Heterologous expression and characterization of mouse spermine oxidase

Polyamine oxidases are key enzymes responsible of the polyamine interconversion metabolism in animal cells. Recently, a novel enzyme belonging to this class of enzymes has been characterized for its capability to oxidize preferentially spermine and designated as spermine oxidase. This is a flavin adenine dinucleotide-containing enzyme, and it has been expressed both in vitro and in vivo systems. The primary structure of mouse spermine oxidase (mSMO) was deduced from a cDNA clone (Image Clone 264769) recovered by a data base search utilizing the human counterpart of polyamine oxidases, PAOh1. The open reading frame predicts a 555-amino acid protein with a calculated M(r) of 61,852.30, which shows a 95.1% identity with PAOh1. To understand the biochemical properties of mSMO and its structure/function relationship, the mSMO cDNA has been subcloned and expressed in secreted and secreted-tagged forms into Escherichia coli BL21 DE3 cells. The recombinant enzyme shows an optimal pH value of 8.0 and is able to oxidize rapidly spermine to spermidine and 3-aminopropanal and fails to act upon spermidine and N(1)-acetylpolyamines. The purified recombinant-tagged form enzyme (M(r) approximately 68,000) has K(m) and k(cat) values of 90 microm and 4.5 s(-1), respectively, using spermine as substrate at pH 8.0. Molecular modeling of mSMO protein based on maize polyamine oxidase three-dimensional structure suggests that the general features of maize polyamine oxidase active site are conserved in mSMO.     

A lysine conserved in the monoamine oxidase family is involved in oxidation of the reduced flavin in mouse polyamine oxidase

Lysine 315 of mouse polyamine amine oxidase corresponds to a lysine residue that is conserved in the flavoprotein amine oxidases of the monoamine oxidase structural family. In several structures, this lysine residue forms a hydrogen bond to a water molecule that is hydrogen-bonded to the flavin N(5). Mutation of Lys315 in polyamine oxidase to methionine was previously shown to have no effect on the kinetics of the reductive half-reaction of the enzyme (M. Henderson Pozzi, V. Gawandi, P.F. Fitzpatrick, Biochemistry 48 (2009) 1508-1516). In contrast, the mutation does affect steps in the oxidative half-reaction. The k_cat value is unaffected by the mutation; this kinetic parameter likely reflects product release. At pH 10, the k_cat/K_m value for oxygen is 25-fold lower in the mutant enzyme. The k_cat/K_O2 value is pH-dependent for the wild-type enzyme, decreasing below a pK_a of 7.0, while this kinetic parameter for the mutant enzyme is pH-independent. This is consistent with the neutral form of Lys315 being required for more rapid flavin oxidation. The solvent isotope effect on the k_cat/K_O2 value increases from 1.4 in the wild-type enzyme to 1.9 in the mutant protein, and the solvent inventory changes from linear to bowed. The effects of the mutation can be explained by the lysine orienting the bridging water so that it can accept the proton from the flavin N(5) during flavin oxidation. In the mutant enzyme the lysine amine would be replaced by a water chain.     

Purification and some properties of cholesterol oxidase from Schizophyllum commune with covalently bound flavin.

Cholesterol oxidase [EC 1.1.3.6] from Schizophyllum commune was purified by an affinity chromatography using 3-O-succinylcholesterol-ethylenediamine (3-cholesteryl-3-[2-aminoethylamido]propionate) Sepharose gels. The resulting preparation was homogeneous as judged by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 53,000 by SDS-gel electrophoresis and 46,000 by sedimentation equilibrium. The enzyme contained 483 amino acid residues as calculated on the basis of the molecular weight of 53,000. The enzyme consumed 60 mumol of O2/min per mg of protein with 1.3 mM cholesterol at 37 degrees C. The enzyme showed the highest activity with cholesterol; 3 beta-hydroxysteroids, such as dehydroepiandrosterone, pregnenolone, and lanosterol, were also oxidized at slower rates. Ergosterol was not oxidized by the enzyme. The Km for cholesterol was 0.33 mM and the optimal pH was 5.0. The enzyme is a flavoprotein which shows a visible absorption spectrum having peaks at 353 nm and 455 nm in 0.1 M acetate buffer, pH 4.0. The spectrum was characterized by the hypsochromic shift of the second absorption peak of the bound flavin. The bound flavin was reduced on anaerobic addition of a model substrate, dehydroepiandrosterone. Neither acid not heat treatment released the flavin coenzyme from the enzyme protein. The flavin of the enzyme could be easily released from the enzyme protein in acid-soluble form as flavin peptides when the enzyme protein was digested with trypsin plus chymotrypsin. The mobilities of the aminoacyl flavin after hydrolysis of the flavin peptides on thin layer chromatography and high voltage electrophoresis differed from those of free FAD, FMN, and riboflavin. A pKa value of 5.1 was obtained from pH-dependent fluorescence quenching process of the aminoacyl flavin. AMP was detected by hydrolysis of the flavin peptides with nucleotide pyrophosphatase. The results indicate strongly that cholesterol oxidase from Schizophyllum commune contains FAD as the prothetic group, which is covalently linked to the enzyme protein. The properties of the bound FAD were comparable to those of N (1)-histidyl FAD.     

Bis(benzyl)polyamine analogs as novel substrates for polyamine oxidase

N,N'-Bis(benzyl)polyamine analogs were found to be substrates for highly purified polyamine oxidase. Metabolism of these analogs was apparently dependent on molecular O2 and resulted in the formation of benzaldehyde, H2O2, and a polyamine analog with free terminal amines. The debenzylation reaction was optimal between pH 9 and 10, identical to the pH optimum for polyamine oxidase activity when N1-acetylspermine was used as the substrate. On a molecular sieve column the debenzylating activity co-eluted with N1-acetylspermine oxidizing activity, at an apparent molecular mass of approximately 65 kDa. The purified enzyme also appeared to have a molecular mass of approximately 65 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Debenzylation of the bis(benzyl)polyamines was competitively inhibited by N1-acetylspermine and N1-acetylspermidine. The specific irreversible inhibitor of polyamine oxidase, N1,N4-bis(buta-2,3-dienyl)butanediamine also inhibited the debenzylation, whereas inhibitors of diamine and monoamine oxidases did not. The evolution of benzaldehyde from bis(benzyl)polyamine analogs by polyamine oxidase allowed the development of a simple rapid spectrophotometric assay for use in the measurement of polyamine oxidase activity in partially purified tissue or cell extracts. Further, metabolism of a bis(benzyl)polyamine analog by polyamine oxidase was found to be an important element in the growth inhibitory properties of the compound in a mouse model of malaria.     

The effects of reversible freezing inactivation and inhibitor binding on redox properties of l-amino-acid oxidase

We measured the redox potentials of frozen inactivated l-amino-acid oxidase (l-amino-acid:oxygen oxidoreductase (deaminating), EC 1.4.3.2) and inhibitor-bound (anthranilic acid) enzyme, and compared these redox properties to those of active l-amino-acid oxidase and benzoate-bound d-amino-acid oxidase (EC 1.4.3.3), respectively. The redox properties of the inactive enzyme are similar to the properties of free flavin; the potential is within 0.015 V of free flavin and no radical stabilization is seen. This corresponds to the loss of most interactions between apoprotein and flavin. In contrast, the anthranilic acid lowers the amount of radical stabilized from 85% to 35%. The potentials are still 0.150 V positive of free flavin, indicating that in the presence of inhibitor, many flavin-protein interactions remain intact. The difference between this behavior and that of d-amino-acid oxidase bound to benzoate, where the amount of radical declined from 95% to 5%, is explained on the basis of the relative tightness of binding of apoprotein to FAD. d-Amino-acid oxidase apoprotein has a relatively low K_a (10⁶) for FAD, and benzoate has a relatively high K_a (10⁵) for the enzyme. Therefore, the binding of benzoate increases the tightness of FAD binding to apo-d-amino-acid oxidase (10¹¹), indicating significant changes in flavin-protein interactions. In contrast, apo-l-amino-acid oxidase binds flavin tightly (the K_a is greater than 10⁷) and the enzyme binds to anthranilate much less tightly, with a K_a of 10³. The l-amino-acid oxidase apoprotein binding to FAD is tight initially, and the binding of anthranilate changes it only slightly. Therefore, redox studies indicate that the ability of a flavoprotein to be regulated may be influenced by the strength of the interaction of flavin with the apoprotein, as well as the strength of interaction of the substrate or activator.     

Properties of D-amino-acid oxidase from Rhodotorula gracilis

The flavoprotein D-amino-acid oxidase was purified to homogeneity from the yeast Rhodotorula gracilis by a highly reproducible procedure. The amino acid composition of the protein was determined; the protein monomer had a molecular mass of 39 kDa and contained one molecule of FAD. The ratio between A274/A455 was about 8.2. D-Amino-acid oxidase from yeast showed typical flavin spectral perturbations on binding of the competitive inhibitor benzoate and was reduced by D-alanine under anaerobiosis. The enzyme reacted readily with sulfite to form a covalent reversible adduct and stabilized the red anionic form of the flavin semiquinone on photoreduction in the presence of 5-deazariboflavin; the 3,4-dihydro-FAD form was not detectable after reduction with sodium borohydride. Thus D-amino-acid oxidase from yeast exhibited most of the general properties of the dehydrogenase/oxidase class of flavoproteins; at the same time, the enzyme showed some peculiar features with respect to the same protein from pig kidney.     

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.     

Cholesterol oxidase from Brevibacterium sterolicum. The relationship between covalent flavinylation and redox properties

Brevibacterium sterolicum possesses two forms of cholesterol oxidase, one containing noncovalently bound FAD, the second containing a FAD covalently linked to His(69) of the protein backbone. The functional role of the histidyl-FAD bond in the latter cholesterol oxidase was addressed by studying the properties of the H69A mutant in which the FAD is bound tightly, but not covalently, and by comparison with native enzyme. The mutant retains catalytic activity, but with a turnover rate decreased 35-fold; the isomerization step of the intermediate 3-ketosteroid to the final product is also preserved. Stabilization of the flavin semiquinone and binding of sulfite are markedly decreased, this correlates with a lower midpoint redox potential (-204 mV compared with -101 mV for wild-type). Reconstitution with 8-chloro-FAD led to a holoenzyme form of H69A cholesterol oxidase with a midpoint redox potential of -160 mV. In this enzyme form, flavin semiquinone is newly stabilized, and a 3.5-fold activity increase is observed, this mimicking the thermodynamic effects induced by the covalent flavin linkage. It is concluded that the flavin 8alpha-linkage to a (N1)histidine is a pivotal factor in the modulation of the redox properties of this cholesterol oxidase to increase its oxidative power.     

Inhibition of plant amine oxidases by a novel series of diamine derivatives

A series of N,N'-bis(2-pyridinylmethyl)diamines was synthesized and characterized for their inhibition effects towards plant copper-containing amine oxidase (EC 1.4.3.6) and polyamine oxidase (EC 1.5.3.11), which mediate the catabolic regulation of cellular polyamines. Even though these enzymes catalyze related reactions and, among others, act upon two common substrates (spermidine and spermine), their molecular and kinetic properties are different. They also show a different spectrum of inhibitors. It is therefore of interest to look for compounds providing a dual inhibition (i.e. inhibiting both enzymes with the same inhibition potency), which would be useful in physiological studies involving modulations of polyamine catabolism. The synthesized diamine derivatives comprised from two to eight carbon atoms in the alkyl spacer chain. Kinetic measurements with pea (Pisum sativum) diamine oxidase and oat (Avena sativa) polyamine oxidase demonstrated reversible binding of the compounds at the active sites of the enzymes as they were almost exclusively competitive inhibitors with K(i) values ranging from 10(-5) to 10(-3)M. In case of oat polyamine oxidase, the K(i) values were significantly influenced by the number of methylene groups in the inhibitor molecule. The measured inhibition data are discussed with respect to enzyme structure. For that reason, the oat enzyme was analyzed by de novo peptide sequencing using mass spectrometry and shown to be homologous to polyamine oxidases from barley (isoform 1) and maize. We conclude that some of the studied N,N'-bis(2-pyridinylmethyl)diamines might have a potential to be starting structures in design of metabolic modulators targeted to both types of amine oxidases.     

Structural bases for inhibitor binding and catalysis in polyamine oxidase

Polyamine oxidase (PAO) carries out the FAD-dependent oxidation of the secondary amino groups of spermidine and spermine, a key reaction in the polyamine catabolism. The active site of PAO consists of a 30 A long U-shaped catalytic tunnel, whose innermost part is located in front of the flavin ring. To provide insight into the PAO substrate specificity and amine oxidation mechanism, we have investigated the crystal structure of maize PAO in the reduced state and in complex with three different inhibitors, guazatine, 1,8-diaminooctane, and N(1)-ethyl-N(11)-[(cycloheptyl)methyl]-4,8-diazaundecane (CHENSpm). In the reduced state, the conformation of the isoalloxazine ring and the surrounding residues is identical to that of the oxidized enzyme. Only Lys300 moves away from the flavin to compensate for the change in cofactor protonation occurring upon reduction. The structure of the PAO.inhibitor complexes reveals an exact match between the inhibitors and the PAO catalytic tunnel. Inhibitor binding does not involve any protein conformational change. Such lock-and-key binding occurs also in the complex with CHENSpm, which forms a covalent adduct with the flavin N5 atom. Comparison of the enzyme complexes hints at an "out-of-register" mechanism of inhibition, in which the inhibitor secondary amino groups are not properly aligned with respect to the flavin to allow oxidation. Except for the Glu62-Glu170 pair, no negatively charged residues are involved in the recognition of substrate and inhibitor amino groups, which is in contrast to other polyamine binding proteins. This feature may be exploited in the design of drugs specifically targeting PAO.     

Polyamine metabolism and cancer

Polyamines are aliphatic cations present in all cells. In normal cells, polyamine levels are intricately controlled by biosynthetic and catabolic enzymes. The biosynthetic enzymes are ornithine decarboxylase, S-adenosylmethionine decarboxylase, spermidine synthase, and spermine synthase. The catabolic enzymes include spermidine/spermine acetyltransferase, flavin containing polyamine oxidase, copper containing diamine oxidase, and possibly other amine oxidases. Multiple abnormalities in the control of polyamine metabolism and uptake might be responsible for increased levels of polyamines in cancer cells as compared to that of normal cells. This review is designed to look at the current research in polyamine biosynthesis, catabolism, and transport pathways, enumerate the functions of polyamines, and assess the potential for using polyamine metabolism or function as targets for cancer therapy.     

Structure of the flavocoenzyme of two homologous amine oxidases: monomeric sarcosine oxidase and N-methyltryptophan oxidase

Monomeric sarcosine oxidase (MSOX) and N-methyltryptophan oxidase (MTOX) are homologous enzymes that catalyze the oxidative demethylation of sarcosine (N-methylglycine) and N-methyl-L-tryptophan, respectively. MSOX is induced in various bacteria upon growth on sarcosine. MTOX is an E. coli enzyme of unknown metabolic function. Both enzymes contain covalently bound flavin. The covalent flavin is at the FAD level as judged by electrospray mass spectrometry. The data provide the first evidence that MTOX is a flavoprotein. The following observations indicate that 8alpha-(S-cysteinyl)FAD is the covalent flavin in MSOX from Bacillus sp. B-0618 and MTOX. FMN-containing peptides, prepared by digestion of MSOX or MTOX with trypsin, chymotrypsin, and phosphodiesterase, exhibited absorption and fluorescence properties characteristic of an 8alpha-(S-cysteinyl)flavin and could be bound to apo-flavodoxin. The thioether link in the FMN-containing peptides was converted to the sulfone by performic acid oxidation, as judged by characteristic absorbance changes and an increase in flavin fluorescence. The sulfone underwent a predicted reductive cleavage reaction upon treatment with dithionite, releasing unmodified FMN. Cys315 was identified as the covalent FAD attachment site in MSOX from B. sp. B-0618, as judged by the sequence obtained for a flavin-containing tryptic peptide (GAVCMYT). Cys315 aligns with a conserved cysteine in MSOX from other bacteria, MTOX (Cys308) and pipecolate oxidase, a homologous mammalian enzyme known to contain covalently bound flavin. There is only one conserved cysteine found among these enzymes, suggesting that Cys308 is the covalent flavin attachment site in MTOX.     

L-amino-acid oxidase from the Malayan pit viper Calloselasma rhodostoma. Comparative sequence analysis and characterization of active and inactive forms of the enzyme.

Here we report the cDNA-deduced amino-acid sequence of L-amino-acid oxidase (LAAO) from the Malayan pit viper Calloselasma rhodostoma, which shows 83% identity to LAAOs from the Eastern and Western diamondback rattlesnake (Crotalus adamanteus and Crotalus atrox, respectively). Phylogenetic comparison of the FAD-dependent ophidian LAAOs to FAD-dependent oxidases such as monoamine oxidases, D-amino-acid oxidases and tryptophan 2-monooxygenases reveals only distant relationships. Nevertheless, all LAAOs share a highly conserved dinucleotide-binding fold with monoamine oxidases, tryptophan 2-monooxygenases and various other proteins that also may have a requirement for FAD. In order to characterize Ca. rhodostoma LAAO biochemically, the enzyme was purified from snake venom to apparent homogeneity. It was found that the enzyme undergoes inactivation by either freezing or increasing the pH to above neutrality. Both inactivation processes are fully reversible and are associated with changes in the UV/visible range of the flavin absorbance spectrum. In addition, the spectral characteristics of the freeze-and pH-induced inactivated enzyme are the same, indicating that the flavin environments are similar in the two inactive conformational forms. Monovalent anions, such as Cl(-), prevent pH-induced inactivation. LAAO exhibits typical flavoprotein oxidase properties, such as thermodynamic stabilization of the red flavin semiquinone radical and formation of a sulfite adduct. The latter complex as well as the complex with the competitive substrate inhibitor, anthranilate, were only formed with the active form of the enzyme indicating diminished accessibility of the flavin binding site in the inactive form(s) of the enzyme.     

Inhibition of polyamine oxidase enhances the cytotoxicity of polyamine oxidase substrates. A model study with N1-(n-octanesulfonyl)spermine and human colon cancer cells

N(1)-(n-octanesulfonyl)spermine (N(1) OSSpm) is a substrate of polyamine oxidase. It shares several properties with spermine, such as antagonism of NMDA-type glutamate receptors, calmodulin antagonism, and cytotoxicity, but it is more potent by orders of magnitude in these regards than spermine. The human colon carcinoma-derived cell line CaCo-2 was used as a model to study the toxicity of N(1) OSSpm as a function of polyamine oxidase (PAO) activity and differentiation. If the formation of hydrogen peroxide and aminoaldehyde by the PAO-catalysed reactions was prevented by selective inactivation of the enzyme with MDL 72527, cytotoxicity of N(1)OSSpm was not diminished, but on the contrary, enhanced. Exponentially growing CaCo-2 cells were considerably more sensitive to N(1)OSSpm than differentiating cells. The results suggest that cytotoxic substrates of PAO exhibit enhanced cytotoxicity in cells, if PAO activity is inhibited. Since tumour cells are known to have lower polyamine oxidase activities than their normal counterparts, it will be interesting to explore whether cytotoxic substrates of polyamine oxidase, for which N(1)OSSpm is an example, are suited to preferentially kill tumour cells.     

Chemical and catalytic properties of the peroxisomal acyl-coenzyme A oxidase from Candida tropicalis

The peroxisomal acyl-CoA oxidase has been purified from extracts of the yeast Candida tropicalis grown with alkanes as the principal energy source. The enzyme has a molecular weight of 552,000 and a subunit molecular weight of 72,100. Using an experimentally determined molar extinction coefficient for the enzyme-bound flavin, a minimum molecular weight of 146,700 was determined. Based on these data, the oxidase contains eight perhaps identical subunits and four equivalents of FAD. No other β-oxidation enzyme activities are detected in purified preparations of the oxidase. The oxidase flavin does not react with sulfite to form an N(5) flavin-sulfite complex. Photochemical reduction of the oxidase flavin yields a red semiquinone; however, the yield of semiquinone is strongly pH dependent. The yield of semiquinone is significantly reduced below pH 7.5. The flavin semiquinone can be further reduced to the hydroquinone. The behavior of the oxidase flavin during photoreduction and its reactivity toward sulfite are interpreted to reflect the interaction in the N(1)-C(2)O region of the flavin with a group on the protein which acts as a hydrogen-bond acceptor. Like the acyl-CoA dehydrogenases which catalyze the same transformation of acyl-CoA substrates, the oxidase is inactivated by the acetylenic substrate analog, 3-octynoyl-CoA, which acts as an active site-directed inhibitor.     

A 30-angstrom-long U-shaped catalytic tunnel in the crystal structure of polyamine oxidase.

BACKGROUND: Polyamines are essential for cell growth and differentiation; compounds interfering with their metabolism are potential anticancer agents. Polyamine oxidase (PAO) plays a central role in polyamine homeostasis. The enzyme utilises an FAD cofactor to catalyse the oxidation of the secondary amino groups of spermine and spermidine. RESULTS: The first crystal structure of a polyamine oxidase has been determined to a resolution of 1.9 Angstroms. PAO from Zea mays contains two domains, which define a remarkable 30 Angstrom long U-shaped catalytic tunnel at their interface. The structure of PAO in complex with the inhibitor MDL72527 reveals the residues forming the catalytic machinery and unusual enzyme-inhibitor CH.O H bonds. A ring of glutamate and aspartate residues surrounding one of the two tunnel openings contributes to the steering of the substrate towards the inside of the tunnel. CONCLUSIONS: PAO specifically oxidizes substrates that have both primary and secondary amino groups. The complex with MDL72527 shows that the primary amino groups are essential for the proper alignment of the substrate with respect to the flavin. Conservation of an N-terminal sequence motif indicates that PAO is member of a novel family of flavoenzymes. Among these, monoamine oxidase displays significant sequence homology with PAO, suggesting a similar overall folding topology.     

Fumarate reductase of Escherichia coli. Elucidation of the covalent-flavin component.

Fumarate reductase is a membrane-bound terminal oxidase which is induced when Escherichia coli is grown anaerobically. The purified enzyme is composed of two polypeptide chains of 69,000 and 24,000 daltons and contains 1 mol of covalently bound flavin adenine dinucleotide per mol of enzyme. Fluorescence scanning of SDS-polyacrylamide gels of the protein shows that the flavin is attached to the large subunit. The hypsochromic shift of the 372 nm band of riboflavin to 350 nm in both native fumarate reductase and a flavin peptide released by proteolytic digestion indicates that the flavin is attached via position 8 alpha of riboflavin. Based on the spectral properties and pH-fluorescence dependence we have identified the linkage as 8 alpha-[N(3)-histidyl]FAD.     

Bacterial sarcosine oxidase: comparison of two multisubunit enzymes containing both covalent and noncovalent flavin

Sarcosine oxidase was purified to homogeneity from Corynebacterium sp. P-1, a soil organism isolated by a serial enrichment technique. The enzyme contains 1 mol of noncovalently bound flavin [flavin adenine dinucleotide (FAD)] plus 1 mol of covalently bound flavin [8 alpha-(N3-histidyl)-FAD] per mole of enzyme (Mr 168,000). The two flavins appear to have different roles in catalysis. The enzyme has an unusual subunit composition, containing four dissimilar subunits (Mr 100,000, 42,000, 20,000, and 6000). The same subunits are detected in Western blot analysis of cell extracts prepared in the presence of trichloroacetic acid, indicating that the subunits are a genuine property of the enzyme as it exists in vivo. The presence of both covalent and noncovalent flavin in a single enzyme is extremely unusual and has previously been observed only with a sarcosine oxidase from a soil Corynebacterium isolated in Japan. The enzymes exhibit many similarities but are distinguishable in electrophoretic studies. Immunologically, the enzymes are cross-reactive but not identical. The results indicate that the synthesis of a sarcosine oxidase containing both covalent and noncovalent flavin is not a particularly unusual event in corynebacteria.     

Effects of reversing the protein positive charge in the proximity of the flavin N(1) locus of choline oxidase

A protein positive charge near the flavin N(1) locus is a distinguishing feature of most flavoprotein oxidases, with mechanistic implications for the modulation of flavin reactivity. A recent study showed that in the active site of choline oxidase the protein positive charge is provided by His(466). Here, we have reversed the charge by substitution with aspartate (CHO-H466D) and, for the first time, characterized a flavoprotein oxidase with a negative charge near the flavin N(1) locus. CHO-H466D formed a stable complex with choline but lost the ability to oxidize the substrate. In contrast to the wild-type enzyme, which binds FAD covalently in a 1:1 ratio, CHO-H466D contained approximately 0.3 FAD per protein, of which 75% was not covalently bound to the enzyme. Anaerobic reduction of CHO-H466D resulted in the formation of a neutral hydroquinone, with no stabilization of the flavin semiquinone; in contrast, the anionic semiquinone and hydroquinone species were observed with the wild type and a H466A variant of the enzyme. The midpoint reduction potential for the oxidized-reduced couple in CHO-H466D was approximately 160 mV lower than that of the wild-type enzyme. Finally, CHO-H466D lost the ability to form complexes with glycine betaine or sulfite. Thus, with a reversal of the protein charge near the FAD N(1) locus, choline oxidase lost the ability to stabilize negative charges in the active site, irrespective of whether they develop on the flavin or are borne on ligands, resulting in defective flavinylation of the protein, the decreased electrophilicity of the flavin, and the consequent loss of catalytic activity.