Role of the tetrameric structure of Escherichia coli pyruvate oxidase in enzyme activation and lipid binding

Pyruvate oxidase of Escherichia coli, an enzyme greatly activated by phospholipids, is a tetramer of a Mr 62,000 subunit. We have utilized the differing electrophoretic mobilities of several mutant oxidases on native polyacrylamide gels to study the role of the quaternary structure of the enzyme in the activation process. We found that when two poxB gene alleles coexisted in cells, heterotetrameric species were formed in addition to homotetramers. The concentration of each tetrameric species varied according to the concentration of the different subunits present, and the distribution seemed virtually identical to those expected from random mixing. We showed that the intrinsic activity of pyruvate oxidase was not affected by interactions among the four subunits. However, binding of the enzyme to lipids, a property required for function in vivo, required that a tetramer contain at least two subunits capable of lipid binding. Our data fit the model proposed previously (Grabau, C., Chang, Y.-Y., and Cronan, J. E., Jr. (1989) J. Biol. Chem. 264, 12510-12519) in which the carboxyl termini of two subunits interact to form a functional lipid-binding domain. We also have detected oxidase activity in a form of oxidase of unusually high electrophoretic mobility. This form seems to be either a monomeric or a dimeric form (more probably the former) of the oxidase subunit.     

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.     

Characterization of monoclonal antibodies directed against pyruvate oxidase from Escherichia coli: modulation of antibody-induced inhibition by enzyme conformation

Monoclonal antibodies have been prepared against pyruvate oxidase, a flavoprotein dehydrogenase isolated from Escherichia coli. Six monoclonals were obtained, but only one was found to bind to the native form of the enzyme. This monoclonal, 1I1, was a potent inhibitor. Although this antibody inhibited the unactivated and lipid-activated forms of the enzyme, it had much less of an inhibitory effect on the protease-activated form of the enzyme, although the antibody still bound to this form. Hence, the coupling between antibody binding and the conformation at the active site can itself be modulated by the conformation of the protein.     

丙酮酸氧化酶激活机制的研究——α-肽与磷脂囊的结合

 丙酮酸氧化酶既能被两性脂类激活,也能被蛋白酶从羧基端切下一个23肽(称α-肽)激活,而且这两种激活方式相互排斥。本文用分离纯化的α-肽,通过测定色氨酸荧光能量转移的方法,证明α-肽能与磷脂囊(vesicle)结合。我们选用两种不同类型的荧光探针,发现α-肽对它们表现不同的色氨酸能量转移效率。α-肽对Dan-syl-DPPE为7.4±1.0％对Dansyl-UAPC为12.5±2.0％。由此推测α-肽的色氨酸残基易与磷脂双层内部疏水区结合,说明α-肽是一种较为疏水的肽。我们推测α-肽在这种外膜酶的激活过程中,提供了与膜结合的部位,在生理过程中起着重要作用。     

D-aspartate oxidase from beef kidney. Purification and properties

The flavoprotein D-aspartate oxidase (EC 1.4.3.1) has been purified to homogeneity from beef kidney cortex. The protein is a monomer with a molecular weight of 39,000 containing 1 molecule of flavin. The enzyme as isolated is a mixture of a major active form containing FAD and a minor inactive form containing 6-hydroxy-flavin adenine dinucleotide (6-OH-FAD). The absorption and fluorescence spectral properties of the two forms have been studied separately after reconstitution of the apoprotein with FAD or 6-OH-FAD, respectively. FAD-reconstituted D-aspartate oxidase has flavin fluorescence, shows characteristic spectral perturbation upon binding of the competitive inhibitor tartaric acid, is promptly reduced by D-aspartic acid under anaerobiosis, reacts with sulfite to form a reversible covalent adduct, stabilizes the red anionic form of the flavin semiquinone upon photoreduction, and yields the 3,4-dihydro-FAD-form after reduction with borohydride. A Kd of 5 X 10(-8) M was calculated for the binding of FAD to the apoprotein. 6-OH-FAD-reconstituted D-aspartate oxidase has no flavin fluorescence, shows no spectral perturbation in the presence of tartaric acid, is not reduced by D-aspartic acid under anaerobiosis, does not stabilize any semiquinone upon photoreduction, and does not yield the 3,4-dihydro-form of the coenzyme when reduced with borohydride; the enzyme stabilizes the p-quinoid anionic form of 6-OH-FAD and lowers its pKa more than two pH units below the value observed for the free flavin. The general properties of the enzyme thus resemble those of the dehydrogenase/oxidase class of flavoprotein, particularly those of the amino acid oxidases.     

Allosteric regulation of the biotin-dependent enzyme pyruvate carboxylase by acetyl-CoA

The activity of the biotin-dependent enzyme pyruvate carboxylase from many organisms is highly regulated by the allosteric activator acetyl-CoA. A number of X-ray crystallographic structures of the native pyruvate carboxylase tetramer are now available for the enzyme from Rhizobium etli and Staphylococcus aureus. Although all of these structures show that intersubunit catalysis occurs, in the case of the R. etli enzyme, only two of the four subunits have the allosteric activator bound to them and are optimally configured for catalysis of the overall reaction. However, it is apparent that acetyl-CoA binding does not induce the observed asymmetrical tetramer conformation and it is likely that, under normal reaction conditions, all of the subunits have acetyl-CoA bound to them. Thus the activation of the enzyme by acetyl-CoA involves more subtle structural effects, one of which may be to facilitate the correct positioning of Arg353 and biotin in the biotin carboxylase domain active site, thereby promoting biotin carboxylation and, at the same time, preventing abortive decarboxylation of carboxybiotin. It is also apparent from the crystal structures that there are allosteric interactions induced by acetyl-CoA binding in the pair of subunits not optimally configured for catalysis of the overall reaction.     

Characterization of the alpha-peptide released upon protease activation of pyruvate oxidase

The pyruvate oxidase of Escherichia coli is a homo-tetrameric enzyme which can be activated greater than 500-fold (kcat/Km) by limited proteolytic digestion with alpha-chymotrypsin in the presence of pyruvate and thiamine pyrophosphate. The cleavage produces an Mr 2000 peptide (the alpha-peptide) from each subunit and mimics the physiologically important activation of the enzyme by phospholipids. Moreover, the proteolytic cleavage results in the loss of the high affinity lipid-binding site of the enzyme. We now report the isolation and characterization of the alpha-peptide fragment which is cleaved from the carboxyl terminus of each subunit by protease activation. Both the site of cleavage and the sequence of the alpha-peptide have been determined by a combination of Edman degradation of the purified peptide and DNA sequence analysis of the gene encoding the oxidase. The cleavage site lies within a sequence of hydrophobic amino acids predicted to form a beta-sheet. Another segment of the alpha-peptide is predicted to form an amphipathic alpha-helix. Quantitative assessment of the amphipathic nature of this alpha-helix (Eisenberg, D. (1984) Annu. Rev. Biochem. 53, 595-623) gives a value very similar to the values for several helical peptides which spontaneously bind to the surface of phospholipid vesicles. From these analyses, we propose that the alpha-peptide may play a role in binding pyruvate oxidase to cell membrane phospholipids in vivo.     

Proteolysis of L-(+)-lactate cytochrome c oxidoreductase (cytochrome b2) extracted from Saccharomyces cerevisiae and Hansenula anomala yeasts.

The L-(+)-Lactate:cytochrome c oxidoreductase or cytochrome b2 from the yeasts Saccharomyces cerevisiae and Hansenula anomala were partially hydrolysed in various concentrations of trypsin. Conditions were found which allowed the isolation from the Hansenula enzyme of a 140 000 +/- 10 000-dalton flavoprotein. The prosthetic flavin groups were still reducible by substrate (spectroscopic evidence) but the flavoprotein was unable to form a complex with cytochrome c, the physiological acceptor in the enzymatic reaction. No such flavoprotein units could be found during proteolysis of the Saccharomyces enzyme. The heme prosthetic group of the Hansenula enzyme remained bound to a 15 500 +/- 1000-dalton protein unit which was larger than, but very similar to, the well known 'cytochrome b2 core' of the Saccharomyces enzyme. Moreover, the degradation of different enzyme samples by contaminated proteases allowed the isolation of a particular form of Hansenula enzyme: each tetramer had, on the mean, four bound flavins and only two heme groups. These molecules completely retained their ability to form a complex with cytochrome c.     

The cooperative binding of fructose-1,6-bisphosphate to yeast pyruvate kinase.

The cooperative binding of the allosteric activator fructose-1,6-bisphosphate [Fru(1,6)P2] to yeast pyruvate kinase was investigated by equilibrium dialysis and fluorescence quench titration. The results show that yeast pyruvate kinase binds four molecules of Fru(1,6)P2 per tetramer and the observed fluorescence quench follows the binding of the ligand and not the cooperative T to R state transition. Additionally it is shown that the binding of Fru(1,6)P2 to yeast pyruvate kinase is compatible with the model of cooperativity that has been proposed and incorporates an intermediate state, R', with properties between those of the T and R states.     

Kinetic studies of the lipid-activated pyruvate oxidase flavoprotein of Escherichia coli

Pyruvate oxidase is a flavoprotein dehydrogenase isolated from Escherichia coli which catalyzes the oxidative decarboxylation of pyruvate to acetate and CO2. In vivo, the enzyme can bind to the bacterial membrane and reduce ubiquinone-8, feeding electrons into the respiratory chain. The purified enzyme has been shown previously to bind to phospholipids and detergents and, upon doing so, is activated. The turnover with ferricyanide as an electron acceptor increases 20- to 30-fold upon lipid binding. In this work, initial velocity and stop-flow kinetics are used to investigate the activation of this enzyme. It is shown that the unactivated form of the enzyme is markedly hysteretic. Progress curves at low substrate concentrations show an initial acceleration in enzyme turnover. This is consistent with the results of stop-flow experiments. Rates obtained for either the reduction of the unactivated flavoprotein by pyruvate or its reoxidation by ferricyanide in single turnover experiments are much slower than the rates predicted by observed turnover in initial velocity studies, in some cases by more than 2 orders of magnitude. The data are best explained by the slow interconversion between two forms of the enzyme, one with low turnover and one which rapidly turns over. As isolated, the enzyme is highly unreactive, as revealed by the stop-flow experiments. During turnover, even in the absence of lipid activators, some of the enzyme converts to the rapid-turnover form. This slow interconversion is shown by kinetic simulation to preclude a steady state from being established. Lipid activators appear to shift the equilibrium to favor the rapid-turnover form of the enzyme. Once the enzyme is "locked" into an activated conformation, the hysteresis is no longer observed, and the stop-flow results are in agreement with data obtained from initial velocity experiments. Activation appears to result in both increased rates of electron transfer into and out of the flavin.     

The crystal structure of pyruvate decarboxylase from Kluyveromyces lactis. Implications for the substrate activation mechanism of this enzyme

The crystal structure of pyruvate decarboxylase from Kluyveromyces lactis has been determined to 2.26 A resolution. Like other yeast enzymes, Kluyveromyces lactis pyruvate decarboxylase is subject to allosteric substrate activation. Binding of substrate at a regulatory site induces catalytic activity. This process is accompanied by conformational changes and subunit rearrangements. In the nonactivated form of the corresponding enzyme from Saccharomyces cerevisiae, all active sites are solvent accessible due to the high flexibility of loop regions 106-113 and 292-301. The binding of the activator pyruvamide arrests these loops. Consequently, two of four active sites become closed. In Kluyveromyces lactis pyruvate decarboxylase, this half-side closed tetramer is present even without any activator. However, one of the loops (residues 105-113), which are flexible in nonactivated Saccharomyces cerevisiae pyruvate decarboxylase, remains flexible. Even though the tetramer assemblies of both enzyme species are different in the absence of activating agents, their substrate activation kinetics are similar. This implies an equilibrium between the open and the half-side closed state of yeast pyruvate decarboxylase tetramers. The completely open enzyme state is favoured for Saccharomyces cerevisiae pyruvate decarboxylase, whereas the half-side closed form is predominant for Kluyveromyces lactis pyruvate decarboxylase. Consequently, the structuring of the flexible loop region 105-113 seems to be the crucial step during the substrate activation process of Kluyveromyces lactis pyruvate decarboxylase.     

Purification and characterization of vanillyl-alcohol oxidase from Penicillium simplicissimum. A novel aromatic alcohol oxidase containing covalently bound FAD.

Vanillyl-alcohol oxidase was purified 32-fold from Penicillium simplicissimum, grown on veratryl alcohol as its sole source of carbon and energy. SDS/PAGE of the purified enzyme reveals a single fluorescent band of 65 kDa. Gel filtration and sedimentation-velocity experiments indicate that the purified enzyme exists in solution as an octamer, containing 1 molecule flavin/subunit. The covalently bound prosthetic group of the enzyme was identified as 8 alpha-(N3-histidyl)-FAD from pH-dependent fluorescence quenching (pKa = 4.85) and no decrease in fluorescence upon reduction with sodium borohydride. The enzyme shows a narrow substrate specificity, only vanillyl alcohol and 4-hydroxybenzyl alcohol are substrates for the enzyme. Cinnamyl alcohol is a strong competitive inhibitor of vanillyl-alcohol oxidation. The visible absorption spectrum of the oxidized enzyme shows maxima at 354 nm and 439 nm, and shoulders at 370, 417 and 461 nm. Under anaerobic conditions, the enzyme is easily reduced by vanillyl alcohol to the two-electron reduced form. Upon mixing with air, rapid reoxidation of the flavin occurs. Both with dithionite reduction and photoreduction in the presence of EDTA and 5-deazaflavin the red semiquinone flavin radical is transiently stabilized. Opposite to most flavoprotein oxidases, vanillyl-alcohol oxidase does not form a flavin N5-sulfite adduct. Photoreduction of the enzyme in the presence of the competitive inhibitor cinnamyl alcohol gives rise to a complete, irreversible bleaching of the flavin spectrum.     

Lipid binding by Escherichia coli pyruvate oxidase is disrupted by small alterations of the carboxyl-terminal region

Escherichia coli pyruvate oxidase is a membrane-associated flavoprotein dehydrogenase which is greatly activated by lipids and detergents. The carboxyl-terminal region of the protein has been shown to play a critical role in the interaction with lipids. We report mutations generated by chemical and oligonucleotide-mediated site-directed mutagenesis of the poxB gene which result in enzymes defective in lipid activation. Nine mutants were isolated which encode enzymes with point mutations in the carboxyl-terminal segment of the protein. Two mutant lesions introduced termination codons giving enzymes lacking the last nine or three amino acids of the protein which were unable to interact with detergents in vitro and were unable to function in vivo. Of the missense mutants isolated, two were most informative. One was the substitution of Glu-564 with proline in the PoxB16 oxidase. This residue lies in the center of a putative lipid-binding amphipathic alpha-helix (Arg-558 to Thr-568) located close to the carboxyl terminus. Strains producing the PoxB16 oxidase were devoid of oxidase activity in vivo, the enzymes could not be activated by Triton X-100, and were activated poorly by phospholipids in vitro. These results indicated that the PoxB16 oxidase lacked normal lipid-binding ability. Another mutant oxidase (PoxB15) in which proline was substituted for Asp-560 at the beginning of the amphipathic alpha-helix had normal oxidase activity. These findings indicate that the amphipathic alpha-helix structure plays an essential role in the activation and lipid-binding properties of the enzyme. The second informative missense mutation was the substitution of the carboxyl-terminal arginine with glycine. This enzyme showed normal activation in vitro by phospholipids and some detergents, and somewhat reduced activity in vivo. This mutant enzyme appeared to dissociate from detergent vesicles more readily than does the normal enzyme. A model for the membrane interaction of the carboxyl terminus based on the properties of these mutant proteins is presented.     

Binding of pyruvate oxidase alpha-peptide to phospholipid vesicles

The alpha-peptide of pyruvate oxidase is a 23 residue peptide which is cleaved from the carboxy terminus of the enzyme during proteolytic activation by chymotrypsin (M. Recny et al. (1985) J. Biol. Chem. 260, 14287-14291). Cleavage of alpha-peptide results in the loss of the high affinity lipid-binding site in the enzyme. The beta-peptide of pyruvate oxidase is a 101 residue peptide which also is cleaved from the carboxy terminus of pyruvate oxidase. Cleavage of the beta-peptide from pyruvate oxidase results in the inactivation of the enzyme. The beta-peptide includes the alpha-peptide amino acid sequences at its carboxyl terminus. We now report on the binding of the alpha- and beta-peptides to phospholipid vesicles. Both peptides bind with equal and high affinity to phosphatidylcholine vesicles. We conclude from these results that the alpha-peptide furnishes the membrane-binding site which plays the physiologically important role in the activation of this peripheral membrane enzyme.     

Human plasma dopamine beta-hydroxylase. Purification and properties

Dopamine beta-hydroxylase was isolated from normal human plasma. The major form of the active enxyme in plasma was purified to apparent homogeneity and is a 300,000-dalton tetramer containing 4 atoms of tightly bound copper. About 20% of the enzyme activity in plasma was isolated as a dimeric form of this enzyme. Sodium dodecyl sulfate gel electrophoresis of the purified form gave a polypeptide subunit molecular weight of 72,000 and disulfide-linked dimers of this component were observed. Both forms of the enzyme are apparently glycoproteins and interact with immobilized concanavalin A. Furthermore, the enzyme is capable of binding to alkyl-substituted agarose by hydrophobic interaction. Advantage was taken of these properties to purify the enzyme. Both purified tetramer and partially purified dimer were further characterized by kinetic analysis and the Stokes radii and S20,W of these species were compared. Rabbit antiserum to the purified tetramer revealed no immunochemical differences between the two enzyme forms by using a method of immunotitration.     

Purification and characterization of Haemophilus influenzae D-lactate dehydrogenase

Haemophilus influenzae D(-)-lactate dehydrogenase (D(-)-lactate:NAD oxidoreductase; EC 1.1.1.28) was purified to electrophoretic homogeneity using salt fractionation, hydrophobic and dye affinity chromatography. The enzyme was purified 2100-fold with a 14% recovery and a final specific activity of 300 units/mg protein. The enzyme was demonstrated to be a tetramer of Mr 135,000. The enzyme catalyzed the reduction of pyruvate to give exclusively D(-)-lactate using NADH as coenzyme. The reaction catalyzed was essentially unidirectional, with the oxidation of D-lactate in the presence of NAD proceeding at less than 0.2% the rate of pyruvate reduction. Kinetic parameters for the reduction of pyruvate were determined for NADH and four structural analogs of the coenzyme. Coenzyme-competitive inhibition by adenosine derivatives indicated the presence of regions in the coenzyme binding site interacting with the adenosine and pyrophosphate moieties of the coenzyme. The purified enzyme was sensitive to oxidation and was effectively inactivated by sulfhydryl reagents. Conversion of D-lactate to pyruvate catalyzed by a membrane-bound D-lactate oxidase was demonstrated in cell-free extracts of H. influenzae.     

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.     

Factor VII binding to tissue factor in reconstituted phospholipid vesicles: induction of cooperativity by phosphatidylserine

The binding of factor VII and tissue factor produces a membrane-associated proteolytic complex which may be the primary biological initiator of coagulation. Homogeneous tissue factor, a glycoprotein purified from bovine brain, was reconstituted into phospholipid vesicles ranging from neutral (100% phosphatidylcholine) to highly charged (40% phosphatidylserine) with octyl glucoside. The vesicles were characterized with respect to size and tissue factor content and orientation. Employing data from protease digestion, we deduced that tissue factor is randomly oriented; thus, its effective concentration in these vesicles was half its total concentration. In all binding experiments, 1 mol of enzyme was bound per mole of available activator at saturation. This stoichiometry was not affected by the form of the enzyme employed or the phospholipid composition of the vesicles. With tissue factor incorporated into phosphatidylcholine vesicles, the Kd was 13.2 +/- 0.72 nM for factor VII and 4.54 +/- 1.37 nM for factor VIIa. Thus, the one-chain zymogen binds to the activator with only slightly less affinity than the more active two-chain enzyme. Active-site modification of factor VII and factor VIIa with diisopropyl fluorophosphate resulted in tighter binding of the derivatized molecules. Inclusion of phosphatidylserine in the vesicles altered the binding both quantitatively and qualitatively. With increasing acidic phospholipid, the concentration of enzyme required to occupy half the activator sites was decreased. In addition, positive cooperativity was observed, the degree of which depended on the vesicle charge and the form of the enzyme. An explicit two-site cooperative binding model is presented which fits these complex data. In this model, tissue factor is at least a dimer with two interacting enzyme binding sites.     

A single-step large-scale purification of pyruvate oxidase

Pyruvate oxidase is an Escherichia coli peripheral membrane flavoprotein which catalyzes the oxidative decarboxylation of pyruvate to acetate and CO2. Pyruvate oxidase, like several other peripheral membrane enzymes, can be activated either by binding to lipid amphiphiles or by limited protease digestion. This paper reports a rapid and convenient method for effecting the large-scale purification of pyruvate oxidase from crude enzyme preparations using a Triton X-114 phase separation technique. It appears likely that this purification procedure can be used successfully with the family of enzymes which respond to both lipid and protease activation.     

ADP competes with FAD binding in putrescine oxidase

Putrescine oxidase from Rhodococcus erythropolis NCIMB 11540 (PuO(Rh)) is a soluble homodimeric flavoprotein of 100 kDa, which catalyzes the oxidative deamination of putrescine and some other aliphatic amines. The initial characterization of PuO(Rh) uncovered an intriguing feature: the enzyme appeared to contain only one noncovalently bound FAD cofactor per dimer. Here we show that this low FAD/protein ratio is the result of tight binding of ADP, thereby competing with FAD binding. MS analysis revealed that the enzyme is isolated as a mixture of dimers containing two molecules of FAD, two molecules ADP, or one FAD and one ADP molecule. In addition, based on a structural model of PuO(Rh) that was built using the crystal structure of human monoamine oxidase B (MAO-B), we constructed an active mutant enzyme, PuO(Rh) A394C, that contains covalently bound FAD. These findings show that the covalent FAD-protein linkage can be formed autocatalytically and hint to a new-found rationale for covalent flavinylation: covalent flavinylation may have evolved to prevent binding of ADP or related cellular compounds, which would prohibit formation of flavinylated and functional enzyme.