Identification of the covalently bound flavin prosthetic group of cholesterol oxidase.

Highly purified preparations of cholesterol oxidase from Schizophyllum commune contain a covalently bound flavin component. A flavin peptide has been obtained by digestion with trypsin-chymotrypsin and purification on a column of phosphocellulose. Digestion with nucleotide pyrophosphatase results in increased fluorescence at pH 3.4 and release of 5'-adenylate, showing that the flavin is in the dinucleotide form. The absorption spectrum of the flavin peptide shows the hypsochromic shift of the second absorption band characteristic of 8 alpha-substituted flavins. The fluorescence at pH 7 is extensively quenched even in the mononucleotide form, with a pKa at pH 5.8 in the flavin peptide and at 5.05 following acid hydrolysis to the aminoacyl flavin level. This suggests that histidine is the amino acid substituted at the 8 alpha position of the flavin and that N(1) of the imidazole ring is the site of attachment. These data, the reduction of the flavin by borohydride, and comparison of the mobilities in high voltage electrophoresis at two pH values with N(1)- and N(3)-histidyl riboflavin and their 2',5'-anhydro forms shows that the prosthetic group of cholesterol oxidase is 8 alpha-[N(1)-histidyl]-FAD.     

Identification of the covalently bound flavin of thiamin dehydrogenase.

Thiamin dehydrogenase, a flavoprotein isolated from an unidentified soil bacterium, contains 1 mol of covalently bound FAD/mol of enzyme. A flavin peptide, isolated from tryptic-chymotryptic digests of the enzyme and hydrolyzed to the FMN level, shows a pH-dependent fluorescence yield being maximal at pH 3.5 to 4.0 and decreasing over 90% at pH 7.5 with a pKa of 5.8. Acid hydrolysis of the peptide results in an aminoacylflavin which shows a pKa of fluorescence quenching of 5.2. Absorption and electron paramagnetic resonance spectral data show the covalent substituent to be at the 8alpha position of the flavin as is the case with all known enzymes containing covalently bound flavin. The aminoacylflavin gives a negative Pauly reaction but yields 1 mol of histidine on drastic acid hydrolysis thus showing an imidazole ring nitrogen as the 8alpha substituent of the flavin. The aminoacylflavin differs from synthetic 8alpha-[N(3)-histidyl]riboflavin or its acid-modified form in pKa of fluorescence quenching, in electrophoretic mobility, in being reduced by borohydride, and in being labile to storage, yielding 8-formylriboflavin. In all of these properties, however, the 8alpha-histidylriboflavin isolated from thiamin dehydrogenase is indistinguishable from 8alpha-[N(1)-histidyl]riboflavin. It is therefore concluded that the FAD moiety of thiamin dehydrogenase is covalently linked via the 8alpha-methylene group to the N(1) position of the imidazole ring of histidine.     

Identification and properties of the covalently bound flavin of beta-cyclopiazonate oxidocyclase.

Beta-Cyclopiazonate oxidocyclase from Penicillium cyclopium has been previously shown to contain flavin dinucleotide in covalent linkage to the protein. In the present study, a pure flavin mononucleotide peptide was isolated from the enzyme by tryptic-chymotryptic digestion, chromatography on Florisil and on diethylaminoethylcellulose, and hydrolysis with nucleotide pyrophosphatase. The flavin peptide contains 9 amino acids, including histidine in linkage to the flavin, and Asx as the N-terminal residue. The fluorescence of the flavin in the FMN peptide is profoundly quenched even at pH 3.2, where protonation of the imidazole prevents queching of the flavin fluorescence by histidine. This quenching appears to be due to interaction of the flavin with a tryptophan residue, as the quenching is abolished by oxidation of the tryptophan with performic acid. Similarly, the fluorescence of the tryptophan in the peptide is quenched, presumably by the flavin. The flavin of beta-cyclopiazonate oxidocylcase is attached, by the way of the 8alpha-methylene group, to the imidazole ring of a histidine. The aminoacylflavin isolated from the enzyme is identical in the pKa of its imidazole group, in reduction by NaBH4, and in other properties with synthetic 8alpha-(N1-histidyl)riboflavin. The pKa of the histidylriboflavin component of the oxidocyclase is 5.2 before and 5.0 after acid modification of the ribityl chain, as is found in the synthetic derivative. It is concluded that the enzyme contains the N1 isomer of histidylriboflavin and that acid hydrolysis of flavin peptides isolated from the oxidocyclase, while liberating histidylriboflavin, also causes acid modification of the ribityl chain of the flavin moiety.     

Dissecting the structural determinants of the stability of cholesterol oxidase containing covalently bound flavin

Cholesterol oxidase from Brevibacterium sterolicum is a monomeric flavoenzyme catalyzing the oxidation and isomerization of cholesterol to cholest-4-en-3-one. This protein is a class II cholesterol oxidases, with the FAD cofactor covalently linked to the enzyme through the His(69) residue. In this work, unfolding of wild-type cholesterol oxidase was compared with that of a H69A mutant, which does not covalently bind the flavin cofactor. The two protein forms do not show significant differences in their overall topology, but the urea-induced unfolding of the H69A mutant occurred at significant lower urea concentrations than wild-type (approximately 3 versus approximately 5 M, respectively), and the mutant protein had a melting temperature approximately 10-15 degrees C lower than wild-type in thermal denaturation experiments. The different sensitivity of the various spectroscopic features used to monitor protein unfolding indicated that in both proteins a two-step (three-state) process occurs. The presence of an intermediate was more evident for the H69A mutant at 2 m urea, where catalytic activity and tertiary structure were lost, and new hydrophobic patches were exposed on the protein surface, resulting in protein aggregation. Comparative analysis of the changes occurring upon urea and thermal treatment of the wild-type and H69A protein showed a good correlation between protein instability and the elimination of the covalent link between the flavin and the protein. This covalent bond represents a structural device to modify the flavin redox potentials and stabilize the tertiary structure of cholesterol oxidase, thus pointing to a specific meaning of the flavin binding mode in enzymes that carry out the same reaction in pathogenic versus non-pathogenic bacteria.     

Spectroscopic studies on the photoreaction of choline oxidase, a flavoprotein, with covalently bound flavin

Formation of the anionic flavosemiquinone was observed spectrophotometrically during the anaerobic photo-irradiation of Alcaligenes sp. choline oxidase in the presence of EDTA. Further irradiation slowly converted the semiquinone form into the fully reduced state. The presence of a catalytic amount of riboflavin greatly enhances the photoreduction rate not only to the semiquinone state but also to the fully reduced state. This semiquinone species has low reactivity toward the substrate, choline or betaine aldehyde, as well as toward oxygen. This low reactivity toward oxygen is unique to the semiquinone form of a flavoprotein oxidase. The oxidized enzyme forms a complex with betaine, the product of the enzymatic reaction of choline oxidase. The dissociation constant for this complex was found to be 17 mM by spectroscopic titration. Anaerobic photo-irradiation of the enzyme with a saturating amount of betaine in the absence of EDTA produces, with no detectable semiquinone formation, an absorption spectrum which resembles (but significantly differs from) that of the fully reduced form. This species was found to comprise two flavin species. One of them is rapidly oxidized to the oxidized form by oxygen and is thus assigned as the fully reduced state. The other is converted slowly to the oxidized form upon aerobic standing in the dark. We tentatively assigned this latter species as a C(4a)-adduct. Formaldehyde was detected as a product of this photoreaction. The amount of formaldehyde formed coincided with that of the fully reduced enzyme. On the basis of the results obtained we propose a mechanism of the photoreaction of the enzyme in the presence of betaine where a C(4a)-adduct and the fully reduced enzyme via an N(5)-adduct are formed. Betaine also affects the dithionite reduction. In the dithionite reduction of the oxidized enzyme, the semiquinone species is an intermediate in the conversion of the oxidized to the fully reduced form, while the reduction of the oxidized enzyme-betaine complex with dithionite produces the fully reduced form without any significant formation of the semiquinone species.     

Biosynthesis of covalently bound flavin: isolation and in vitro flavinylation of the monomeric sarcosine oxidase apoprotein

The covalently bound FAD in native monomeric sarcosine oxidase (MSOX) is attached to the protein by a thioether bond between the 8alpha-methyl group of the flavin and Cys315. Large amounts of soluble apoenzyme are produced by controlled expression in a riboflavin-dependent Escherichia coli strain. A time-dependent increase in catalytic activity is observed upon incubation of apoMSOX with FAD, accompanied by the covalent incorporation of FAD to approximately 80% of the level observed with the native enzyme. The spectral and catalytic properties of the reconstituted enzyme are otherwise indistinguishable from those of native MSOX. The reconstitution reaction exhibits apparent second-order kinetics (k = 139 M(-)(1) min(-)(1) at 23 degrees C) and is accompanied by the formation of a stoichiometric amount of hydrogen peroxide. A time-dependent reduction of FAD is observed when the reconstitution reaction is conducted under anaerobic conditions. The results provide definitive evidence for autoflavinylation in a reaction that proceeds via a reduced flavin intermediate and requires only apoMSOX and FAD. Flavinylation of apoMSOX is not observed with 5-deazaFAD or 1-deazaFAD, an outcome attributed to a decrease in the acidity of the 8alpha-methyl group protons. Covalent flavin attachment is observed with 8-nor-8-chloroFAD in an aromatic nucleophilic displacement reaction that proceeds via a quininoid intermediate but not a reduced flavin intermediate. The reconstituted enzyme contains a modified cysteine-flavin linkage (8-nor-8-S-cysteinyl) as compared with native MSOX (8alpha-S-cysteinyl), a difference that may account for its approximately 10-fold lower catalytic activity.     

Thiols liberate covalently bonded flavin from monoamine oxidase

Although it was recommended that 2-mercaptoethanol should be added to all buffers used in monoamine oxidase purification, purification of the enzyme in the absence of thiols has yielded double the amount of enzyme. In fact the presence of 0.1 M 2-mercaptoethanol or dithioerythritol-SDS¹ was found to liberate about 50% of the covalently bonded FAD. This observation is surprising since the covalently bonded flavin has been reported to be 8-α-cysteinyl-FAD. There is a thioether linkage between the flavin and the protein and mild reducing agents such as mercaptoethanol would not normally cleave the thioether linkage. The importance of the present finding is that the thiols may be used possibly to liberate suicide substrate-flavin adducts from the enzyme for structural studies without isolating pure flavin peptides. The flavin can be removed simply by treatment of the enzyme with 0.1 M thiol solution containing 1% sodium dodecylsulfate followed by chromatography on Sephadex G-25.     

Micro-determination of L-gulono-gamma-lactone oxidase activity.

Highly sensitive assay method of L-gulono-gamma-lactone oxidase (GLO) was constructed. In this method, L-ascorbic acid formed in the enzymatic reaction was converted to its bis(dinitrophenyl)hydrazone derivative, and the amount of the latter was determined by high-performance liquid chromatography. Twenty picomoles of ascorbic acid was detected, which makes this method 25 times more sensitive than the previously used dipyridyl one. By the present method, a minute activity of GLO in liver microsomes prepared from rats of the Osteogenic Disorder Shionogi strain (ODS-od/od) could be measured.     

Covalently bound flavin as prosthetic group of choline oxidase.

Highly purified choline oxidase of $\text{Arthrobacter globiformis}$ fluoresced as a yellow band on SDS gel in 7% acetic acid. The absorption spectrum of the enzyme showed marked hypsochromic shift of the second absorption band. Aminoacyl flavin obtained from this enzyme was identified with 8α-[N(3)-histidyl]FAD.     

Fumarate reductase mutants of Escherichia coli that lack covalently bound flavin

Menaquinol-fumarate oxidoreductase of Escherichia coli is a four-subunit membrane-bound complex that catalyzes the final step in anaerobic respiration when fumarate is the terminal electron acceptor. The catalytic domain of fumarate reductase consists of the FrdA subunit, which contains the active site, and a FAD prosthetic group covalently attached to His44, plus the FrdB subunit which contains at least two of the three nonidentical iron-sulfur clusters of the enzyme. To examine the role of covalently bound FAD in enzyme activity and electron transfer during anaerobic cell growth, site-directed mutagenesis was used to alter His44 of the FrdA subunit to a Ser, Cys, or Tyr residue. The resulting mutant enzyme complexes that were synthesized associated normally with the cytoplasmic membrane, but had decreased ability (greater than 70%) to reduce fumarate with reduced benzyl viologen, an artificial electron donor of low redox potential (Em = -359 mV; Clark, W. M. (1972) Oxidation-Reduction Potentials of Organic Systems, Robert E. Kreiger Publishing Co., Melbourne, FL). Even lower activities were measured when the higher potential, natural electron donor menaquinol was used, which, however, correlated with the slower growth rates of the different mutant complexes. In contrast to the normal enzyme, the mutant enzyme complexes were unable to oxidize succinate. Substitution of Arg for His44 produced a totally inactive enzyme complex that permitted no cell growth on nonfermentable substrates with fumarate as electron acceptor. All four mutant complexes contained noncovalently bound FAD in stoichiometric amounts. These data indicate a unique role of the 8 alpha-[N(3)-histidyl] FAD linkage in enzyme activity, by raising the redox potential of free FAD to permit reduction by both menaquinol and succinate.     

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.