Kinetics of electron entry, exit, and interflavin electron transfer during catalysis by sarcosine oxidase.

Sarcosine oxidase contains 1 mol of covalently bound plus 1 mol of noncovalently bound FAD per active site. The first phase of the anaerobic reduction of the enzyme with sarcosine converts oxidized enzyme to an equilibrium mixture of two-electron-reduced forms (EH2) and occurs at a rate (2700 min-1, pH 8.0) similar to that determined for the maximum rate of aerobic turnover in steady-state kinetic studies (2600 min-1). The second phase of the anaerobic half-reaction converts EH2 to the four-electron-reduced enzyme (EH4) and occurs at a rate (k = 350 min-1) which is 7-fold slower than aerobic turnover. Reaction of EH2 with oxygen is 1.7-fold faster (k = 4480 min-1) than aerobic turnover and 13-fold faster than the anaerobic conversion of EH2 to EH4. The results suggest that the enzyme cycles between fully oxidized and two-electron-reduced forms during turnover with sarcosine. The long wavelength absorbance observed for EH2 is attributable to a flavin biradical (FADH.FAD.-) which is generated in about 50% yield at pH 8.0 and in nearly quantitative yield at pH 7.0. The rate of biradical formation is determined by the rate of electron transfer from sarcosine to the noncovalent flavin since electron equilibration between the two flavins (k = 750 s-1 or 45,000 min-1, pH 8.0) is nearly 20-fold faster, as determined in pH-jump experiments. Only two of the three possible isoelectronic forms of EH2 are likely to transfer electrons to oxygen since the reaction is known to occur at the covalent flavin. However, equilibration among EH2 forms is probably maintained during reoxidation, consistent with the observed monophasic kinetics, since interflavin electron transfer is 10-fold faster than electron transfer to oxygen.     

Monomeric sarcosine oxidase: role of histidine 269 in catalysis

Conservative mutation of His269 (to Asn, Ala, or Gln) does not-significantly affect the expression of monomeric sarcosine oxidase (MSOX), covalent flavinylation, the physicochemical properties of bound FAD, or the overall protein structure. Turnover with sarcosine and the limiting rate of the reductive half-reaction with L-proline at pH 8.0 are, however, nearly 2 orders of magnitude slower than that with with wild-type MSOX. The crystal structure of the His269Asn complex with pyrrole-2-carboxylate shows that the pyrrole ring of the inhibitor is displaced as compared with wild-type MSOX. The His269 mutants all form charge-transfer complexes with pyrrole-2-carboxylate or methylthioacetate, but the charge-transfer bands are shifted to shorter wavelengths (higher energy) as compared with wild-type MSOX. Both wild-type MSOX and the His269Asn mutant bind the zwitterionic form of L-proline. The E(ox).L-proline complex formed with the His269Asn mutant or wild-type MSOX contains an ionizable group (pK(a) = 8.0) that is required for conversion of the zwitterionic L-proline to the reactive anionic form, indicating that His269 is not the active-site base. We propose that the change in ligand orientation observed upon mutation of His269 results in a less than optimal overlap of the highest occupied orbital of the ligand with the lowest unoccupied orbital of the flavin. The postulated effect on orbital overlap may account for the increased energy of charge-transfer bands and the slower rates of electron transfer observed for mutant enzyme complexes with charge-transfer ligands and substrates, respectively.     

A mutant sarcosine oxidase in which activity depends on flavin adenine dinucleotide

The covalent flavin attachment site in the Arthrobacter sarcosine oxidase (cysteine at position 318) was replaced with serine, and the mutational effect of C318S was analyzed. Wild type and C318S with a C-terminal 6-histidine tag were constructed and homogeneously purified by the single step. The covalently binding to flavin was not essential to the enzyme activity because the C318S mutant exhibited extremely weak activity. Moreover, the activity of the mutant was recovered in the presence of flavin adenine dinucleotide (FAD), and significantly increased as the concentration of FAD increased. This dependence of the mutant on FAD indicates that the noncovalent binding of free FAD to the mutant enzyme is reversible.     

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.     

Kinetic studies on the reaction mechanism of sarcosine oxidase

A sarcosine oxidase (sarcosine: oxygen oxidoreductase (demethylating), EC 1.5.3.1) isolated from Corynebacterium sp. U-96 contains both covalently bound FAD and noncovalently bound FAD. The noncovalent FAD reacts with sarcosine, the covalent FAD with molecular oxygen (Jorns, M.S. (1985) Biochemistry 24, 3189-3194). To clarify the reaction mechanism of the enzyme, kinetic investigations were performed by the stopped-flow method as well as by analysis of the overall reaction. The absorption spectrum of the enzyme in the steady state was very similar to that of the oxidized enzyme, and no intermediate enzyme species, such as a semiquinoid flavin, was detected. The rate for anaerobic reduction of the noncovalently bound FAD and the covalently bound FAD by sarcosine were 31 and 6.7 s-1, respectively. The latter value was smaller than the value of respective Vmax/e0 obtained by the overall reaction kinetics (Vmax/e0: the maximum velocity per enzyme concentration). Both rate constants for oxidation of the two FADs by molecular oxygen were 100 s-1. A reaction scheme of sarcosine oxidase is proposed to account for the data obtained; 70% of the enzyme functions via a fully reduced enzyme, and 30% of the enzyme goes along a side-path, without forming the fully reduced enzyme. In addition, it is suggested that the reactivity of noncovalently bound FAD with sarcosine is affected by the oxidation-reduction state of the covalently bound FAD, in contrast to the reactivity of the covalently bound FAD with molecular oxygen, which is independent of the oxidation-reduction state of the noncovalently bound FAD.     

Covalent flavinylation of monomeric sarcosine oxidase: identification of a residue essential for holoenzyme biosynthesis

FAD in monomeric sarcosine oxidase (MSOX) is covalently linked to the protein by a thioether linkage between its 8alpha-methyl group and Cys315. Covalent flavinylation of apoMSOX has been shown to proceed via an autocatalytic reaction that requires only FAD and is blocked by a mutation of Cys315. His45 and Arg49 are located just above the si-face of the flavin ring, near the site of covalent attachment. His45Ala and His45Asn mutants contain covalently bound FAD and exhibit catalytic properties similar to wild-type MSOX. The results rule out a significant role for His45 in covalent flavinylation or sarcosine oxidation. In contrast, Arg49Ala and Arg49Gln mutants are isolated as catalytically inactive apoproteins. ApoArg49Ala forms a stable noncovalent complex with reduced 5-deazaFAD that exhibits properties similar to those observed for the corresponding complex with apoCys315Ala. The results show that elimination of a basic residue at position 49 blocks covalent flavinylation but does not prevent noncovalent flavin binding. The Arg49Lys mutant contains covalently bound FAD, but its flavin content is approximately 4-fold lower than wild-type MSOX. However, most of the apoprotein in the Arg49Lys preparation is reconstitutable with FAD in a reaction that exhibits kinetic parameters similar to those observed for flavinylation of wild-type apoMSOX. Although covalent flavinylation is scarcely affected, the specific activity of the Arg49Lys mutant is only 4% of that observed with wild-type MSOX. The results show that a basic residue at position 49 is essential for covalent flavinylation of MSOX and suggest that Arg49 also plays an important role in sarcosine oxidation.     

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.     

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.     

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.     

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.     

Properties and catalytic function of the two nonequivalent flavins in sarcosine oxidase

Sarcosine oxidase from Corynebacterium sp. U-96 contains 1 mol of noncovalently bound flavin and 1 mol of covalently bound flavin per mole of enzyme. Anaerobic titrations of the enzyme with either sarcosine or dithionite show that both flavins are reducible and that two electrons per flavin are required for complete reduction. Absorption increases in the 510-650-nm region, attributed to the formation of a blue neutral flavin radical, are observed during titration of the enzyme with dithionite or substrate, during photochemical reduction of the enzyme, and during reoxidation of substrate-reduced enzyme. Fifty percent of the enzyme flavin forms a reversible, covalent complex with sulfite (Kd = 1.1 X 10(-4) M), accompanied by a complete loss of catalytic activity. Sulfite does not prevent reduction of the sulfite-unreactive flavin by sarcosine but does interfere with the reoxidation of reduced enzyme by oxygen. The stability of the sulfite complex is unaffected by excess acetate (an inhibitor competitive with sarcosine) or by removal of the noncovalent flavin to form a semiapoprotein preparation where 75% of the flavin reacts with sulfite (Kd = 9.4 X 10(-5) M) while only 3% remains reducible with sarcosine. The results indicate that oxygen and sulfite react with the covalently bound flavin and suggest that sarcosine is oxidized by the noncovalently bound flavin.     

Mechanistic aspects and redox properties of hyperthermophilic L-proline dehydrogenase from Pyrococcus furiosus related to dimethylglycine dehydrogenase/oxidase

Two ORFs encoding a protein related to bacterial dimethylglycine oxidase were cloned from Pyrococcus furiosus DSM 3638. The protein was expressed in Escherichia coli, purified, and shown to be a flavoprotein amine dehydrogenase. The enzyme oxidizes the secondary amines L-proline, L-pipecolic acid and sarcosine, with optimal catalytic activity towards L-proline. The holoenzyme contains one FAD, FMN and ATP per alphabeta complex, is not reduced by sulfite, and reoxidizes slowly following reduction, which is typical of flavoprotein dehydrogenases. Isolation of the enzyme in a form containing only FAD cofactor allowed detailed pH dependence studies of the reaction with L-proline, for which a bell-shaped dependence (pK(a) values 7.0 +/- 0.2 and 7.6 +/- 0.2) for k(cat)/K(m) as a function of pH was observed. The pH dependence of k(cat) is sigmoidal, described by a single macroscopic pK(a) of 7.7 +/- 0.1, tentatively attributed to ionization of L-proline in the Michaelis complex. The preliminary crystal structure of the enzyme revealed active site residues conserved in related amine dehydrogenases and potentially implicated in catalysis. Studies with H225A, H225Q and Y251F mutants ruled out participation of these residues in a carbanion-type mechanism. The midpoint potential of enzyme-bound FAD has a linear temperature dependence (- 3.1 +/- 0.05 mV x C degrees (-1)), and extrapolation to physiologic growth temperature for P. furiosus (100 degrees C) yields a value of - 407 +/- 5 mV for the two-electron reduction of enzyme-bound FAD. These studies provide the first detailed account of the kinetic/redox properties of this hyperthermophilic L-proline dehydrogenase. Implications for its mechanism of action are discussed.     

Monomeric sarcosine oxidase: evidence for an ionizable group in the E.S complex

Monomeric sarcosine oxidase (MSOX) contains covalently bound FAD and catalyzes the oxidation of sarcosine (N-methylglycine) and other secondary amino acids, including L-proline. The reductive half-reaction with L-proline proceeds via a rapidly attained equilibrium (K(d)) between free E(ox) and the E(ox).S complex, followed by a practically irreversible reduction step (E(ox).S --> E(red).P) associated with a rate constant, k(lim). The effect of pH on the reductive half-reaction shows that the K(d) for L-proline binding is pH-independent (pH 6.46-9.0). This indicates that MSOX binds the zwitterionic form of L-proline, the predominant species in solution at neutral pH (pK(a) = 10.6). Values for the limiting rate of reduction (k(lim)) are, however, strongly pH-dependent and indicate that an ionizable group in the E(ox).L-proline complex (pK(a) = 8.02) must be unprotonated for conversion to E(red).P. Charge-transfer interaction with L-proline as the donor and FAD as acceptor is possible only with the anionic form of L-proline. The ionizable group in the E(ox).L-proline complex is required for conversion of enzyme-bound L-proline from the zwitterionic to the reactive anionic form, as judged by the independently determined pK(a) for charge-transfer complex formation with the MSOX flavin (pK(a) = 7.94). The observation that the anionic form of L-proline with a neutral amino group is the reactive species in the reduction of MSOX is similar to that observed for other flavoenzymes that oxidize amines, including monoamine oxidase and trimethylamine dehydrogenase.     

Arginine 49 is a bifunctional residue important in catalysis and biosynthesis of monomeric sarcosine oxidase: a context-sensitive model for the electrostatic impact of arginine to lysine mutations

Monomeric sarcosine oxidase (MSOX) contains covalently bound FAD and catalyzes the oxidative demethylation of sarcosine ( N-methylglycine). The side chain of Arg49 is in van der Waals contact with the si face of the flavin ring; sarcosine binds just above the re face. Covalent flavin attachment requires a basic residue (Arg or Lys) at position 49. Although flavinylation is scarcely affected, mutation of Arg49 to Lys causes a 40-fold decrease in k cat and a 150-fold decrease in k cat/ K m sarcosine. The overall structure of the Arg49Lys mutant is very similar to wild-type MSOX; the side chain of Lys49 in the mutant is nearly congruent to that of Arg49 in the wild-type enzyme. The Arg49Lys mutant exhibits several features consistent with a less electropositive active site: (1) Charge transfer bands observed for mutant enzyme complexes with competitive inhibitors absorb at higher energy than the corresponding wild-type complexes. (2) The p K a for ionization at N(3)H of FAD is more than two pH units higher in the mutant than in wild-type MSOX. (3) The reduction potential of the oxidized/radical couple in the mutant is 100 mV lower than in the wild-type enzyme. The lower reduction potential is likely to be a major cause of the reduced catalytic activity of the mutant. Electrostatic interactions with Arg49 play an important role in catalysis and covalent flavinylation. A context-sensitive model for the electrostatic impact of an arginine to lysine mutation can account for the dramatically different consequences of the Arg49Lys mutation on MSOX catalysis and holoenzyme biosysnthesis.     

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.     

Expression and characterization of a thermostable sarcosine oxidase (SOX) from Bacillus sp. in Escherichia coli

A heat-stable sarcosine oxidase produced by Bacillus sp. BSD-8 (SOX) had been studied and its complete gene sequence, which contained 1,164 bp nucleotides and encoded a protein of 387 amino acids, was obtained by DNA Walking method. The sox gene was cloned and functionally overexpressed in E. coli and the recombinant SOX (rSOX) was purified to homogeneity, its properties was studied and compared with the wild type of SOX. The rSOX as well as SOX was stable at 60°C and at pH 7.0∼10.0, respectively. The optimal temperature for this enzyme was 60°C and at pH 8.5, it showed its highest activity. The Km and Kcat of the enzyme was 3.1 mM and 20.3/s, respectively. The difference between the properties of the SOX and rSOX was that the SOX contained noncovalent FAD, whereas the rSOX contained covalent FAD. The study also showed that an increased number of alanine residues in the rSOX might have some contribution in the enzymatic thermostability.     

芽胞杆菌BSD-8肌氨酸氧化酶的纯化与性质

对一株从土壤中分离到的芽胞杆菌Bacillus sp.BSD-8菌株所产生的热稳定性较高的肌氨酸氧化酶进行纯化,并对该酶的特性进行了研究。通过硫酸铵分级沉淀、DEAE-纤维素离子交换柱、Toyopearl疏水层析柱和Sephadex G-75分子筛层析,使酶提纯25倍,比活力达到5.3U/mg。研究了纯化后的酶的生化特性,确定了该酶的主要特性:该酶为黄素蛋白,与黄素以非共价键的方式结合,由单一亚基组成,其亚基分子量为51kDa。酶的最适反应温度及pH分别为60℃与8.5。该酶在60℃及pH8.0~10.0条件下稳定。以Lineveaver-Burk作图法求得该酶米氏常数Km值为3.1mmol/L。Ag+、Hg2+、SDS及Tween80对该酶有强抑制作用,而Tween20和Triton X-100对酶活性无影响。该肌氨酸氧化酶在耐热性质上比以前所报道的肌氨酸氧化酶有很大的提高,在酶法肌酐测定应用中有明显的优势。     

Influence of polymolecular events on inactivation behavior of xylose isomerase from Thermotoga neapolitana 5068

The inactivation behavior of the xylose isomerase from Thermotoga neapolitana (TN5068 XI) was examined for both the soluble and immobilized enzyme. Polymolecular events were involved in the deactivation of the soluble enzyme. Inactivation was biphasic at 95 degrees C, pH 7.0 and 7.9, the second phase was concentration-dependent. The enzyme was most stable at low enzyme concentrations, however, the second phase of inactivation was 3- to 30-fold slower than the initial phase. Both phases of inactivation were more rapid at pH 7.9, relative to 7.0. Differential scanning calorimetry of the TN5068 XI revealed two distinct thermal transitions at 99 degrees and 109 degrees C. The relative magnitude of the second transition was dramatically reduced at pH 7.9 relative to pH 7.0. Approximately 24% and 11% activity were recoverable after the first transition at pH 7.0 and 7.9, respectively. When the TN5068 XI was immobilized by covalent attachment to glass beads, inactivation was monophasic with a rate corresponding to the initial phase of inactivation for the soluble enzyme. The immobilized enzyme inactivation rate corresponded closely to the rate of ammonia release, presumably from deamidation of labile asparagine and/or glutamine residues. A second, slower inactivation phase suggests the presence of an unfolding intermediate, which was not observed for the immobilized enzyme. The concentration dependence of the second phase of inactivation suggests that polymolecular events were involved. Formation of a reversible polymolecular aggregate capable of protecting the soluble enzyme from irreversible deactivation appears to be responsible for the second phase of inactivation seen for the soluble enzyme. Whether this characteristic is common to other hyperthermophilic enzymes remains to be seen.     

Identification of the oxygen activation site in monomeric sarcosine oxidase: role of Lys265 in catalysis

Monomeric sarcosine oxidase (MSOX) catalyzes the oxidation of N-methylglycine and contains covalently bound FAD that is hydrogen bonded at position N(5) to Lys265 via a bridging water. Lys265 is absent in the homologous but oxygen-unreactive FAD site in heterotetrameric sarcosine oxidase. Isolated preparations of Lys265 mutants contain little or no flavin but can be covalently reconstituted with FAD. Mutation of Lys265 to a neutral residue (Ala, Gln, Met) causes a 6000- to 9000-fold decrease in apparent turnover rate whereas a 170-fold decrease is found with Lys265Arg. Substitution of Lys265 with Met or Arg causes only a modest decrease in the rate of sarcosine oxidation (9.0- or 3.8-fold, respectively), as judged by reductive half-reaction studies which show that the reactions proceed via an initial enzyme.sarcosine charge transfer complex and a novel spectral intermediate not detected with wild-type MSOX. Oxidation of reduced wild-type MSOX (k = 2.83 x 10(5) M(-1) s(-1)) is more than 1000-fold faster than observed for the reaction of oxygen with free reduced flavin. Mutation of Lys265 to a neutral residue causes a dramatic 8000-fold decrease in oxygen reactivity whereas a 250-fold decrease is observed with Lys265Arg. The results provide definitive evidence for Lys265 as the site of oxygen activation and show that a single positively charged amino acid residue is entirely responsible for the rate acceleration observed with wild-type enzyme. Significantly, the active sites for sarcosine oxidation and oxygen reduction are located on opposite faces of the flavin ring.     

Molecular characterization of NikD,a new flavoenzyme important in the biosynthesis of nikkomycin antibiotics

Nikkomycin antibiotics are potent inhibitors of chitin synthase, effective as therapeutic antifungal agents in humans and easily degradable insecticides in agriculture. NikD is a novel flavoprotein that catalyzes the oxidation of Delta(1)- or Delta(2)-piperideine-2-carboxylate, a key step in the biosynthesis of nikkomycin antibiotics. The resulting dihydropicolinate product may be further oxidized by nikD or converted to picolinate in a nonenzymic reaction. Saturated nitrogen heterocycles (L-pipecolate, L-proline) and 3,4-dehydro-L-proline act as alternate substrates. The ability of nikD to oxidize 3,4-dehydro-L-proline, but not 1-cyclohexenoate, suggests that the enzyme is specific for the oxidation of a carbon-nitrogen bond. An equivalent reaction is possible with the enamine (Delta(2)), but not the imine (Delta(1)), form of the natural piperideine-2-carboxylate substrate. Apparent steady-state kinetic parameters for the reaction of nikD with Delta(1)- or Delta(2)-piperideine-2-carboxylate (k(cat) = 64 min(-1); K(m) = 5.2 microM) or 3,4-dehydro-L-proline (k(cat) = 18 min(-1); K(m) = 13 mM) were determined in air-saturated buffer by measuring hydrogen peroxide formation in a coupled assay. NikD appears to be a new member of the monomeric sarcosine oxidase (MSOX) family of amine oxidizing enzymes. The enzyme contains 1 mol of flavin adenine dinucleotide (FAD) covalently linked to Cys321. The covalent flavin attachment site and two residues that bind substrate carboxylate in MSOX are conserved in nikD. NikD, however, exhibits an unusual long-wavelength absorption band, attributed to charge-transfer interaction between FAD and an ionizable (pK(a) = 7.3) active-site residue. Similar long-wavelength absorption bands have been observed for flavoproteins containing an active site cysteine or cysteine sulfenic acid. Interestingly, Cys273 in nikD aligns with an active-site histidine in MSOX (His269) that is, otherwise, a highly conserved residue within the MSOX family.