Role of the covalent flavin linkage in monomeric sarcosine oxidase

Monomeric sarcosine oxidase (MSOX) is a prototypical member of a recently recognized family of amine-oxidizing enzymes that all contain covalently bound flavin. Mutation of the covalent flavin attachment site in MSOX produces a catalytically inactive apoprotein (apoCys315Ala) that forms an unstable complex with FAD (K(d) = 100 muM), similar to that observed with wild-type apoMSOX where the complex is formed as an intermediate during covalent flavin attachment. In situ reconstitution of sarcosine oxidase activity is achieved by assaying apoCys315Ala in the presence of FAD or 8-nor-8-chloroFAD, an analogue with an approximately 55 mV higher reduction potential. After correction for an estimated 65% reconstitutable apoprotein, the specific activity of apoCys315Ala in the presence of excess FAD or 8-nor-8-chloroFAD is 14% or 80%, respectively, of that observed with wild-type MSOX. Unlike oxidized flavin, apoCys315Ala exhibits a high affinity for reduced flavin, as judged by results obtained with reduced 5-deazaFAD (5-deazaFADH(2)) where the estimated binding stoichiometry is unaffected by dialysis. The Cys315Ala.5-deazaFADH(2) complex is also air-stable but is readily oxidized by sarcosine imine, a reaction accompanied by release of weakly bound oxidized 5-deazaFAD. The dramatic difference in the binding affinity of apoCys315Ala for oxidized and reduced flavin indicates that the protein environment must induce a sizable increase in the reduction potential of noncovalently bound flavin (DeltaE(m) approximately 120 mV). The covalent flavin linkage prevents loss of weakly bound oxidized FAD and also modulates the flavin reduction potential in conjunction with the protein environment.     

Studies on a sarcosine oxidase of bacterial origin

A "sarcosine oxidase" was prepared from a creatinine-decomposing strain of Pseudomonas aeruginosa. The enzyme is inactivated by drying, lyophilization, and dialysis against distilled water. No dialyzable cofactor was found. Optimal activity of the enzyme is reached at pH 7.8. Enzyme activity is directly proportional to enzyme concentration and also to substrate concentration up to the point of saturation of enzyme with substrate molecules. One molecule of enzyme combines with one molecule of substrate. Data concerning the effect of temperature and of a variety of chemical compounds on the enzyme are presented. Its inactivation by heat follows the course of a first order reaction, and the critical thermal increment between 48° and 52°C. was calculated to be 103,000 calories per mol. The relationship of enzyme concentration to heat inactivation rates is illustrated.     

Characterization of glycine sarcosine N-methyltransferase and sarcosine dimethylglycine N-methyltransferase

Glycine betaine is accumulated in cells living in high salt concentrations to balance the osmotic pressure. Glycine sarcosine N-methyltransferase (GSMT) and sarcosine dimethylglycine N-methyltransferase (SDMT) of Ectothiorhodospira halochloris catalyze the threefold methylation of glycine to betaine, with S-adenosylmethionine acting as the methyl group donor. These methyltransferases were expressed in Escherichia coli and purified, and some of their enzymatic properties were characterized. Both enzymes had high substrate specificities and pH optima near the physiological pH. No evidence of cofactors was found. The enzymes showed Michaelis-Menten kinetics for their substrates. The apparent K(m) and V(max) values were determined for all substrates when the other substrate was present in saturating concentrations. Both enzymes were strongly inhibited by the reaction product S-adenosylhomocysteine. Betaine inhibited the methylation reactions only at high concentrations.     

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 of a covalently bound flavoprotein in rat liver mitochondria with sarcosine dehydrogenase.

A covalently bound flavoprotein having highest molecular weight among four covalently bound flavoproteins found in rat liver mitochondria was partially purified and characterized. Its subunit molecular weight was estimated to be 94,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Its absorption maxima were observed at 353 and 460 nm. Since this flavoprotein was reduced by either sarcosine or dimethylglycine and oxidized by phenazine methosulfate, it was identified with sarcosine dehydrogenase.     

Analysis of interaction between the Arthrobacter sarcosine oxidase and the coenzyme flavin adenine dinucleotide by site-directed mutagenesis.

Sarcosine oxidase from Arthrobacter sp. TE1826 (SoxA) tightly binds with the coenzyme flavin adenine dinucleotide (FAD). The amino-terminal region of this enzyme was recognized as a part of the FAD-binding domain by homology search analysis. Comparison with other structurally well-known flavoproteins suggested that the aspartate residue at position 35 (D-35) and the motif sequence (six residues at positions 12 to 17) were important for the interaction with FAD. Site-directed mutagenesis of each position was performed, and mutant SoxAs were purified and characterized. When D-35 was substituted with glutamate, asparagine, and alanine, it was indicated that the carboxyl group of the side chain interacted with FAD. Changes in the enzyme-bound FAD were also observed from the altered spectral profiles. Thirteen mutant SoxAs were obtained by replacing amino acids in the motif sequence. Most of them showed inhibited or remarkably decreased sarcosine oxidase activity, and their spectral profiles were altered. However, some of them were reactivated by chloride ion. Their spectral profiles also became close to that of wild type in the presence of chloride ion. These results strongly suggest that the inhibition of interaction of enzyme with FAD was caused by the substitution in the motif and that it could be recovered under different conditions.     

Mutations in the sarcosine dehydrogenase gene in patients with sarcosinemia

Sarcosinemia is an autosomal recessive metabolic trait manifested by relatively high concentrations of sarcosine in blood and urine. Sarcosine is a key intermediate in 1-carbon metabolism and under normal circumstances is converted to glycine by the enzyme sarcosine dehydrogenase. We encountered six families from two different descents (French and Arab), each with at least one individual with elevated levels of sarcosine in blood and urine. Using the “candidate gene approach” we sequenced the gene encoding sarcosine dehydrogenase (SARDH), which plays an important role in the conversion of sarcosine to glycine, and found four different mutations (P287L, V71F, R723X, R514X) in three patients. In an additional patient, we found a uniparental disomy in the region of SARDH gene. In two other patients, we did not find any mutations in this gene. We have shown for the first time that mutations in the SARDH gene are associated with sarcosinemia. In addition, our results indicate that other genes are most probably involved in the pathogenesis of this condition.     

LuxG is a functioning flavin reductase for bacterial luminescence

The luxG gene is part of the lux operon of marine luminous bacteria. luxG has been proposed to be a flavin reductase that supplies reduced flavin mononucleotide (FMN) for bacterial luminescence. However, this role has never been established because the gene product has not been successfully expressed and characterized. In this study, luxG from Photobacterium leiognathi TH1 was cloned and expressed in Escherichia coli in both native and C-terminal His₆-tagged forms. Sequence analysis indicates that the protein consists of 237 amino acids, corresponding to a subunit molecular mass of 26.3 kDa. Both expressed forms of LuxG were purified to homogeneity, and their biochemical properties were characterized. Purified LuxG is homodimeric and has no bound prosthetic group. The enzyme can catalyze oxidation of NADH in the presence of free flavin, indicating that it can function as a flavin reductase in luminous bacteria. NADPH can also be used as a reducing substrate for the LuxG reaction, but with much less efficiency than NADH. With NADH and FMN as substrates, a Lineweaver-Burk plot revealed a series of convergent lines characteristic of a ternary-complex kinetic model. From steady-state kinetics data at 4°C pH 8.0, K_m for NADH, K_m for FMN, and k_cat were calculated to be 15.1 μM, 2.7 μM, and 1.7 s⁻¹, respectively. Coupled assays between LuxG and luciferases from P. leiognathi TH1 and Vibrio campbellii also showed that LuxG could supply FMNH⁻ for light emission in vitro. A luxG gene knockout mutant of P. leiognathi TH1 exhibited a much dimmer luminescent phenotype compared to the native P. leiognathi TH1, implying that LuxG is the most significant source of FMNH⁻ for the luminescence reaction in vivo.     

Characterization of a new flavin metabolite from human urine

A new flavin metabolite comprising approximately 5% of the total flavin of human urine was isolated and characterized using absorption and fluorescence spectra, oxidation-reduction and hydrolysis data, and ninhydrin reactions. The flavin is a derivative associated with a peptide residue in ester linkage from an amino acid carboxyl to the ribityl chain of riboflavin, probably at the 5'-terminus.     

Spectral evidence for a flavin adduct in a monoalkylated derivative of pig heart lipoamide dehydrogenase.

A derivative of the flavoprotein pig heart lipoamide dehydrogenase has been described recently (Thorpe, C., and Williams, C.H. (1976) J. Biol. Chem. 251, 3553-3557), in which 1 of the 2 cysteine residues generated on reduction of the intrachain active center disulfide bridge is selectively alkylated with iodoacetamide. This monolabeled enzyme exhibits a spectrum of oxidized bound flavin. The addition of 1 mM NAD+ to this derivative at pH 8.3 causes a decrease in absorbance of approximately 50% at 448 nm, with a concomitant increase at 380 nm. These spectral changes are complete within 3 ms and are reversible. NAD+ titrations generate isosbestic points at 408, 374, and 327 nm; allowing values for the apparent dissociation constant for NAD+ and the extent of bleaching at infinite ligand to be obtained from double reciprocal plots. Between pH 6.1 and 8.8, the apparent KD decreases from 320 to 35 muM, whereas the extrapolated delta epsilon 448 values remain approximately constant at 1/2 epsilon 448. Direct measurement of NAD+ binding by gel filtration at pH 8.8 indicates that the spectral changes are associated with a stoichiometry of 1.2 mol of NAD+ bound/2 mol of FAD. The modified protein is a dimer containing 1 FAD and 1 alkylated cysteine residue/subunit; the native enzyme is also dimeric. The visible spectrum of the species absorbing at 380 nm, approximated by correction for the residual oxidized FAD, shows a single maximum at 384 nm, epsilon 384 = 8.7 mM-1cm-1. Comparison of this spectrum with that of model compounds of known structure suggests that it may represent a reversible covalent flavin adduct induced on binding NAD+.     

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.     

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.     

Characterization of the solution conformations of unbound and Tat peptide-bound forms of HIV-1 TAR RNA.

Basic peptides from the carboxy terminus of the HIV-1 Tat protein bind to the apical stem-loop region of TAR RNA with high affinity and moderate specificity. The conformations of the unbound and 24 residue Tat peptide (Tfr24)-bound forms of TAR RNA have been characterized by NMR spectroscopy. The unbound form of TAR exists in major and minor forms having different trinucleotide bulge conformations. A specific TAR RNA conformational change is observed upon complex formation with Tfr24, consisting of coaxial stacking of helical stems and base triple formation. A U23-A27-U38 base triple is proposed based on exchangeable proton NMR data, where U23 forms a base pair with A27 in the major groove. No evidence for base triple formation was found for Tat peptides in which lysine residues are extensively substituted for arginine.     

The amino acid sequences of the flavin-peptides of dimethylglycine dehydrogenase and sarcosine dehydrogenase from rat liver mitochondria

The flavoenzymes dimethylglycine dehydrogenase (EC 1.5.99.2) and sarcosine dehydrogenase (EC 1.5.99.1) contain covalently bound FAD linked via the 8 alpha-position of the isoalloxazine ring to the imidazole N(3) of a histidine residue (Cook, R. J., Misono, K. S., and Wagner, C. (1984) J. Biol. Chem. 259, 12475-12480). The flavin-peptides from tryptic digests of these two enzymes have been isolated and sequenced. Automated sequence analysis showed that the flavin-peptide from dimethylglycine dehydrogenase contained 25 amino acid residues in the following sequence: Ser-Glu-Leu-Thr-Ala-Gly-Ser- Thr-Trp-His(flavin)-Ala-Ala-Gly-Leu-Thr-Thr-Tyr-Phe-His-Pro-Gly-Ile-A sn-Leu-Lys. The sequence determined for the flavin-peptide from sarcosine dehydrogenase contained 14 amino acid residues Leu-Thr-Ser-Gly-Thr-Thr-Trp-His(flavin)-Thr-Ala-Gly-Leu-Gly-Arg.     

Characterization of the bacterial community of a zinc-polluted soil.

The bacterial community of a zinc-contaminated soil (Maatheide soil in Lommel, Belgium) was studied using cultivation as well as cultivation-independent techniques. Colony-forming units (CFU) were determined by plating on media with or without metals. Dominant isolates were characterized by fatty acid methyl ester analysis (FAME analysis) and PCR fingerprinting using repetitive extragenic palindromic sequences as primers. DNA was directly extracted from soil samples and used as a template for the PCR amplification of the 16S rDNA (8-1511) or a 16S rDNA fragment (968-1401). Clones resulting from cloning the 16S rDNA from soil DNA were sequenced. Temperature gradient gel electrophoresis (TGGE analysis) was performed for 16S rDNA fragments (968-1401) amplified from the dominant isolates, the clones, and the total soil DNA extracted according to two protocols differing in strength of lysis. Total CFU ranged from 10(4) to 10(5)/g soil. The majority of the isolates were identified by FAME analysis as Arthrobacter spp. (18 out of 23). None of the isolates were identified as a Ralstonia eutropha like strain (formerly Alcaligenes eutrophus). Metalloresistant Rastomia eutropha like strains were previously shown to be dominant in the analyzed biotope. Most of the isolates were zinc tolerant but only seven could be considered zinc resistant. Sequences of the 16S rDNA clones obtained from total soil DNA were affiliated with genes of different bacteria such as alpha-proteobacteria, beta-proteobacteria, and the Cytophaga-Flexibacter-Bacteroides group. None of the sequenced clones aligned with the Ralstonia eutropha 16S rRNA gene. TGGE analysis of the 16S rDNA fragments (968-1401) amplified from the dominant strains, the clones, and the total soil DNA showed that isolates and clones represented only a part of the bands present in the TGGE pattern from total DNA. The 968-1401 fragment amplified from all Arthrobacter strains had a similar electrophoretic mobility. This band was seen as a major band in the pattern of DNA extracted from soil using a harsh cell lysis, whereas it did not appear, or appeared only as a weak band, in patterns obtained from soil DNA extracted using gentle lysis. The previously reported predominance of a Ralstonia eutropha like strain in this soil was no longer observed. This may suggest a population replacement by less resistant bacteria, concomitant with a progressive decrease of the zinc toxicity in the Maatheide soil.     

Differential transfers of reduced flavin cofactor and product by bacterial flavin reductase to luciferase

It is believed that the reduced FMN substrate required by luciferase from luminous bacteria is provided in vivo by NAD(P)H-FMN oxidoreductases (flavin reductases). Our earlier kinetic study indicates a direct flavin cofactor transfer from Vibrio harveyi NADPH-preferring flavin reductase P (FRP(H)) to the luciferase (L(H)) from the same bacterium in the in vitro coupled luminescence reaction. Kinetic studies were carried out in this work to characterize coupled luminescence reactions using FRP(H) and the Vibrio fischeri NAD(P)H-utilizing flavin reductase G (FRG(F)) in combination with L(H) or luciferase from V. fischeri (L(F)). Comparisons of K(m) values of reductases for flavin and pyridine nucleotide substrates in single-enzyme and luciferase-coupled assays indicate a direct transfer of reduced flavin, in contrast to free diffusion, from reductase to luciferase by all enzyme couples tested. Kinetic mechanisms were determined for the FRG(F)-L(F) and FRP(H)-L(F) coupled reactions. For these two and the FRG(F)-L(H) coupled reactions, patterns of FMN inhibition and effects of replacement of the FMN cofactor of FRP(H) and FRG(F) by 2-thioFMN were also characterized. Similar to the FRP(H)-L(H) couple, direct cofactor transfer was detected for FRG(F)-L(F) and FRP(H)-L(F). In contrast, despite the structural similarities between FRG(F) and FRP(H) and between L(F) and L(H), direct flavin product transfer was observed for the FRG(F)-L(H) couple. The mechanism of reduced flavin transfer appears to be delicately controlled by both flavin reductase and luciferase in the couple rather than unilaterally by either enzyme species.     

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.