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.

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.

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.

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)

Expression of rat tyrosine hydroxylase in insect tissue culture cells and purification and characterization of the cloned enzyme.

Rat tyrosine hydroxylase has been expressed at high levels in Spodoptera frugiperda cells using a baculovirus expression system. A cDNA containing the coding region for PC12 tyrosine hydroxylase was inserted into the unique EcoRI site of the transfer vector pLJC8 to yield the recombinant vector pLJC9. Spodoptera frugiperda cells were then co-infected with pLJC9 and wild type Autographa californica nuclear polyhedrosis virus. Recombinant virus particles containing the cDNA for tyrosine hydroxylase were selected by hybridization with authentic tyrosine hydroxylase cDNA. Three recombinant viruses were plaque-purified. All expressed a protein of Mr = 55,000 which reacted with antibodies to tyrosine hydroxylase. Forty-eight h after infection of cells with recombinant virus, the specific activity of tyrosine hydroxylase in the cell lysate was 30-100 nmol of dihydroxyphenylalanine produced/min/mg, consistent with 5-10% of the cell protein being tyrosine hydroxylase. Purification from 2.1 g of cells gave 5.8 mg of enzyme with a specific activity of 1.7 mumol of dihydroxyphenylalanine/min/mg. The purified enzyme is a tetramer of identical subunits, containing one covalently bound phosphoryl residue and 0.1 iron atom/subunit. No carbohydrate was detectable. Steady state kinetic results with tetrahydrobiopterin as substrate are consistent with a sequential mechanism for binding of tyrosine and tetrahydrobiopterin. Substrate inhibition occurs at tyrosine concentrations above 50 microM. Steady state kinetic parameters at pH 6.5 are Vmax = 74 min-1, KBH4 = 21 microM, KTyr = 9.4 microM, and Ko2 less than or equal to 6 microM. The Vmax value shows a broad pH optimum around pH 7. The KBH4 value is pH-dependent, increasing from about 20 microM below pH 7 to about 100 microM above pH 8. The KTyr value is independent of pH between pH 6 and pH 8.5.

Site-directed mutagenesis of bacterial luciferase: analysis of the 'essential' thiol.

It has been appreciated for many years that the luciferase from the luminous marine bacterium Vibrio harveyi has a highly reactive cysteinyl residue which is protected from alkylation by binding of flavin. Alkylation of the reactive thiol, which resides in a hydrophobic pocket, leads to inactivation of the enzyme. To determine conclusively whether the reactive thiol is required for the catalytic mechanism, we have constructed a mutant by oligonucleotide directed site-specific mutagenesis in which the reactive cysteinyl residue, which resides at position 106 of the alpha subunit, has been replaced with a seryl residue. The resulting alpha 106Ser luciferase retains full activity in the bioluminescence reaction, although the mutant enzyme has a ca 100-fold increase in the FMNH2 dissociation constant. The alpha 106Ser luciferase is still inactivated by N-ethylmaleimide, albeit at about 1/10 the rate of the wild-type (alpha 106Cys) enzyme, demonstrating the existence of a second, less reactive, cysteinyl residue that was obscured in the wild-type enzyme by the highly reactive cysteinyl residue at position alpha 106. An alpha 106Ala variant luciferase was also active, but the alpha 106Val mutant enzyme was about 50-fold less active than the wild type. All three variants (Ser, Ala and Val) appeared to have somewhat reduced affinities for the aldehyde substrate, the valine mutant being the most affected. It is interesting to note that the alpha 106 mutant luciferases are much less subject to aldehyde substrate inhibition than is the wild-type V. harveyi luciferase, suggesting that the molecular mechanism of aldehyde substrate inhibition involves the Cys at alpha 106.