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 a stable flavin-thiolate adduct in heterotetrameric sarcosine oxidase

Heterotetrameric sarcosine oxidase (TSOX) is a complex bifunctional flavoenzyme that contains two flavins. Most of the FMN in recombinant TSOX is present as a covalent adduct with an endogenous ligand. Enzyme denaturation disrupts the adduct, accompanied by release of a stoichiometric amount of sulfide. Enzyme containing>or=90% unmodified FMN is prepared by displacement of the endogenous ligand with sulfite, a less tightly bound competing ligand. Reaction of adduct-depleted TSOX with sodium sulfide produces a stable complex that resembles the endogenous TSOX adduct and known 4a-S-cysteinyl flavin adducts. The results provide definitive evidence for sulfide as the endogenous TSOX ligand and strongly suggest that the modified FMN is a 4a-sulfide adduct. A comparable reaction with sodium sulfide is not detected with other flavoprotein oxidases. A model of the postulated TSOX adduct suggests that it is stabilized by nearby residues that may be important in the electron transferase/oxidase function of the coenzyme.     

Heterotetrameric sarcosine oxidase: structure of a diflavin metalloenzyme at 1.85 A resolution

The crystal structure of heterotetrameric sarcosine oxidase (TSOX) from Pseudomonas maltophilia has been determined at 1.85 A resolution. TSOX contains three coenzymes (FAD, FMN and NAD+), four different subunits (alpha, 103 kDa; beta, 44 kDa; gamma, 21 kDa; delta, 11 kDa) and catalyzes the oxidation of sarcosine (N-methylglycine) to yield hydrogen peroxide, glycine and formaldehyde. In the presence of tetrahydrofolate, the oxidation of sarcosine is coupled to the formation of 5,10-methylenetetrahydrofolate. The NAD+ and putative folate binding sites are located in the alpha-subunit. The FAD binding site is in the beta-subunit. FMN is bound at the interface of the alpha and beta-subunits. The FAD and FMN rings are separated by a short segment of the beta-subunit with the closest atoms located 7.4 A apart. Sulfite, an inhibitor of oxygen reduction, is bound at the FMN site. 2-Furoate, a competitive inhibitor with respect to sarcosine, is bound at the FAD site. The sarcosine dehydrogenase and 5,10-methylenetetrahydrofolate synthase sites are 35 A apart but connected by a large internal cavity (approximately 10,000 A3). An unexpected zinc ion, coordinated by three cysteine and one histidine side-chains, is bound to the delta-subunit. The N-terminal half of the alpha subunit of TSOX (alphaA) is closely similar to the FAD-binding domain of glutathione reductase but with NAD+ replacing FAD. The C-terminal half of the alpha subunit of TSOX (alphaB) is similar to the C-terminal half of dimethylglycine oxidase and the T-protein of the glycine cleavage system, proteins that bind tetrahydrofolate. The beta-subunit of TSOX is very similar to monomeric sarcosine oxidase. The gamma-subunit is similar to the C-terminal sub-domain of alpha-TSOX. The delta-subunit shows little similarity with any PDB entry. The alphaA domain/beta-subunit sub-structure of TSOX closely resembles the alphabeta dimer of L-proline dehydrogenase, a heteroctameric protein (alphabeta)4 that shows highest overall similarity to TSOX.     

Kinetic studies of the mechanism of carbon-hydrogen bond breakage by the heterotetrameric sarcosine oxidase of Arthrobacter sp. 1-IN

The reaction of heterotetrameric sarcosine oxidase (TSOX) of Arthrobactor sp. 1-IN has been studied by stopped-flow spectroscopy, with particular emphasis on the reduction of the enzyme by sarcosine. Expression of the cloned gene encoding TSOX in Escherichia coli enables the production of TSOX on a scale suitable for stopped-flow studies. Treatment of the enzyme with sulfite provides the means for selective formation of a flavin-sulfite adduct with the covalent 8alpha-(N(3)-histidyl)-FMN. Formation of the sulfite-flavin adduct suppresses internal electron transfer between the noncovalent FAD (site of sarcosine oxidation) and the covalent FMN (site of enzyme oxidation) and thus enables detailed characterization of the kinetics of FAD reduction by sarcosine using stopped-flow methods. The rate of FAD reduction displays a simple hyperbolic dependence on sarcosine concentration. Studies in the pH range 6.5-10 indicate there are no kinetically influential ionizations in the enzyme-substrate complex. A plot of the limiting rate of flavin reduction/the enzyme-substrate dissociation constant (k(lim)/K(d)) versus pH is bell-shaped and characterized by two macroscopic pK(a) values of 7.4 +/- 0.1 and 10.4 +/- 0.2: potential candidates for the two ionizable groups are discussed with reference to the structure of monomeric sarcosine oxidase (MSOX). The kinetic data are discussed with reference to potential mechanisms for the oxidation of amine molecules by flavoenzymes. Additionally, kinetic isotope effect studies of the rate of C-H bond breakage suggest that a ground-state quantum tunneling mechanism for H-transfer, facilitated by the low-frequency thermal motions of the protein molecule, accounts for C-H bond cleavage by TSOX. TSOX thus provides another example of C-H bond breakage by ground-state quantum tunneling, driven by protein dynamics [vibrationally enhanced ground-state quantum tunneling (VEGST)], for the oxidation of amines by enzymes.     

Architecture and characterization of sarcosine oxidase from Thermococcus kodakarensis KOD1

Sarcosine oxidase (SOX) catalyzes the oxidation of the methyl group in sarcosine and transfer of the oxidized methyl group into the one-carbon metabolic pool. Here, we separately cloned and expressed α and β subunit of SOX from Thermococcus kodakarensis KOD1 (TkSOX) in Escherichia coli and the recombinant proteins were purified to homogeneity. Gel filtration chromatography and transmission electron microscopy analysis showed that the α subunit formed a dimeric structure and behaved as an NADH dehydrogenase; β subunit was a tetramer that had sarcosine oxidase and l-proline dehydrogenase activity. The TkSOX complex assembled into the hetero-octameric (αβ)₄ form and had NADH dehydrogenase activity. Gold-label analysis indicated that α and β subunits were oriented in the alternative form. Based on these results, we suggested that TkSOX was a multifunctional enzyme and that each subunit and (αβ)₄ complex may separately exist as a function enzyme in different conditions.     

Formate dehydrogenase from Pseudomonas oxalaticus

Formate dehydrogenase (EC 1.2.1.2) from Pseudomonas oxalaticus has been isolated and characterized. The enzyme (molecular weight 315000) is a complex flavoprotein containing 2 FMN, 18--25 non-heme iron atoms and 15--20 acid-labile sulphides. In the last step of the purification, a sucrose gradient centrifugation, a second catalytically active species has been found apparently originating from a dissociation of the enzyme into two equal subunits. The enzyme is specific toward its natural substrate formate. It transfers electrons to NAD+, oxygen, ferricyanide, and a lot of nonphysiological acceptors (dyes). In addition electrons are transferred from NADH to these acceptors. The (reversible) removal of FMN requires a reduction step. Reincorporation has been followed by the reappearance of the reactivity against formate and by fluorescence titration. The deflavo enzyme also binds FAD and riboflavin. The resulting enzyme species show characteristic catalytic abilities. Activity against formate is peculiar to the FMN species.     

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.     

Esters of mandelic acid as substrates for (S)-mandelate dehydrogenase from Pseudomonas putida: implications for the reaction mechanism

(S)-Mandelate dehydrogenase (MDH) from Pseudomonas putida is a flavin mononucleotide (FMN)-dependent enzyme that oxidizes (S)-mandelate to benzoylformate. In this work, we show that the ethyl and methyl esters of (S)-mandelic acid are substrates for MDH. Although the binding affinity of the neutral esters is 25-50-fold lower relative to the negatively charged (S)-mandelate, they are oxidized with comparable k(cat)s. Substrate analogues in which the carbonyl group on the C-1 carbon is replaced by other electron-withdrawing groups were not substrates. The requirement of a carbonyl group on the C-1 carbon in a substrate suggests that the negative charge developed during the reaction is stabilized by delocalization to the carbonyl oxygen. Arg277, a residue that is important in both binding and transition state stabilization for the activity with (S)-mandelate, is also critical for transition state stabilization for the esters, but not for their binding affinity. We previously showed that the substrate oxidation half-reaction with (S)-mandelate has two rate-limiting steps of similar activation energies and proceeds through the formation of a charge-transfer complex of an electron-rich donor and oxidized FMN [Dewanti, A. R., and Mitra, B. (2003) Biochemistry 42, 12893-12901]. This charge-transfer intermediate was observed with the neutral esters as well. The observation of this electron-rich intermediate for the oxidation of an uncharged substrate to an uncharged product, as well as the critical role of Arg277 in the reaction with the esters, provides further evidence that the MDH reaction mechanism is not a concerted transfer of a hydride ion from the substrate to the FMN, but involves the transient formation of a carbanion/ene(di)olate intermediate.     

An aspartate transcarbamylase lacking catalytic subunit interactions. Study of conformational changes by ultraviolet absorbance and circular dichroism spectroscopy.

A modified form of aspartate transcarbamylase is synthesized by Escherichia coli in the presence of 2-thiouracil which does not exhibit homotropic cooperative interactions between active sites yet retains heterotropic cooperative interactions due to nucleotide binding. The conformational changes induced in the modified enzyme by the binding of different ligands (substrates, substrate analogs, a transition state analog, and nucleotide effectors) were studied using ultraviolet absorbance and circular dichroism difference spectroscopy. Comparison of the results for the modified enzyme and its isolated subunits to those for the native enzyme and its isolated subunits showed that the conformational changes detected by these methods are qualitatively similar in the two enzymes. Comparison of the absorbance difference spectra due to the binding of a transition substrate analog to the intact native or modified enzymes to the corresponding results for the isolated subunits suggested that ligand binding causes an increased exposure to solvent of certain tyrosyl and phenylalanyl residues in the intact enzymes but not in the isolated subunits. This result is consistent with a diminution of subunit contacts due to substrate binding in the course of homotropic interactions in the native enzyme. Such conformational changes, though perhaps necessary for homotropic cooperativity, are not sufficient to cause homotropic cooperativity since the modified enzyme gave identical perturbations. Interactions of the transition state analog, N-(phosphonacetyl)-L-aspartate, with the modified enzyme were studied. Enzyme kinetic data obtained at low aspartate concentrations showed that this transition state analog does not stimulate activity, but rather exhibits the inhibition predicted for the total absence of homotropic cooperative interactions in the modified enzyme. Spectrophotometric titrations of the number of catalytic sites with the transition state analog showed that the modified enzyme and its isolated subunits possess, respectively, four and two high affinity sites for the inhibitor instead of six and three observed in the case of the normal enzyme and its isolated catalytic subunits. These results are correlated with the lower specific enzymatic activities of the modified enzyme and its catalytic subunits compared to the normal corresponding enzymatic species.     

Purification and properties of a NAD(P)H:flavin oxidoreductase from the luminous bacterium, Beneckea harveyi.

A NAD(P)H:flavin oxidoreductase, which produces FMNH2, one of the substrates for the luciferase reaction in bioluminescent bacteria, has been purified with the aid of affinity chromatography on epsilon-aminohexanoyl-FMN-Sepharose. The purified enzyme, isolated from Beneckea harveyi, had a specific activity of 89 mumol of NADH oxidized/min/mg of protein at 23 degrees in the presence of saturating FMN and NADH and appeared homogeneous by several criteria on polyacrylamide gel electrophoresis. A molecular weight of 24,000 was estimated both by gel filtration and and sodium dodecyl sulfate gel electrophoresis indicating that the enzyme is composed of a single polypeptide chain. Kinetic studies showed that the higher specificity of the enzyme for NADH than NADPH and for riboflavin and FMN than FAD was primarily due to variations in the Michaelis constants for the different substrates. Initial velocity studies with all pairs of substrates gave intersecting patterns supporting a sequential mechanism for the NAD(P)H:flavin oxidoreductase.     

Ferredoxin-dependent iron-sulfur flavoprotein glutamate synthase (GlsF) from the Cyanobacterium synechocystis sp. PCC 6803: expression and assembly in Escherichia coli

The unicellular cyanobacterium Synechocystis sp. PCC 6803 contains two different glutamate synthases whose genes, gltB and glsF (previously known as gltS), have been cloned (F. Navarro et al., 1995, Plant Mol. Biol. 27, 753-767). The glsF gene has been expressed in the glutamate auxotrophic Escherichia coli strain CLR207 RecA, but the corresponding protein does not complement the auxotrophy. The transformed strain showed ferredoxin-dependent glutamate synthase (Fd-GOGAT) activity, demonstrating the capability of E. coli for providing and correctly assembling both the iron-sulfur center and the flavin cofactor of the enzyme. Fd-GOGAT (GlsF) is correctly cleaved at Cys37 to form the mature enzyme in E. coli, as occurs with the large subunit of its own NADPH-GOGAT. The recombinant Fd-GOGAT has been purified to electrophoretic homogeneity, using as the main purification step a ferredoxin-affinity chromatography. The pure enzyme, with a molecular mass of about 180 kDa, shows an absorption spectrum characteristic of iron-sulfur flavoproteins. The analyses of the prosthetic groups indicate that Fd-GOGAT contains only one FMN, but no FAD, and one [3Fe-4S](+,0) cluster per molecule. Oxidation-reduction titration, using absorbance changes of the FMN group in the visible region, gave a midpoint redox potential of -200 +/- 25 mV at pH 7.5. The recombinant enzyme is strictly ferredoxin-dependent and shows apparent K(M) values similar to those of the native Synechocystis protein: 4.5 vs 3.5 microM, 2.2 vs 2.5 mM, and 0.6 vs 0.5 mM for ferredoxin, glutamine, and 2-oxoglutarate, respectively. The addition of the reductant dithionite to the enzyme resulted in the loss of the absorption peak at 436 nm, characteristic of oxidized flavins, which was restored by the anaerobic addition of 2-oxoglutarate, in the presence of glutamine.     

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.     

A new flavin radical signal in the Na(+)-pumping NADH:quinone oxidoreductase from Vibrio cholerae. An EPR/electron nuclear double resonance investigation of the role of the covalently bound flavins in subunits B and C

The Na(+)-pumping NADH-ubiquinone oxidoreductase has six polypeptide subunits (NqrA-F) and a number of redox cofactors, including a noncovalently bound FAD and a 2Fe-2S center in subunit F, covalently bound FMNs in subunits B and C, and a noncovalently bound riboflavin in an undisclosed location. The FMN cofactors in subunits B and C are bound to threonine residues by phosphoester linkages. A neutral flavin-semiquinone radical is observed in the oxidized enzyme, whereas an anionic flavin-semiquinone has been reported in the reduced enzyme. For this work, we have altered the binding ligands of the FMNs in subunits B and C by replacing the threonine ligands with other amino acids, and we studied the resulting mutants by EPR and electron nuclear double resonance spectroscopy. We conclude that the sodium-translocating NADH:quinone oxidoreductase forms three spectroscopically distinct flavin radicals as follows: 1) a neutral radical in the oxidized enzyme, which is observed in all of the mutants and most likely arises from the riboflavin; 2) an anionic radical observed in the fully reduced enzyme, which is present in wild type, and the NqrC-T225Y mutant but not the NqrB-T236Y mutant; 3) a second anionic radical, seen primarily under weakly reducing conditions, which is present in wild type, and the NqrB-T236Y mutant but not the NqrC-T225Y mutant. Thus, we can tentatively assign the first anionic radical to the FMN in subunit B and the second to the FMN in subunit C. The second anionic radical has not been reported previously. In electron nuclear double resonance spectra, it exhibits a larger line width and larger 8alpha-methyl proton splittings, compared with the first anionic radical.     

Formate dehydrogenase from the methane oxidizer Methylosinus trichosporium OB3b.

Formate dehydrogenase (NAD+ dependent) was isolated from the obligate methanotroph Methylosinus trichosporium OB3b. When the enzyme was isolated anaerobically, two forms of the enzyme were seen on native polyacrylamide gels, DE-52 cellulose and Sephacryl S-300 columns; they were approximately 315,000 and 155,000 daltons. The enzyme showed two subunits on sodium dodecyl sulfate-polyacrylamide gels. The Mr of the alpha-subunit was 53,800 +/- 2,800, and that of the beta-subunit was 102,600 +/- 3,900. The enzyme (Mr 315,000) was composed of these subunits in an apparent alpha 2 beta 2 arrangement. Nonheme iron was present at a concentration ranging from 11 to 18 g-atoms per mol of enzyme (Mr 315,000). Similar levels of acid-labile sulfide were detected. No other metals were found in stoichiometric amounts. When the enzyme was isolated aerobically, there was no cofactor requirement for NAD reduction; however, when isolated anaerobically, activity was 80 to 90% dependent on the addition of flavin mononucleotide (FMN) to the reaction mixture. Furthermore, the addition of formate to an active, anoxic solution of formate dehydrogenase rapidly inactivated it in the absence of an electron acceptor; this activity could be reconstituted approximately 85% by 50 nM FMN. Flavin adenine dinucleotide could not replace FMN in reconstituting enzyme activity. The Kms of formate dehydrogenase for formate, NAD, and FMN were 146, 200, and 0.02 microM, respectively. "Pseudomonas oxalaticus" formate dehydrogenase, which has physical characteristics nearly identical to those of the M. trichosporium enzyme, was also shown to be inactivated under anoxic conditions by formate and reactivated by FMN. The evolutionary significance of this similarity is discussed.     

Kinetic studies on the role of Lys-171 and Lys-358 in the beta subunit of sarcosine oxidase from Corynebacterium sp. U-96

Heterotetrameric sarcosine oxidase from Corynebacterium sp.U-96(SO-U96) contains non-covalent and covalent flavins. Lys-358 and Lys-171 in the beta subunit is present at non-covalent flavin adenine dinucleotide (FAD)- and covalent flavin monodinucleotide (FMN)-binding sites, respectively. The Lys-358 mutant, K358R showed 0.07% activity and higher apparent K(m) for sarcosine than the wild-type enzyme, but K358A and K358D mutants showed no activity, suggesting the importance of amino group of Lys358 in the sarcosine-binding to the enzyme. The Lys171 mutants, K171R, K171A and K171D showed 58, 39 and 32% activity of the wild-type enzyme, respectively. An apparent K(m) for oxygen and K(d) of enzyme-sulphite complex increased by the mutation. The rate of reduction of the FAD of K171 mutants with sarcosine did not change by the mutation. The stopped-flow photodiode array analyses of the anaerobic reduction with sarcosin of the wild-type and K171 mutant enzymes showed characteristic spectra of neutral and anionic semiquinones, especially for K171A enzyme. On the basis of these results, the reductive-half reaction of the wild-type and K171 mutant enzymes is explained by a mechanism involving the semiquinones. Low activity of K171 mutants is suggested to be derived from the low rate of oxidation of the reduced FMN in the enzyme.     

Active site analysis and stabilization of sarcosine oxidase by the substitution of cysteine residues.

Two cysteine residues (C-265 and C-318) in the putative hydrophilic regions of sarcosine oxidase were substituted by using site-directed mutagenesis. Since the mutant with the C-to-S mutation at position 318 (C318S) lost the enzyme activity, C-318 (conserved among sarcosine oxidases) is most likely a part of the active site. C265S, C265A, C265D, and C265R showed nearly the same enzymatic properties as those of the wild type. However, they were much more stable than the wild type in the presence of inhibitors that modified the thiol group. Moreover, they were extremely stable throughout the cultivation of the recombinant strains or even in cell extracts.     

Crystal structure of the type II isopentenyl diphosphate:dimethylallyl diphosphate isomerase from Bacillus subtilis

Two types of isopentenyl diphosphate:dimethylallyl diphosphate isomerases (IDI) have been characterized at present. The long known IDI-1 is only dependent on divalent metals for activity, whereas IDI-2 requires a metal, FMN and NADPH. Here, we report the first structure of an IDI-2 from Bacillus subtilis at 1.9A resolution in the ligand-free form and of the FMN-bound form at 2.8A resolution. The enzyme is an octamer that forms a D4 symmetrical open, cage-like structure. The monomers of 45 kDa display a classical TIM barrel fold. FMN is bound only with very moderate affinity and is therefore completely lost during purification. However, the enzyme can be reconstituted in the crystals by soaking with FMN. Three glycine-rich sequence stretches that are characteristic for IDI-2 participate in FMN binding within the interior of the cage. Regions harboring strictly conserved residues that are implicated in substrate binding or catalysis remain largely disordered even in the presence of FMN.     

Content and localization of FMN, Fe-S clusters and nickel in the NAD-linked hydrogenase of Nocardia opaca 1b

By preparative polyacrylamide gel electrophoresis at pH 8.5, and in the absence of nickel ions, two types of subunit dimers of the NAD-linked hydrogenase from Nocardia opaca 1b were separated and isolated, and their properties were compared with each other as well as with the properties of the native enzyme. The intact hydrogenase contained 14.3 +/- 0.4 labile sulphur, 13.6 +/- 1.1 iron and 3.8 +/- 0.1 nickel atoms and approximately 1 FMN molecule per enzyme molecule. The oxidized hydrogenase showed an absorption spectrum with maxima (shoulders) at 380 nm and 420 nm and an electron spin resonance (ESR) spectrum with a signal at g = 2.01. The midpoint redox potential of the Fe-S cluster giving rise to this signal was +25 mV. In the reduced state, hydrogenase gave characteristic low-temperature (10-20 K) and high-temperature (greater than 40 K) ESR spectra which were interpreted as due to [4Fe-4S] and [2Fe-2S] clusters, respectively. The midpoint redox potentials of these clusters were determined to be -420 mV and -285 mV, respectively. The large hydrogenase dimer, consisting of subunits with relative molecular masses Mr, of 64000 and 31000, contained 9.9 +/- 0.4 S2- and 9.3 +/- 0.5 iron atoms per protein molecule. This dimer contained the FMN molecule, but no nickel. The absorption and ESR spectra of the large dimer were qualitatively similar to the spectra of the whole enzyme. This dimer did not show any hydrogenase activity, but reduced several electron acceptors with NADH as electron donor (diaphorase activity). The small hydrogenase dimer, consisting of subunits with Mr of 56000 and 27000, was demonstrated to have substantially different properties. For iron and labile sulphur average values of 3.9 and 4.3 atoms/dimer molecule have been determined, respectively. The dimer contained, in addition, about 2 atoms of nickel and was free of flavins. In the oxidized state this dimer showed an absorption spectrum with a broad band in the 400-nm region and a characteristic ESR signal at g = 2.01. The reduced form of the dimer was ESR-silent. The small dimer alone was diaphorase-inactive and did not reduce NAD with H2, but it displayed high H2-uptake activities with viologen dyes, methylene blue and FMN, and H2-evolving activity with reduced methyl viologen. Hydrogen-dependent NAD reduction was fully restored by recombining both subunit dimers, although the reconstituted enzyme differed from the original in its activity towards artificial acceptors and the ESR spectrum in the oxidized state.     

Histone demethylase LSD1 is a folate-binding protein

Methylation of lysine residues in histones has been known to serve a regulatory role in gene expression. Although enzymatic removal of the methyl groups was discovered as early as 1973, the enzymes responsible for their removal were isolated and their mechanism of action was described only recently. The first enzyme to show such activity was LSD1, a flavin-containing enzyme that removes the methyl groups from lysines 4 and 9 of histone 3 with the generation of formaldehyde from the methyl group. This reaction is similar to the previously described demethylation reactions conducted by the enzymes dimethylglycine dehydrogenase and sarcosine dehydrogenase, in which protein-bound tetrahydrofolate serves as an accepter of the formaldehyde that is generated. We now show that nuclear extracts of HeLa cells contain LSD1 that is associated with folate. Using the method of back-scattering interferometry, we have measured the binding of various forms of folate to both full-length LSD1 and a truncated form of LSD1 in free solution. The 6R,S form of the natural pentaglutamate form of tetrahydrofolate bound with the highest affinity (K(d) = 2.8 μM) to full-length LSD1. The fact that folate participates in the enzymatic demethylation of histones provides an opportunity for this micronutrient to play a role in the epigenetic control of gene expression.     

Purification of the two-enzyme system catalyzing the oxidation of the D-proline residue of pristinamycin IIB during the last step of pristinamycin IIA biosynthesis.

High levels of conversion of 14C-labelled pristinamycin IIB (PIIB) to pristinamycin IIA (PIIA) were obtained in vivo in Streptomyces pristinaespiralis and in some other streptogramin A producers. This established that PIIB was an intermediate on the pathway to PIIA. In addition, in vitro studies with cell-free protein preparations demonstrated that the oxidation of PIIB to PIIA is a complex process requiring NADH, riboflavin 5'-phosphate (FMN), and molecular oxygen. Two enzymes were shown to be necessary to catalyze this reaction. Both were purified to homogeneity from S. pristinaespiralis by a coupled enzyme assay based on the formation of PIIA and by requiring addition of the complementing enzyme. One enzyme was purified about 3,000-fold by a procedure including a decisive affinity chromatography step on FMN-agarose. It was shown to be a NADH:FMN oxidoreductase (E.C. 1.6.8.1.) (hereafter called FMN reductase), providing reduced FMN (FMNH2) to the more abundant second enzyme. The latter was purified only 160-fold and was called PIIA synthase. Our data strongly suggest that this enzyme catalyzes a transient hydroxylation of PIIB by molecular oxygen immediately followed by a dehydration leading to PIIA. The native PIIA synthase consists of two different subunits with Mrs of around 50,000 and 35,000, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while the FMN reductase seems to be a monomer with a Mr of around 28,000 and containing one molecule of tightly bound FMN. Stepwise Edman degradation of the entire polypeptides or some of their trypsin-digested fragments provided amino acid sequences for the two isolated proteins.