Organization of the multiple coenzymes and subunits and role of the covalent flavin link in the complex heterotetrameric sarcosine oxidase.

Heterotetrameric (alphabetagammadelta) sarcosine oxidase from Corynebacterium sp. P-1 (cTSOX) contains noncovalently bound FAD and NAD(+) and covalently bound FMN, attached to beta(His173). The beta(His173Asn) mutant is expressed as a catalytically inactive, labile heterotetramer. The beta and delta subunits are lost during mutant enzyme purification, which yields a stable alphagamma complex. Addition of stabilizing agents prevents loss of the delta but not the beta subunit. The covalent flavin link is clearly a critical structural element and essential for TSOX activity or preventing FMN loss. The alpha subunit was expressed by itself and purified by affinity chromatography. The alpha and beta subunits each contain an NH(2)-terminal ADP-binding motif that could serve as part of the binding site for NAD(+) or FAD. The alpha subunit and the alphagamma complex were each found to contain 1 mol of NAD(+) but no FAD. Since NAD(+) binds to alpha, FAD probably binds to beta. The latter could not be directly demonstrated since it was not possible to express beta by itself. However, FAD in TSOX from Pseudomonas maltophilia (pTSOX) exhibits properties similar to those observed for the covalently bound FAD in monomeric sarcosine oxidase and N-methyltryptophan oxidase, enzymes that exhibit sequence homology with beta. A highly conserved glycine in the ADP-binding motif of the alpha(Gly139) or beta(Gly30) subunit was mutated in an attempt to generate NAD(+)- or FAD-free cTSOX, respectively. The alpha(Gly139Ala) mutant is expressed only at low temperature (t(optimum) = 15 degrees C), but the purified enzyme exhibited properties indistinguishable from the wild-type enzyme. The much larger barrier to NAD(+) binding in the case of the alpha(Gly139Val) mutant could not be overcome even by growth at 3 degrees C, suggesting that NAD(+) binding is required for TSOX expression. The beta(Gly30Ala) mutant exhibited subunit expression levels similar to those of the wild-type enzyme, but the mutation blocked subunit assembly and covalent attachment of FMN, suggesting that both processes require a conformational change in beta that is induced upon FAD binding. About half of the covalent FMN in recombinant preparations of cTSOX or pTSOX is present as a reversible covalent 4a-adduct with a cysteine residue. Adduct formation is not prevented by mutating any of the three cysteine residues in the beta subunit of cTSOX to Ser or Ala. Since FMN is attached via its 8-methyl group to the beta subunit, the FMN ring must be located at the interface between beta and another subunit that contains the reactive cysteine residue.

Inactivation of monomeric sarcosine oxidase by reaction with N-(cyclopropyl)glycine.

Monomeric sarcosine oxidase (MSOX) catalyzes the oxidative demethylation of sarcosine (N-methylglycine) and contains covalently bound flavin adenine dinucleotide (FAD). The present study demonstrates that N-(cyclopropyl)glycine (CPG) is a mechanism-based inhibitor. CPG forms a charge transfer complex with MSOX that reacts under aerobic conditions to yield a covalently modified, reduced flavin (lambda(max) = 422 nm, epsilon(422) = 3.9 mM(-1) cm(-1)), accompanied by a loss of enzyme activity. The CPG-modified flavin is converted at an 8-fold slower rate to 1,5-dihydro-FAD (EFADH(2)), which reacts rapidly with oxygen to regenerate unmodified, oxidized enzyme. As a result, CPG-modified MSOX reaches a CPG-dependent steady-state concentration under aerobic conditions and reverts back to unmodified enzyme upon removal of excess reagent. No loss of activity is observed under anaerobic conditions where EFADH(2) is formed in a reaction that goes to completion at low CPG concentrations. Aerobic denaturation of CPG-modified enzyme yields unmodified, oxidized flavin at a rate similar to the anaerobic denaturation reaction, which yields 1,5-dihydro-FAD. The CPG-modified flavin can be reduced with borohydride, a reaction that blocks conversion to unmodified flavin upon removal of excess CPG or enzyme denaturation. The possible chemical mechanism of inactivation and structure of the CPG-modified flavin are discussed.

Monomeric sarcosine oxidase: 1. Flavin reactivity and active site binding determinants.

Monomeric sarcosine oxidase (MSOX) is an inducible bacterial flavoenzyme that catalyzes the oxidative demethylation of sarcosine (N-methylglycine) and contains covalently bound FAD [8alpha-(S-cysteinyl)FAD]. This paper describes the spectroscopic and thermodynamic properties of MSOX as well as the X-ray crystallographic characterization of three new enzyme.inhibitor complexes. MSOX stabilizes the anionic form of the oxidized flavin (pK(a) = 8.3 versus 10.4 with free FAD), forms a thermodynamically stable flavin radical, and stabilizes the anionic form of the radical (pK(a) < 6 versus pK(a) = 8.3 with free FAD). MSOX forms a covalent flavin.sulfite complex, but there appears to be a significant kinetic barrier against complex formation. Active site binding determinants were probed in thermodynamic studies with various substrate analogues whose binding was found to perturb the flavin absorption spectrum and inhibit MSOX activity. The carboxyl group of sarcosine is essential for binding since none is observed with simple amines. The amino group of sarcosine is not essential, but binding affinity depends on the nature of the substitution (CH(3)XCH(2)CO(2)(-), X = CH(2) < O < S < Se < Te), an effect which has been attributed to differences in the strength of donor-pi interactions. MSOX probably binds the zwitterionic form of sarcosine, as judged by the spectrally similar complexes formed with dimethylthioacetate [(CH(3))(2)S(+)CH(2)CO(2)(-)] and dimethylglycine (K(d) = 20.5 and 17.4 mM, respectively) and by the crystal structure of the latter. The methyl group of sarcosine is not essential but does contribute to binding affinity. The methyl group contribution varied from -3.79 to -0.65 kcal/mol with CH(3)XCH(2)CO(2)(-) depending on the nature of the heteroatom (NH(2)(+) > O > S) and appeared to be inversely correlated with heteroatom electron density. Charge-transfer complexes are formed with MSOX and CH(3)XCH(2)CO(2)(-) when X = S, Se, or Te. An excellent linear correlation is observed between the energy of the charge transfer bands and the one-electron reduction potentials of the ligands. The presence of a sulfur, selenium, or telurium atom identically positioned with respect to the flavin ring is confirmed by X-ray crystallography, although the increased atomic radius of S < Se < Te appears to simultaneously favor an alternate binding position for the heavier atoms. Although L-proline is a poor substrate, aromatic heterocyclic carboxylates containing a five-membered ring and various heteroatoms (X = NH, O, S) are good ligands (K(d, X=NH) = 1.37 mM) and form charge-transfer complexes with MSOX. The energy of the charge-transfer bands (S > O >> NH) is linearly correlated with the one-electron ionization potentials of the corresponding heterocyclic rings.

Monomeric sarcosine oxidase: 2. Kinetic studies with sarcosine, alternate substrates, and a substrate analogue.

Monomeric sarcosine oxidase (MSOX) is a flavoenzyme that catalyzes the oxidative demethylation of sarcosine (N-methylglycine) to yield glycine, formaldehyde, and hydrogen peroxide. MSOX can oxidize other secondary amino acids (N-methyl-L-alanine, N-ethylglycine, and L-proline), but N,N-dimethylglycine, a tertiary amine, is not a substrate. N-Methyl-L-alanine is a good alternate substrate, exhibiting a k(cat) value (8700 min(-)(1)) similar to sarcosine (7030 min(-)(1)). Turnover with L-proline (k(cat) = 25 min(-)(1)) at 25 degrees C occurs at less than 1% of the rate observed with sarcosine. MSOX is converted to a two-electron reduced form upon anaerobic reduction with sarcosine or L-proline. No evidence for a spectrally detectable intermediate was obtained in reductive half-reaction studies with L-proline. The reductive half-reaction with L-proline at 4 degrees C exhibited saturation kinetics (k(lim) = 6.0 min(-)(1), K(d) = 260 mM) and other features consistent with a mechanism in which a practically irreversible reduction step (E(ox). S --> E(red).P) with a rate constant, k(lim), is preceded by a rapidly attained equilibrium (K(d)) between free E and the E.S complex. Steady-state kinetic studies with sarcosine and N-methyl-L-alanine in the absence or presence of a dead-end inhibitor (pyrrole-2-carboxylate) indicate that catalysis proceeds via a "modified" ping pong mechanism in which oxygen reacts with E(red).P prior to the dissociation of the imino acid product. In this mechanism, double reciprocal plots will appear nearly parallel (as observed) if the reduction step is nearly irreversible. A polar mechanism, involving formation of a covalent 4a-flavin-substrate adduct is one of several plausible mechanisms for sarcosine oxidation. Thiols are known to form similar 4a-flavin adducts. MSOX does not form a 4a-adduct with thioglycolate but does form a charge-transfer complex that undergoes an unanticipated one-electron-transfer reaction to yield the anionic flavin radical.

Cloning and mapping of the cDNA for human sarcosine dehydrogenase, a flavoenzyme defective in patients with sarcosinemia.

Sarcosine dehydrogenase is a liver mitochondrial matrix flavoenzyme that is defective in patients with sarcosinemia, a rare autosomal metabolic defect characterized by elevated levels of sarcosine in blood and urine. Some patients also exhibit mental retardation and growth failure. A full-length cDNA for human sarcosine dehydrogenase was isolated from an adult liver cDNA library. The first 22 residues in the deduced amino acid sequence exhibit features expected for a mitochondrial targeting sequence. The predicted mass of the mature human liver sarcosine dehydrogenase (99,505 Da) is in good agreement with that observed for rat liver sarcosine dehydrogenase ( approximately 100,000 Da). Human sarcosine dehydrogenase exhibits 89% identity with rat liver sarcosine dehydrogenase and strong homology ( approximately 35% identity) with rat liver dimethylglycine dehydrogenase, a sarcosine dehydrogenase-related protein from Rhodobacter capsulatus, and the regulatory subunit from bovine pyruvate dehydrogenase phosphatase. The human sarcosine dehydrogenase gene is at least 75.3 kb long and located on chromosome 9q34. The adult human liver clone is assembled from 21 exons (1-6, 7a, 8a, 9-21). Two smaller cDNA clones, isolated from adult liver and infant brain libraries, were assembled from the same sarcosine dehydrogenase gene by the use of alternate polyadenylation and splice sites. This is the first report of the genomic structure of the sarcosine dehydrogenase gene in any species. The observed chromosomal location is consistent with genetic studies with a mouse model for sarcosinemia that map the mouse gene to a region of mouse chromosome 2 syntenic with human 9q33-q34. The availability of the SDH gene sequence will enable characterization of the genotypes of sarcosinemia patients with different phenotypes.

Monomeric sarcosine oxidase: structure of a covalently flavinylated amine oxidizing enzyme.

BACKGROUND: Monomeric sarcosine oxidases (MSOXs) are among the simplest members of a recently recognized family of eukaryotic and prokaryotic enzymes that catalyze similar oxidative reactions with various secondary or tertiary amino acids and contain covalently bound flavins. Other members of this family include heterotetrameric sarcosine oxidase, N-methyltryptophan oxidase and pipecolate oxidase. Mammalian sarcosine dehydrogenase and dimethylglycine dehydrogenase may be more distantly related family members. RESULTS: The X-ray crystal structure of MSOX from Bacillus sp. B-0618, expressed in Escherichia coli, has been solved at 2.0 A resolution by multiwavelength anomalous dispersion (MAD) from crystals of the selenomethionine-substituted enzyme. Fourteen selenium sites, belonging to two MSOX molecules in the asymmetric unit, were used for MAD phasing and to define the local twofold symmetry axis for electron-density averaging. The structures of the native enzyme and of two enzyme-inhibitor complexes were also determined. CONCLUSIONS: MSOX is a two-domain protein with an overall topology most similar to that of D-amino acid oxidase, with which it shares 14% sequence identity. The flavin ring is located in a very basic environment, making contact with sidechains of arginine, lysine, histidine and the N-terminal end of a helix dipole. The flavin is covalently attached through an 8alpha-S-cysteinyl linkage to Cys315 of the catalytic domain. Covalent attachment is probably self-catalyzed through interactions with the positive sidechains and the helix dipole. Substrate binding is probably stabilized by hydrogen bonds between the substrate carboxylate and two basic sidechains, Arg52 and Lys348, located above the re face of the flavin ring.

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.

Identification of the covalent flavin attachment site in sarcosine oxidase.

Sarcosine oxidase from Corynebacterium sp. P-1 is a heterotetrameric enzyme (alphabetagammadelta) that contains two noncovalently bound coenzymes (FAD, NAD+) and covalently bound FMN [8alpha-(N3-histidyl)FMN] which is attached to the beta subunit. Chlumsky et al. [(1995) J. Biol. Chem. 270, 18252-18259] tentatively identified His175 as the covalent FMN attachment site in the beta subunit, based on an alignment of the sequence of C. sp. P-1 beta subunit with a highly homologous flavin-containing peptide from another corynebacterial sarcosine oxidase (C. sp. U-96). To test this hypothesis, His175 in the C. sp. P-1 beta subunit was mutated to an alanine. Unexpectedly, the mutant enzyme was found to contain 1 mol of covalently bound flavin and to exhibit catalytic activity similar to wild-type enzyme. Covalent flavin-containing peptides were isolated from wild-type and mutant enzymes and analyzed by electrospray mass spectrometry. The mass observed for the mutant peptide (1152.4 Da) matched that predicted for an FMN-containing hexapeptide, corresponding to residues 173-178 (1152.1 Da). In the mutant, this region (HDAVAW) contains a single histidine (His173) which must be the covalent flavin attachment site. The mass observed for the wild-type peptide (1218.6 Da) matched that predicted for an FMN-containing hexapeptide, also corresponding to residues 173-178 in the beta subunit (1218.2 Da). This region in the wild-type enzyme includes two histidine residues (HDHVAW). Attempts to sequence the wild-type or mutant peptides by automated Edman degradation were unsuccessful. Instead, the peptide sequences were investigated by collisional-activated dissociation (CAD) and tandem mass spectrometry. The CAD mass spectral data with the mutant peptide confirmed the sequence deduced based on the mass of the intact peptide. The CAD mass spectral results with the wild-type peptide showed that FMN was covalently attached to the N-terminal histidine in the hexapeptide, which corresponds to His173 in the beta subunit.

Electrospray ionization-mass spectrometry characterization of heterotetrameric sarcosine oxidase.

Electrospray ionization (ESI) Fourier transform ion cyclotron resonance (FTICR) mass spectrometry has been used to characterize heterotetrameric corynebacterial sarcosine oxidase. By using a conventional quadrupole mass spectrometer, no spectra for the intact complex could be obtained (i.e., electrospraying protein at neutral pH), but spectra showing the four protein subunits were obtained when electrospraying from acidic solution. Initial low resolution ESI-FTICR mass spectra of the intact heterotetramer revealed a typical narrow charge state distribution in the range 6000 < m/z < 9000, consistent with retention of a compact structure in the gas phase, and gave a mass measurement about 1000 u higher than predicted. Efficient in-trap clean up, based upon low energy collisionally induced dissociation of adducts, allowed significant improvement in mass measurement accuracy. The present results represent the largest heteromultimeric protein complex successfully analyzed using FTICR mass spectrometry, and clearly illustrate the importance of sample clean up methods for large molecule characterization.

An unnatural folate stereoisomer is catalytically competent in DNA photolyase.

The folate chromophore in native Escherichia coli DNA photolyase ([6R]-5,10-CH+-H4Pte(Glu)n=3-6) serves as an antenna, transferring light energy to the fully reduced flavin (FADH2) reaction center at high efficiency (EET = 0.92). Apophotolyase reconstituted after an overnight incubation with [6R,S]-5,10-CH+-H4folate (a monoglutamate analogue of the native cofactor) contains equimolar amounts of the [6R]- and [6S]-isomers, suggesting similar binding affinities. A rapid, biphasic increase in fluorescence (approximately 100-fold) is observed upon binding of 5,10-CH+-H4folate to apophotolyase at 5 degrees C; the [6S]-isomer binds about 25-fold faster than the [6R]-isomer. Although identical absorption and fluorescence emission maxima are observed for enzyme reconstituted with [6S]-, [6R]-, or [6R,S]-5,10-CH+-H4folate, folate fluorescence quantum yield values vary depending on the stereochemical configuration at the 6 position (theta = 0.18, 0.82, or 0.46, respectively, at 5 degrees C), a feature not seen with free folate. The fluorescence of enzyme-bound folate is quenched upon flavin binding; the efficiency of quenching by flavin radical (EQ = 0.96) or FADH2 (EQ = 0.89) is the same for both folate isomers. In contrast, energy transfer from folate to FADH2 is sensitive to the stereochemical configuration at the 6 position. The efficiency of energy transfer observed for enzyme containing FADH2 and [6S]-, [6R]-, or [6R,S]-5,10-CH+-H4folate (theta = 0.26, 0.66, or 0.44, respectively) is directly proportional to the fluorescence quantum yield observed for folate in the absence of FADH2, as expected for Förster-type energy transfer. Although less efficient, the unnatural [6S]-isomer is catalytically functional, a feature not previously observed with other folate-dependent enzymes. Fluorescence quantum yield studies at 77 K with free (theta = 0.67) and enzyme-bound (theta = 1.0) folate suggest that differences in solvent exposure may contribute to the fluorescence efficiency differences observed with the enzyme-bound folate isomers at 5 degrees C.

Sarcosine oxidase contains a novel covalently bound FMN.

Sarcosine oxidase from Corynebacterium sp. P-1 is a heterotetrameric protein containing three different enzymes: noncovalent FAD, noncovalent NAD+, and covalently bound flavin which is released as 8 alpha-(N3-histidyl)riboflavin upon complete hydrolysis of the protein. The following results show that the covalent flavin is not at the FAD level, as previously proposed, but it is rather as 8 alpha-(N3- histidyl)FMN coenzyme. First, no AMP is released when the protein moiety is treated with phosphodiesterase or subjected to mild acid hydrolysis. The enzyme contains a total of 5 mol of phosphate. Only one phosphate is covalently bound. The other four phosphates are noncovalent and attributed to noncovalently bound FAD and NAD+. The 31P NMR spectrum of native enzyme exhibits resonances due to a single phosphate monoester an two pyrophosphates. Only a resonance due to phosphate monoester is observed after removal of the noncovalent cofactors and proteolytic digestion of the protein moiety. The 8 alpha-(N3-histidyl)FMN found in corynebacterial sarcosine oxidase represents a novel type of covalent flavin. Studies with sarcosine oxidases from Arthrobacter sp. and Pseudomonas sp. show that these heterotetrameric enzymes also contain covalently bound FMN plus noncovalently bound FAD and NAD+, similar to corynebacterial sarcosine oxidase. In contrast, two monomeric sarcosine oxidases (from Bacillus sp. and an unidentified microorganism) were found to contain only covalently bound FAD.

Discovery of a third coenzyme in sarcosine oxidase.

Denaturation of recombinant sarcosine oxidase or the natural enzyme isolated from Corynebacterium sp. P-1 with guanidine hydrochloride releases noncovalently bound FAD and a second UV-absorbing component (peak 2) which comigrates with NAD+ during reversed-phase HPLC. Both FAD and peak 2 are also found in extracts prepared by incubating sarcosine oxidase at 37 degrees C for 30 min, a procedure which causes partial (approximately 50%) release of the enzyme's noncovalently bound FAD. Peak 2 in the 37 degrees C extract is heat labile and decomposes upon boiling for 5 min at pH 8.0. A similar instability was observed with NAD+. Reaction of the 37 degrees C extract from sarcosine oxidase with phosphodiesterase yields nicotinamide mononucleotide, AMP, and FMN, as expected for a mixture containing NAD+ and FAD. Peak 2 was converted to NADH upon reaction of the 37 degrees C extract with yeast alcohol dehydrogenase in the presence of ethanol. Guanidine hydrochloride extracts, prepared from recombinant or natural enzyme, contain 1 mol of NAD+/mol of FAD. Since sarcosine oxidase contains 1 mol of noncovalently bound FAD, the results show that the enzyme also contains 1 mol of NAD+. The NAD+ is tightly bound and is not lost during enzyme purification. It is not susceptible toward hydrolysis by NADase, reduction by alcohol dehydrogenase, or nucleophilic attack by cyanide. Unlike the flavins in sarcosine oxidase, NAD+ is not reduced by sarcosine and is not in redox equilibrium with the flavins.

Stereospecificity of folate binding to DNA photolyase from Escherichia coli.

DNA photolyase from Escherichia coli contains folate ([6S]-5,10-CH(+)-H4Pte(Glu)n = 3-6) and reduced FAD. The folate chromophore acts as an antenna, harvesting light energy which is transferred to the reduced flavin where DNA repair occurs. The folate binding stereospecificity of the enzyme was investigated by reconstituting the apoenzyme with [6R,S]-5,10-CH(+)-H4folate and reduced FAD. The isomer composition of [methyl-3H]-5-CH3-H4folate, released into solution upon reduction of the reconstituted enzyme with [3H]NaBH4, was analyzed by enzymatic and chiral chromatographic methods. Both methods showed that the reconstituted enzyme contained nearly equimolar amounts of [6R]- and [6S]-5,10-CH(+)-H4folate.

Association of flavin adenine dinucleotide with the Arabidopsis blue light receptor CRY1.

The arabidopsis thaliana HY4 gene encodes CRY1, a 75-kilodalton flavoprotein mediating blue light-dependent regulation of seedling development. CRY1 is demonstrated here to noncovalently bind stoichiometric amounts of flavin adenine dinucleotide (FAD). The redox properties of FAD bound by CRY1 include an unexpected stability of the neutral radical flavosemiquinone (FADH.). The absorption properties of this flavosemiquinone provide a likely explanation for the additional sensitivity exhibited by CRY1-mediated responses in the green region of the visible spectrum. Despite the sequence homology to microbial DNA photolyases, CRY1 was found to have no detectable photolyase activity.

Sequence analysis of sarcosine oxidase and nearby genes reveals homologies with key enzymes of folate one-carbon metabolism.

Corynebacterial sarcosine oxidase, a heterotetrameric (alpha beta gamma delta) enzyme containing covalent and noncovalent FAD, catalyzes the oxidative demethylation of sarcosine to yield glycine, H2O2, and 5,10-CH2-tetrahydrofolate (H4folate) in a reaction requiring H4folate and O2. The sarcosine oxidase operon contains at least five closely packed genes encoding sarcosine oxidase subunits and serine hydroxymethyltransferase (glyA), arranged in the order glyAsoxBDAG. The operon status of a putative purU gene, found 340 nucleotides downstream from soxG, is not known. No homology with other proteins is observed for the smallest sarcosine oxidase subunits gamma and delta. The beta subunit (405 residues) contains an ADP-binding motif near its NH2 terminus, the covalent FAD attachment site (H175), and exhibits homology with the NH2-terminal half of dimethylglycine dehydrogenase (857 residues) and monomeric, bacterial sarcosine oxidases (approximately 388 residues), enzymes that contain a single covalent FAD. The alpha subunit (967 residues) contains a second ADP-binding motif within an approximately 280 residue region near the NH2 terminus that exhibits homology with subunit A from octopine and nopaline oxidases, heterodimeric enzymes that catalyze analogous oxidative cleavage reactions with N-substituted arginine derivatives. An approximately 380 residue region near the COOH terminus of alpha exhibits homology with T-protein and the COOH-terminal half of dimethylglycine dehydrogenase. These enzymes catalyze the formation of 5,10-CH2-H4folate, using different one-carbon donors. The results suggest that the alpha subunit and dimethylglycine dehydrogenase contain an NH2-terminal domain that binds noncovalent or covalent FAD, respectively, and a carboxyl-terminal H4folate-binding domain.

Photoinduced spin-polarized radical pair formation in a DNA photolyase.substrate complex at low temperature.

Electron spin polarization is a phenomenon characterized by anomalous line intensities (emission or enhanced absorption) in the EPR spectrum. It is highly diagnostic of radical pairs, such as those formed in photoinduced electron transfer reactions. Electron spin polarization behavior (E/A pattern) is observed in light-modulated EPR spectra obtained at 4 K with fully reduced DNA photolyase.substrate complexes. Similar results are obtained with complexes formed with native enzyme or reconstituted enzyme containing fully reduced flavin as its only chromophore. No signal is observed for fully reduced enzyme or substrate alone. The results suggest that the electron spin polarization signal is due to photoinduced formation of a flavin/substrate radical pair (FADH./T < > T.-); splitting of T < > T.- does not occur at 4 K, and the radical pair can only undergo back-electron-transfer reactions. The data are consistent with the proposal that electron transfer initiates DNA repair in the photolyase reaction.

Affinity probing of flavin binding sites. 1. Covalent attachment of 8-(methylsulfonyl)FAD to pig heart lipoamide dehydrogenase.

8-(Methylsulfonyl)FAD reacts with a single cysteine residue (Cys449) in pig apolipoamide dehydrogenase to generate a flavinylated enzyme containing covalently bound 8-(cysteinyl)FAD. Competitive behavior is observed in reconstitution reactions containing both FAD and 8-(methylsulfonyl)FAD. Covalently bound 8-(cysteinyl)FAD is shielded from solvent, as judged by spectral comparison with model 8-(alkylthio)-flavins in various solvents. Flavinylated lipoamide dehydrogenase is monomeric and catalytically inactive. Cys449 is located in the interface domain, near the active site histidine (His452). As shown previously, Cys449 is oxidized when native enzyme is treated with cupric ions. Cys449 is close to the isoalloxazine ring of FAD in native enzyme, as judged by alignment of the pig sequence with the structure of the homologous enzyme from Azotobacter vinelandii. The residue corresponding to Cys449 in A. vinlandii lipoamide dehydrogenase (Val447) is about 9 A from the carbonyl oxygen at C(2) in the pyrimidine ring of FAD. Approximation of a substituent at position 8 in FAD with Cys449 requires a 180 degrees flip of the isoalloxazine ring as compared with its orientation in the native structure. The different flavin orientation can explain the absence of dimerization and catalytic activity. Using the same method of apoenzyme preparation, noncovalent binding was observed with 8-chloroFAD, a less reactive flavin analogue. Relatively nonspecific covalent incorporation was observed with 8-chloroFAD when apoenzyme was prepared by an older method used in previous studies with this derivative [Moore, E.G., Cardemil, E., & Massey, V. (1978) J. Biol. Chem. 253, 6413-6422].

Affinity probing of flavin binding sites. 2. Identification of a reactive cysteine in the flavin domain of Escherichia coli DNA photolyase.

8-(Methylsulfonyl)FAD reacts with a single cysteine residue (Cys293) in the flavin domain of Escherichia coli DNA photolyase to form an 8-(cysteinyl)FAD derivative covalently bound to the protein. About 80% protection against covalent attachment with 8-(methylsulfonyl)FAD was observed in the presence of an equimolar amount of FAD. Flavinylated photolyase retains the ability to repair pyrimidine dimers (15% of native activity) and to bind its antenna chromophore, 5,10-methenyltetrahydrofolate. Comparison of the properties of flavinylated enzyme with photolyase containing noncovalently bound 8-(methylthio)-FAD indicate that a perturbation is necessary to accommodate covalent bond formation. 8-(Methylthio)-FAD-reconstituted enzyme exhibits 95% of native activity. The aerobic stability of fully reduced and radical forms of 8-(methylthio)FAD enzyme is similar to that of native enzyme, whereas a radical form is not detected with flavinylated enzyme and the fully reduced enzyme is more easily oxidized by oxygen. The flavin in 8-(methylthio)FAD enzyme or flavinylated photolyase is shielded from solvent. However, the flavin environment in flavinylated enzyme is less hydrophobic as judged by spectral comparison with model 8-(alkylthio)flavins in various solvents. Enzyme containing noncovalently bound 8-(methylsulfonyl)-FAD was prepared by reconstitution with the fully reduced flavin which does not undergo covalent attachment. Covalent attachment was observed after reoxidation but probably involved dissociation and rebinding of oxidized 8-(methylsulfonyl)FAD. The results show that 8-(cysteinyl)FAD in flavinylated photolyase is at or near the normal flavin binding site.(ABSTRACT TRUNCATED AT 250 WORDS)

Preparation and properties of recombinant corynebacterial sarcosine oxidase: evidence for posttranslational modification during turnover with sarcosine.

The genes encoding the four subunits of sarcosine oxidase from Corynebacterium sp. P-1 were isolated and overexpressed in a single step by using indicator plates to screen a genomic library for colonies that generated hydrogen peroxide in a sarcosine-dependent reaction. The genomic library was constructed by inserting size-fractionated genomic DNA, previously subjected to partial digestion by Sau3AI, into pBluescript II SK (+). At least 1.0 kb, but less than 4.0 kb, can be deleted from the 3' end of the original cornyebacterial insert (7.3 kb) without affecting sarcosine oxidase expression, consistent with the estimated 5.0-kb operon size. Recombinant sarcosine oxidase is isolated as a heterotetramer containing equimolar amounts of covalent and noncovalent flavin, identical to that observed for enzyme isolated from Corynebacterium sp. P-1. Despite its similar flavin content, recombinant enzyme exhibits significantly different spectral properties than enzyme from Corynebacterium sp. P-1 (values shown in parentheses) [epsilon 450 = 9.7 (12.7) mM-1 cm-1; A368/A450 = 1.0 (0.83); A280/A450 = 16.9 (12.2)]. This difference is due to the fact that about half of the covalent flavin in recombinant enzyme forms a reversible covalent 4a-adduct with a cysteine residue (lambda max = 383 nm; epsilon 383 = 7.3 mM-1 cm-1). The equilibrium is shifted in favor of adduct dissociation by oxidizing the cysteine residue with hydrogen peroxide or by alkylation with methyl methanethiosulfonate in a reaction that is fully reversible upon addition of excess dithiothreitol. The cysteine residue is also oxidized during aerobic turnover with sarcosine. Reaction of the cysteine residue with hydrogen peroxide (or a precursor) formed during turnover partially competes with the release of hydrogen peroxide into solution, as judged by the effect of catalase on this reaction. Although the same specific activity is observed for recombinant enzyme and enzyme from Corynebacterium sp. P-1, the recombinant enzyme exhibits a pronounced lag in an NADH peroxidase-coupled assay. The lag is eliminated by prior disruption of the 4a-thiolate adduct via reaction with hydrogen peroxide or methyl methanethiosulfonate. The results show that the 4a-thiolate adduct is an inactive form of sarcosine oxidase that can be activated by reaction with sarcosine in what appears to be the first example of a posttranslational modification associated with turnover. Complete activation occurs in vivo when sarcosine oxidase is produced in Corynebacterium sp. P-1, where enzyme synthesis is induced by growth of the organism with sarcosine as the source of carbon and energy.(ABSTRACT TRUNCATED AT 400 WORDS)

Properties of a high-potential flavin analogue and its use as an active site probe with clostridial flavodoxin.

The reduction potential of flavin bearing a methylsulfonyl moiety (MeSO2) in place of a methyl group at position 8 is increased by more than 150 mV as compared with normal flavin. This substitution is accompanied by a substantial increase in reactivity with various reductants, including NADH, and greatly (10(3)-fold) enhanced susceptibility toward nucleophilic attack by sulfite at N(5). 1,5-Dihydro-8-(methylsulfonyl)riboflavin exhibits two intense, well-resolved absorption bands (lambda max = 310, 362 nm) in a region where most other reduced flavins exhibit weak, characterless absorption. This unusual spectrum is attributable to a shift of pi-electron density from the N(5) atom into the benzene ring. It is observed only with reduced flavins bearing a strongly electronegative substituent (MeSO2, CN) at the 8-position. The effect is abolished by replacing the hydrogen at N(5) with a bulky group, like sulfite, which interferes with sp2 hybridization at N(5). Reaction of 8-MeSO2-substituted flavins with thiols results in nucleophilic displacement of MeSO2- in a reaction that is about 10(3)-fold faster than an analogous nucleophilic displacement reaction observed with 8-halo-substituted flavins. The flavin ring acts as a redox switch in controlling electrophilicity at the 8-position, as judged by the fact that the displacement reactions are observed only with the oxidized flavins. Initial studies to evaluate 8-MeSO2-substituted flavins as active site probes were conducted with flavodoxin from Clostridium beijerinckii MP. 8-MeSO2FMN is rapidly bound to apoflavodoxin, accompanied by absorbance and fluorescence changes similar to those observed for FMN binding. 1,5-Dihydro-8-MeSO2FMN flavodoxin exhibits spectral properties (lambda max = 323, 382 nm) similar to those of the corresponding free flavin, except for a bathochromic shift due to a change in the polarity of the flavin environment. As judged by peak resolution and intensity, the spectral properties of 1,5-dihydro-FMN flavodoxin (lambda max = 311, 362 nm) appear to lie about midway between those observed for the free 1,5-dihydro forms of FMN versus 8-MeSO2FMN. This suggests that the protein environment may favor enhanced resonance delocalization of pi-electron density into the benzene ring of bound 1,5-dihydro-FMN, as compared with the free flavin. This hypothesis is consistent with previous NMR studies and with a proposal that electron transfer from reduced flavodoxin to other redox proteins occurs through this region of the ring. 8-MeSO2FMN bound to flavodoxin reacts readily with exogenous thiols but does not react with sulfite.(ABSTRACT TRUNCATED AT 400 WORDS)