbocyanide iodide. These changes were accompanied by a marked depolarization of the membrane potential from -51 mV to -36 mV, 1.5 h after addition, followed by a slow repolarization. Tracer flux studies, using 86Rb+ as a radioactive tracer for K+, demonstrated that the depolarization was not caused by changes in (Na+ + K+)-ATPase-mediated K+ influx or in the transmembrane K+ gradient. The permeability ratio (PNa/PK), determined from electrophysiological measurements, however, increased from 0.10 to 0.27 upon supplementation with oleic acid or linoleic acid. This transient rise of PNa/PK was shown by 24Na+ and 86Rb+ flux measurements to be due to both an increase of the Na+ permeability and a decrease of the K+ permeability. None of these effects occurred upon supplementation of the growth medium with stearic acid.     

Molecular cloning of the gene (poxB) encoding the pyruvate oxidase of Escherichia coli, a lipid-activated enzyme.

The pyruvate oxidase structural gene (poxB) of Escherichia coli was cloned into derivatives of plasmid pBR322. The gene was first cloned into a cosmid vector by selection for the tetracycline resistance determinant of a closely linked Tn10 insertion (no direct selection for the gene was available). Subsequent subcloning resulted in localization of the gene to a 3.1-kilobase-pair DNA segment. Two of the smaller poxB plasmids were shown to cause the overproduction of oxidase activity (by six- to eightfold), and one of these plasmids was shown to encode a protein having the size and antigenic determinants of pyruvate oxidase. Introduction of poxB plasmids into strains (aceEF) lacking pyruvate dehydrogenase activity relieved the aerobic growth requirement of the strains for exogenous acetate.     

Activation of Escherichia coli pyruvate oxidase enhances the oxidation of hydroxyethylthiamin pyrophosphate

The catalytic efficiency (kcat/Km) of Escherichia coli flavin pyruvate oxidase can be stimulated 450-fold either by the addition of lipid activators or by limited proteolytic hydrolysis. Previous studies have shown that a functional lipid binding site is a mandatory prerequisite for the in vivo functioning of this enzyme (Grabau, C., and Cronan, J. E., Jr. (1986) Biochemistry 25, 3748-3751). The effect of activation on the transient state kinetics of partial reactions in the overall oxidative conversion of pyruvate to acetate and CO2 has now been examined. The rate of decarboxylation of pyruvate to form CO2 and hydroxyethylthiamin pyrophosphate for both activated and unactivated forms of the enzyme is identical within experimental error. The decarboxylation step was measured using substrate concentrations of the enzyme in the absence of an electron acceptor. The pseudo-first order rate constant for the decarboxylation step is 60-80 s-1. The rate of oxidation of hydroxyethylthiamin pyrophosphate and concomitant enzyme-bound flavin reduction was analyzed by stopped-flow methods utilizing synthetic hydroxyethylthiamin pyrophosphate. The pseudo-first order rate for this step with unactivated enzyme was 2.85 s-1 and increased 145-fold for lipid-activated enzyme to 413 s-1 and 61-fold for the proteolytically activated enzyme to 173 s-1. The analysis of a third reaction step, the reoxidation of enzyme-bound FADH, was also investigated by stopped-flow techniques utilizing ferricyanide as the electron acceptor. The rate of oxidation of enzyme.FADH is very fast for both unactivated (1041 s-1) and activated enzyme (645 s-1). The data indicate that the FAD reduction step is the rate-limiting step in the overall reaction for unactivated enzyme. Alternatively, the rate-limiting step in the overall reaction with the activated enzyme shifts to one of the partial steps in the decarboxylation reaction.     

Identification of the high-affinity lipid binding site in Escherichia coli pyruvate oxidase

Pyruvate oxidase from Escherichia coli is a peripheral membrane associated enzyme which is activated by lipids. We have investigated the high-affinity lipid binding site associated with lipid activation of pyruvate oxidase by covalent attachment of [14C]lauric acid to the enzyme. Lauric acid is bound stoichiometrically (1 mol/mol of active sites), and the enzyme is essentially irreversibly activated. Mild tryptic digestion of the modified enzyme shows that the lauric acid is bound within the last 100 residues of the 572-residue monomer. Digestion with thermolysin releases two closely related peptides, A and B, in approximately equal amounts. Comparison of the amino acid composition of peptide A with the entire sequence of the protein shows that peptide A corresponds to the sequence from Ala-543 to Ile-554. The analysis of peptide B is very similar to that of A. Limited sequence analysis of peptide B shows that residue 1 is Ala and residue 2 is labeled. These results support the assignment of residue 1 in peptide B as Ala-543 and indicate that lauric acid is bound to Lys-544. Previous work in this laboratory has shown that pyruvate oxidase may be activated independently of lipids by mild protease digestion. Proteolytic activation is accompanied by the release of a small peptide (residues 550-572) from the carboxyl terminus of the protein. The present work locates the lipid binding site very close to this peptide. The significance of these results for the mechanism of activation of pyruvate oxidase and other lipid-activated systems is discussed.     

Reconstitution of the Ubiquinone-dependent pyruvate oxidase system of Escherichia coli with the cytochrome o terminal oxidase complex

The aerobic respiratory chain of Escherichia coli is branched and contains two terminal oxidases. The chain predominant when the cells are grown with low aeration terminates with the cytochrome d terminal oxidase complex, and the branch present under high aeration ends with the cytochrome o terminal oxidase complex. Previous work has shown that cytochrome d complex functions as a ubiquinol-8 oxidase, and that a minimal respiratory chain can be reconstituted in proteoliposomes with a flavoprotein dehydrogenase (pyruvate oxidase), ubiquinone-8, and the cytochrome d complex. This paper demonstrates that the cytochrome o complex functions as an efficient ubiquinol-8 oxidase in reconstituted proteoliposomes, and that ubiquinone-8 serves as an electron carrier from the flavoprotein to the cytochrome complex. The maximal turnover (per cytochrome o) achieved in reconstituted proteoliposomes is at least as fast as observed in E. coli membrane preparations. Electron flow from the flavoprotein to oxygen in the reconstituted proteoliposomes generates a transmembrane potential of at least 120 mV, negative inside, which is sensitive to ionophore uncouplers and inhibitors of the terminal oxidase. These data demonstrate the minimal composition of this respiratory chain as a flavoprotein dehydrogenase, ubiquinone-8, and the cytochrome o complex. Previous models have suggested that cytochrome b556, also a component of the E. coli inner membrane, is required for electron flow to cytochrome o. This is apparently not the case. It now is clear that both of the E. coli terminal oxidases act as ubiquinol-8 oxidases and, thus, ubiquinone-8 is the branch point between the two respiratory chains.     

Kinetic and equilibrium studies on the interaction of reduced flavoprotein D-amino acid oxidase with pyridine carboxylates

The equilibrium constants and the rate constants (binding and dissociation constants) between reduced D-amino acid oxidase and pyridine carboxylates were obtained at various pH values (from pH 6.0 to 8.3). The pH dependence of the constants is consistent with the previous conclusion from a resonance Raman study that pyridine carboxylates in the form of a cation protonated at the N atom can bind to the reduced enzyme, but those in the neutral form cannot bind, showing that the positive charge of cationic pyridine carboxylates interacts with the negative charge of the anionic reduced flavin in the reduced enzyme. The binding rate constants of picolinate and nicotinate in the cationic form for the reduced enzyme were quite similar to each other, but the dissociation rate constant of picolinate is several times smaller than that of nicotinate. Thus, it is concluded that the difference in affinity of picolinate and nicotinate for the reduced enzyme is derived from the difference of the dissociation rate constants.     

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.     

Reconstitution of the lipid-depleted pyruvate oxidase system of Escherichia coli: the palmitic acid effect

Previous work has shown that the coupling of the soluble Escherichia coli pyruvate oxidase to a lipid-depleted membrane terminal electron transport system requires the addition of ubiquinone and a neutral lipid fraction (C. Cunningham and L. P. Hager (1975) J. Biol. Chem. 250, 7139-7146). The active factor present in the neutral lipid fraction has now been isolated and characterized. NMR, uv, and mass spectroscopic analysis identifies palmitic acid as the active component. A comparison of palmitic acid with other fatty acids of varying chain lengths indicates that most fatty acids having chain lengths in the range C12 to C20 have comparable activity to palmitic acid. Exceptions are stearic and arachidic acid which have greatly reduced activity. Fatty acids of C6 to C10 chain length showed about one third the activity of palmitic acid. Fatty acids having chain lengths of 2 to 5 carbon atoms are essentially inactive. The carboxyl function of the fatty acid is required for activity. Derivatives of fatty acids in which the carboxyl group had been modified to an alcohol, aldehyde, or methyl ester function show greatly diminished activity. Both the cis and trans forms of unsaturated long-chain fatty acids are active. The stimulation of the electron transfer reaction by fatty acids occurs at the ubiquinone level of the electron transport chain. Ubiquinone-30 is rapidly reduced by pyruvate oxidase only in the presence of palmitic acid.     

Statistical optimization of medium for the production of pyruvate oxidase by the recombinant Escherichia coli

Pyruvate oxidase (PyOD) is a very useful enzyme for clinical diagnostic applications and environmental monitor. Optimization of the fermentation medium for maximization of PyOD constitutively, production by Escherichia coli DH5α/pSMLPyOD was carried out. Response surface methodology (RSM) was used to optimize the medium constituents. A 2^6–2 fractional factorial design (first order model) was carried out to identify the significant effect of medium components towards PyOD production. Statistical analysis of results shows that yeast extract, ammonium sulfate and composite phosphate were significant factors on PyOD production. The optimized values of these three factors were obtained by RSM based on the result of a 2³ central composite rotatable design. Under these proposed optimized medium, the model predicted a PyOD activity of 610 U/L and via experimental rechecking the model, an activity of 670 U/L was attained.     

Escherichia coli DNA photolyase is a flavoprotein

Escherichia coli DNA photolyase (photoreactivating enzyme) was purified to homogeneity from a strain that greatly overproduces the protein. The purified enzyme has absorption peaks at 280 and 380 nm, a fluorescence emission peak at 480 nm and, upon denaturation, releases a chromophore that has the spectroscopic properties of flavin adenine dinucleotide (FAD), indicating that FAD is an intrinsic chromophore of the enzyme.     

Reconstitution of the membrane-bound, ubiquinone-dependent pyruvate oxidase respiratory chain of Escherichia coli with the cytochrome d terminal oxidase

Pyruvate oxidase is a flavoprotein dehydrogenase located on the inner surface of the Escherichia coli cytoplasmic membrane and coupled to the E. coli aerobic respiratory chain. In this paper, the role of quinones in the pyruvate oxidase system is investigated, and a minimal respiratory chain is described consisting of only two pure proteins plus ubiquinone 8 incorporated in phospholipid vesicles. The enzymes used in this reconstitution are the flavoprotein and the recently purified E. coli cytochrome d terminal oxidase. The catalytic velocity of the reconstituted liposome system is about 30% of that observed when the flavoprotein is reconstituted with E. coli membranes. It is also shown that electron transport from pyruvate to oxygen in the liposome system generates a transmembrane potential of at least 180 mV (negative inside), which is sensitive to the uncouplers carbonyl cyanide p-(tri-chloromethoxy)phenylhydrazone and valinomycin. A trans-membrane potential is also generated by the oxidation of ubiquinol 1 by the terminal oxidase in the absence of the flavoprotein. It is concluded that (1) the flavoprotein can directly reduce ubiquinone 8 within the phospholipid bilayer, (2) menaquinone 8 will not effectively substitute for ubiquinone 8 in this electron-transfer chain, and (3) the cytochrome d terminal oxidase functions as a ubiquinol 8 oxidase and serves as a "coupling site" in the E. coli aerobic respiratory chain. These investigations suggest a relatively simple organization for the E. coli respiratory chain.     

Kinetic studies of L-aspartase from Escherichia coli: substrate activation

The enzyme L-aspartase from Escherichia coli was observed to have a time lag during the production of aspartic acid from fumarate and ammonia. This time lag is pH dependent, with little lag observed below pH 7.0 and a very extensive lag observed above pH 8.0. This time lag was also found to be dependent on both substrate and divalent metal ion concentrations and on the degree of proteolysis of L-aspartase. The observed lag, in the reaction examined in the amination direction, has been found to be correlated with the nonlinear kinetics seen at higher pH in the deamination direction. Both phenomena are consistent with a model in which there is a separate activator site for the substrate, L-aspartic acid, that is distinct from the enzyme active site. Occupation of this site by the substrate, or by various substrate analogues, eliminates both the nonlinearity and the time lag. The D isomer of aspartic acid, which does not bind at the active site, can bind at this newly identified activator site.     

Escherichia coli alkaline phosphatase. Kinetic studies with the tetrameric enzyme

1. The stability of the tetrameric form of Escherichia coli alkaline phosphatase was examined by analytical ultracentrifugation. 2. The stopped-flow technique was used to study the hydrolysis of nitrophenyl phosphates by the alkaline phosphatase tetramer at pH7.5 and 8.3. In both cases transient product formation was observed before the steady state was attained. Both transients consisted of the liberation of 1mol of nitrophenol/2mol of enzyme subunits within the dead-time of the apparatus. The steady-state rates were identical with those observed with the dimer under the same conditions. 3. The binding of 2-hydroxy-5-nitrobenzyl phosphonate to the alkaline phosphatase tetramer was studied by the temperature-jump technique. The self-association of two dimers to form the tetramer is linked to a conformation change within the dimer. This accounts for the differences between the transient phases in the reactions of the dimer and the tetramer with substrate. 4. Addition of P_i to the alkaline phosphatase tetramer caused it to dissociate into dimers. The tetramer is unable to bind this ligand. It is suggested that the tetramer undergoes a compulsory dissociation before the completion of its first turnover with substrate. 5. On the basis of these findings a mechanism is proposed for the involvement of the alkaline phosphatase tetramer in the physiology of E. coli.