6-Hydroxy-D-nicotine oxidase of Arthrobacter oxidans. Gene structure of the flavoenzyme and its relationship to 6-hydroxy-L-nicotine oxidase

The nucleotide sequence of the 6-hydroxy-D-nicotine oxidase (6-HDNO) gene of Arthrobacter oxidans is presented. This covalently flavinylated enzyme specifically oxidizes 6-hydroxy-D-nicotine to 6-hydroxy-N-methylmyosmine. Coinduced in the presence of nicotine is a 6-hydroxy-L-nicotine-specific enzyme, 6-hydroxy-L-nicotine oxidase (6-HLNO), with FAD noncovalently bound to the apoprotein. A comparison of the nucleotide-derived amino acid sequence of the 6-HDNO with the amino acid sequence data obtained from the purified 6-HLNO polypeptide suggests that the two enantiozymes expressed within the same cell are genetically unrelated. This conclusion is supported by the finding that the FAD-binding sites of the two enzymes are different. 6-HLNO exhibits at the amino-terminus of the polypeptide chain a dinucleotide-binding site characteristic for many other FAD- and NAD(P)-dependent enzymes. No such sequence was found in the nucleotide-derived amino acid sequence of 6-HDNO.     

Lysine can replace arginine 67 in the mediation of covalent attachment of FAD to histidine 71 of 6-hydroxy-D-nicotine oxidase

The requirements for FAD-attachment to His71 of 6-hydroxy-D-nicotine oxidase (6-HDNO) were investigated by site-directed mutagenesis. The following amino acid replacements were introduced into the sequence Arg67-Ser68-Gly69-Gly70-His71 of the 6-HDNO-polypeptide: 1) Arg67 was replaced with Ala (A1 mutant); 2) Ser68 was replaced with Ala (A2 mutant); and 3) Arg67 was replaced with Lys (K mutant). The substitution in mutant A2 had no effect on flavinylation, measured as [14C]FAD incorporation into apo-6-HDNO. Replacement of Arg67 with Ala prevented, but replacement with Lys permitted the flavinylation of His71. Mutant A1 showed no 6-HDNO activity, whereas the replacement of Ser with Ala in mutant A2 had only a slight effect on 6-HDNO activity. The substitution of Lys for Arg67, however, reduced the specific 6-HDNO activity in extracts of Escherichia coli cells expressing the mutant polypeptide from 50.3 to 17.5 milliunits/mg protein. It is concluded that a basic amino acid residue (Arg67 or Lys67) is required to mediate the attachment of FAD to His71, and while Lys can substitute for Arg67 in this function, it can only partially replace Arg67 in the enzyme reaction mechanism of 6-HDNO.     

Autoflavinylation of apo6-hydroxy-D-nicotine oxidase

6-Hydroxy-D-nicotine oxidase (6-HDNO) was expressed in Escherichia coli JM109 cells from the recombinant plasmid pAX-6-HDNO as a beta-galactosidase-6-HDNO fusion protein. Affinity chromatography of the fusion protein on p-aminobenzyl-1-thio-beta-galactopyranoside-agarose and subsequent digestion with protease Xa yielded highly purified apo6-HDNO. Incubation of the purified protein with [14C]FAD demonstrated that flavinylation of apo6-HDNO proceeds autocatalytically. Phosphorylated three-carbon compounds such as glycerol-3-P, which are known to stimulate the formation of the histidyl (N3)-(8 alpha) FAD between apo6-HDNO and FAD (Brandsch, R., and Bichler, V. (1989) Eur. J. Biochem. 182, 125-128), could be replaced in their action by high concentrations of glycerol (45%) or sucrose (20%). These substances apparently induced and stabilized a conformational state of the apoenzyme compatible with covalent attachment of FAD. In the absence of glycerol the apoenzyme readily lost the ability to form holoenzyme at temperatures above 30 degrees C. Holoenzyme formation protected the 6-HDNO polypeptide from this thermal denaturation. Autoflavinylation of 6-HDNO was inhibited by the sulfhydryl reagents dithionitrobenzoate or N-ethylmaleimide. Inhibition was prevented by mercaptoethanol or FAD, but not 6-hydroxy-D-nicotine, the substrate of the holoenzyme. A cysteine-thiol group may therefore be involved in reactions leading to the covalent attachment of FAD to apo6-HDNO. When flavinylation of apo6-HDNO proceeded under anaerobic conditions, the amount of incorporation of [14C]FAD into the polypeptide was indistinguishable from reactions performed in the presence of O2. None of the FAD-derivatives (8-demethyl-FAD, 8-chloro-FAD, and 5-deaza-FAD) could replace FAD in holoenzyme formation. The failure of covalent attachment of 5-deaza-FAD to apo6-HDNO is in agreement with the assumption that the quinone methide form of the isolloxazine ring is an intermediate in the flavinylation reaction.     

Binding of FAD to 6-hydroxy-D-nicotine oxidase apoenzyme prevents degradation of the holoenzyme.

Expression of the 6-hydroxy-D-nicotine oxidase (6-HDNO) gene from Arthrobacter oxidans cloned into Escherichia coli showed a marked temperature-dependence. Transformed E. coli cells grown at 30 degrees C exhibited a several-fold higher 6-HDNO activity than did cells grown at 37 degrees C. This effect did not depend on the promoter used for expression of the cloned gene in E. coli, nor was it an effect of 6-HDNO mRNA instability at 37 degrees C. Studies performed in vivo and in vitro revealed that an increased susceptibility of apo-6-HDNO to proteolytic attack at 37 degrees C was responsible for the observed phenomenon. Extracts from cells grown at 37 degrees C showed on Western blots a decrease in immunologically detectable 6-HDNO polypeptide when compared with extracts from cells grown at 30 degrees C. The 6-HDNO polypeptide is covalently modified by attachment of the cofactor FAD to a histidine residue. It could be shown that covalent flavinylation of the apoenzyme in vitro, i.e. formation of holoenzyme, by incubation of cell extracts with FAD and phosphoenolpyruvate protected the 6-HDNO polypeptide from degradation at 37 degrees C. Of a variety of proteinase inhibitors tested only the cysteine-proteinase inhibitor L-3-trans-carboxyoxiran-2-carbonyl-L-leucylagmatine (E64) prevented degradation, by up to 70%, of the apoenzyme.     

GroE dependence of refolding and holoenzyme formation of 6-hydroxy-D-nicotine oxidase.

In Escherichia coli cells expressing 6-hydroxy-D-nicotine oxidase (6-HDNO), a flavoprotein with covalently bound FAD, approximately 40% of the polypeptide is in its apoform. We investigated whether in vivo holoenzyme formation was influenced by the association of the apoenzyme with cellular chaperones. Immunoprecipitation of apoenzyme-containing cell extract with protein-A-Sepharose-bound 6-HDNO- or GroEL-specific antibodies failed to reveal the formation of complexes between these proteins. The limiting factor in holoenzyme formation in vivo appeared to be the intracellular supply of phosphorylated tricarbon compounds (e.g. glycerol-3-P) acting as allosteric effectors in the flavinylation reaction. When holoenzyme formation from purified apo6-HDNO was investigated in vitro, addition of GroEL and GroES to the reaction assays increased the yield of holoenzyme formation. The observed increase in apoenzyme to holoenzyme transition was ATP independent, and the effect of GroE could be simulated by high concentrations of glycerol (40%). Apparently, a nonspecific protein-protein interaction between the GroE proteins and the apo6-HDNO favored holoenzyme formation. The refolding of guanidinium hydrochloride-unfolded holoenzyme, however, was catalyzed by GroEL and GroES in an ATP-dependent reaction. Recovery of the native, enzymatically active, conformation ranged from 30 to 40%. When apo6-HDNO was denatured and refolded, the same dependence on GroE and ATP was observed in the recovery of a conformation able to incorporate FAD and to holoenzyme. [14C] FAD in the refolding assay yielded radioactively labeled 6-HDNO demonstrating the autocatalytical covalent incorporation of FAD into the polypeptide during the folding process.     

Purification, characterization and gene cloning of 6-hydroxynicotinate 3-monooxygenase from Pseudomonas fluorescens TN5.

6-Hydroxynicotinate 3-monooxygenase, a membrane-bound, 42-kDa monomeric enzyme from Pseudomonas fluorescens TN5 was purified and characterized. The enzyme catalyzes the oxidative decarboxylation of 6-hydroxynicotinate and depends on O2, NADH and FAD with the holoenzyme containing 1 M of FAD per 1 M of enzyme. The isolated enzyme was used for the synthesis of 2,5-dihydroxypyridine, a precursor for the chemical synthesis of 5-aminolevulinic acid, which is applied as a plant growth hormone, a herbicide and in cancer therapy. A 1.8-kbp DNA fragment, which contains the ORF encoding 6-hydroxynicotinic acid 3-monooxygenase, was cloned, sequenced and expressed in Escherichia coli. The deduced 385 amino acid sequence of the cloned ORF is in agreement with the enzyme molecular mass, amino acid sequence of an internal peptide, contains a putative FAD-binding site and is homologous to similar flavoproteins such as salicylate 1-monoxygenase.     

Cysteine residues in the active site of Corynebacterium sarcosine oxidase

Sarcosine oxidase from Corynebacterium sp. U-96 is inhibited by iodoacetamide (IAM) and the inhibition is prevented by the substrate analog, sodium acetate. To elucidate the mechanism of inhibition of the enzyme by IAM, we determined the amino acid sequences around the IAM-reactive cysteine residues, and the effects of the modification on the enzyme activity and the oxidation-reduction of the FAD moieties of the enzyme. The enzyme was specifically labeled with [14C]IAM, and the labeled subunit B was digested with trypsin and chymotrypsin. The HPLC profiles of the proteolytic digests showed mainly two radioactive peaks. The 14C-labeled peptides were purified, and their N-terminal sequences were determined to be Cys-Gly-Thr-Pro-Gly-Ala-Gly-Tyr (TC-1) and Ala-Gly-Ile-Ala-Cys-Xaa-Asp-Xaa-Val-Ala(-)- (TC-2). Peptide TC-2 contains a covalent FAD-binding sequence [Asx-His-Val-Ala; Shiga et al. (1983) Biochem. Int., 6, 737]. [14C]IAM-incorporation into the TC-1 sequence was strongly inhibited by sodium acetate. The N-terminal amino acid sequence of the CNBr fragment containing the TC-1 sequence (65 residues) was determined. According to the secondary structure predictions, Gly-Thr-Pro-Gly-Ala-Gly of the TC-1 sequence is located between the beta sheet and alpha helix of the sequence, indicating the presence of an AMP-binding site in the TC-1 region. The activity of the enzyme treated with IAM in the presence and absence of sodium acetate was not inhibited by sodium sulfite, which is known to react specifically with covalent FAD.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cofactors in sarcosine oxidase from Corynebacterium sp. U-96

Sarcosine oxidase from Corynebacterium sp. U-96 is a heterotetrameric enzyme that was reported to contain 1 mol of covalently bound FAD and 1 mol of non-covalently-bound FAD. This work describes the result of reinvestigation of the cofactors in this enzyme. The enzyme was found to contain 1 mol of non-covalently-bound NAD⁺, 1 mol of non-covalently-bound FAD, and 1 mol of covalent FMN. The covalent FMN was identified by the mass and amino acid sequence analyses of the flavin peptide.     

FAD binding by ApbE protein from Salmonella enterica: a new class of FAD-binding proteins

The periplasmic protein ApbE was identified through the analysis of several mutants defective in thiamine biosynthesis and was implicated as having a role in iron-sulfur cluster biosynthesis or repair. While mutations in apbE cause decreased activity of several iron-sulfur enzymes in vivo, the specific role of ApbE remains unknown. Members of the AbpE family include NosX and RnfF, which have been implicated in oxidation-reduction associated with nitrous oxide and nitrogen metabolism, respectively. In this work, we show that ApbE binds one FAD molecule per monomeric unit. The structure of ApbE in the presence of bound FAD reveals a new FAD-binding motif. Protein variants that are nonfunctional in vivo were generated by random and targeted mutagenesis. Each variant was substituted in the environment of the FAD and analyzed for FAD binding after reconstitution. The variant that altered a key tyrosine residue involved in FAD binding prevented reconstitution of the protein.     

A monoclonal antibody recognizing the FAD-binding site of 4-aminobenzoate hydroxylase from Agaricus bisporus

A monoclonal antibody against 4-aminobenzoate hydroxylase (EC 1.14.13.27) from Agaricus bisporus, a common edible mushroom, has been produced by the fusion of BALB/c mouse spleen cells immunized with the denatured enzyme and P3x63Ag8U1 myeloma cells in order to locate and characterize the catalytic site of the enzyme. The monoclonal antibody immunoblotted the enzyme and immunoprecipitated its apoenzyme. The immunoprecipitation was inhibited in the presence of FAD, and the monoclonal antibody competitively inhibited the binding of FAD to the apoenzyme. The monoclonal antibody, therefore, recognizes the FAD-binding site of 4-aminobenzoate hydroxylase. Interestingly, it was shown that the monoclonal antibody was cross-reactive with FAD-dependent enzymes such as salicylate hydroxylase (EC 1.14.13.1) and D-amino acid oxidase (EC 1.4.3.3), and that it was specific for the FAD-binding sites of these enzymes. This fact suggests that these FAD-dependent enzymes have immunologically similar structures on their FAD-binding sites.     

6-S-cysteinylation of bi-covalently attached FAD in berberine bridge enzyme tunes the redox potential for optimal activity

A mutagenic analysis of the amino acid residues His-104 and Cys-166, which are involved in the bi-covalent attachment of FAD to berberine bridge enzyme, was performed. Here we present a detailed biochemical characterization of the cysteine link to FAD observed in this recently discovered group of flavoproteins. The C166A mutant protein still has residual activity, but reduced to approximately 6% of the turnover rate observed for wild-type berberine bridge enzyme. A more detailed analysis of single reaction steps by stopped-flow spectrophotometry showed that the reductive half-reaction is greatly influenced by the lack of the 6-S-cysteinyl linkage, resulting in a 370-fold decrease in the rate of flavin reduction. Determination of the redox potentials for both wild type and the C166A mutein revealed that the difference in the redox potential observed can fully account for the change in the kinetic properties. The wild-type protein exhibits a midpoint potential of +132 mV, which is the highest redox potential determined for any flavoenzyme so far. Removal of the cysteine linkage to FAD in the C166A mutein leads to a redox potential of +53 mV, which is in the expected range for flavoproteins with a single covalent attachment of FAD to a His residue via its 8-alpha position. We also show that the biochemical properties of the mutein resemble that of typical flavoprotein oxidases and that deviations from this behavior observed for the wild type are due to the FAD-6-S-cysteinyl bond. In addition, rapid reaction stopped-flow experiments give no indication for a radical mechanism supporting the direct transfer of a hydride from the substrate to the cofactor.     

Determination of the roles of Glu-461 in beta-galactosidase (Escherichia coli) using site-specific mutagenesis

Site-directed substitutions (Asp, Gly, Gln, His, and Lys) were made for Glu-461 of beta-galactosidase (Escherichia coli). All substitutions resulted in loss of most activity. Substrates and a substrate analog inhibitor were bound better by the Asp-substituted enzyme than by the normal enzyme, about the same for enzyme substituted with Gly, but only poorly when Gln, His, or Lys was substituted. This shows that Glu-461 is involved in substrate binding. Binding of the positively charged transition state analog 2-aminogalactose was very much reduced with Gly, Gln, His, and Lys, whereas the Asp-substituted enzyme bound this inhibitor even better than did the wild-type enzyme. Since Asp, like Glu, is negatively charged, this strongly supports the proposal that one role of Glu-461 is to electrostatically interact with a positively charged galactosyl transition state intermediate. The substitutions also affected the ability of the enzyme to bind L-ribose, a planar analog of D-galactose that strongly inhibits beta-galactosidase activity. This indicates that the binding of a planar "galactose-like" compound is somehow mediated through Glu-461. The data indicated that the presence of Glu-461 is highly important for the acid catalytic component of kappa 2 (glycosylic bond cleavage or "galactosylation"), and therefore Glu-461 must be involved in a concerted acid catalytic reaction, presumably by stabilizing a developing carbonium ion. The kappa 2 values with o- and p-nitrophenyl-beta-D-galactopyranoside as substrates varied more or less as did the K8 values, indicating that most of the glycolytic bond breaking activity found for the enzymes from the mutants with these substrates was probably a result of strain or other such effects. The kappa 3 values (hydrolysis or "degalactosylation") of the substituted enzymes were also low, indicating that Glu-461 is important for that part of the catalysis. The enzyme with His substituted for Glu-461 had the highest kappa 3 value. This is probably a result of the formation of a covalent bond between His and the galactosyl part of the substrate.     

Catalytic mechanism of xylose (glucose) isomerase from Clostridium thermosulfurogenes. Characterization of the structural gene and function of active site histidine

The gene coding for thermophilic xylose (glucose) isomerase of Clostridium thermosulfurogenes was isolated and its complete nucleotide sequence was determined. The structural gene (xylA) for xylose isomerase encodes a polypeptide of 439 amino acids with an estimated molecular weight of 50,474. The deduced amino acid sequence of thermophilic C. thermosulfurogenes xylose isomerase displayed higher homology with those of thermolabile xylose isomerases from Bacillus subtilis (70%) and Escherichia coli (50%) than with those of thermostable xylose isomerases from Ampullariella (22%), Arthrobacter (23%), and Streptomyces violaceoniger (24%). Several discrete regions were highly conserved throughout the amino acid sequences of all these enzymes. To identify the histidine residue of the active site and to elucidate its function during enzymatic xylose or glucose isomerization, histidine residues at four different positions in the C. thermosulfurogenes enzyme were individually modified by site-directed mutagenesis. Substitution of His101 by phenylalanine completely abolished enzyme activity whereas substitution of other histidine residues by phenylalanine had no effect on enzyme activity. When His101 was changed to glutamine, glutamic acid, asparagine, or aspartic acid, approximately 10-16% of wild-type enzyme activity was retained by the mutant enzymes. The Gln101 mutant enzyme was resistant to diethylpyrocarbonate inhibition which completely inactivated the wild-type enzyme, indicating that His101 is the only essential histidine residue involved directly in enzyme catalysis. The constant Vmax values of the Gln101, Glu101, Asn101, and Asp101 mutant enzymes over the pH range of 5.0-8.5 indicate that protonation of His101 is responsible for the reduced Vmax values of the wild-type enzyme at pH below 6.5. Deuterium isotope effects by D-[2-2H]glucose on the rate of glucose isomerization indicated that hydrogen transfer and not substrate ring opening is the rate-determining step for both the wild-type and Gln101 mutant enzymes. These results suggest that the enzymatic sugar isomerization does not involve a histidine-catalyzed proton transfer mechanism. Rather, essential histidine functions to stabilize the transition state by hydrogen bonding to the C5 hydroxyl group of the substrate and this enables a metal-catalyzed hydride shift from C2 to C1.     

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.     

Phosphoenolpyruvate-dependent flavinylation of 6-hydroxy-D-nicotine oxidase

The reaction leading to the flavinylation of apo-6-hydroxy-D-nicotine oxidase was investigated in cell-free extracts of Eschericia coli carrying the 6-hydroxy-D-nicotine oxidase (6-HDNO) gene on the expression plasmid pDB222. It was demonstrated that the reaction required phosphoenolpyruvate (P-pyruvate) in addition to FAD. When [32P]P-pyruvate or [14C]P-pyruvate were used in the reaction with apo-6-HDNO, no phosphorylated or pyruvylated apo-protein could be detected, however. In order to drive the reaction to completion, FAD and P-pyruvate had to be present simultaneously in the reaction mixture. When apo-6-HDNO, highly purified by affinity chromatography, was used in the reaction with P-pyruvate and FAD, no additional protein fraction was required. A possible reaction scheme for the formation of holoenzyme from 6-HDNO is discussed.     

Cytochrome c-dependent methacrylate reductase from Geobacter sulfurreducens AM-1.

Geobacter sulfurreducens AM-1 can use methacrylate as a terminal electron acceptor for anaerobic respiration. In this paper, we report on the purification and properties of the periplasmic methacrylate reductase, and show that the enzyme is dependent on the presence of a periplasmic cytochrome c (apparent K(m) = 0.12 microM). The methacrylate reductase was found to be composed of only one polypeptide with an apparent molecular mass of 50 kDa and to contain, bound tightly but not covalently, 1 mol of FAD per mol. The N-terminal amino acid sequence showed sequence similarity to a periplasmic fumarate reductase from Shewanella putrefaciens. However, methacrylate reductase did not catalyze the reduction of fumarate. The periplasmic cytochrome c, which was also purified, had an apparent molecular mass of 30 kDa and contained approximately 4 mol of heme.mol(-1). Cells of G. sulfurreducens AM-1 grown on acetate and methacrylate as an energy source were found to contain all the enzymes required for the oxidation of acetate to CO(2) via the citric acid cycle.     

Chimeric forms of neuronal nitric oxide synthase identify different regions of the reductase domain that are essential for dimerization and activity.

Nitric oxide synthase (NOS) is the enzyme responsible for the conversion of L-arginine to L-citrulline and nitric oxide. Dimerization of the enzyme is an absolute requirement for catalytic activity. Each NOS monomer contains an N-terminal heme-binding domain and a C-terminal reductase domain. It is unclear how the reductase domain is involved in controlling dimerization and whether dimer formation alone controls enzyme activity. Our initial studies demonstrated that no dimerization or activity could be detected when the reductase domain of rat neuronal NOS (nNOS) was expressed either separately or in combination with the heme domain. To further evaluate the reductase domain, a set of expression plasmids was created by replacing the reductase domain of nNOS with other electron-transport proteins, thereby creating nNOS chimeric fusion proteins. The rat nNOS heme domain was linked with either cytochrome P450 reductase, adrenodoxin reductase, or the reductase domain from Bacillus megaterium cytochrome P450, BM-3. All the chimeric enzymes retained the ability to dimerize but were unable to metabolize L-arginine (<8% of wildtype activity levels), indicating that dimerization alone is insufficient to produce an active enzyme. Because the greatest regions of homology between electron-transport proteins are in the flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and nicotinamide adenine dinucleotide phosphate (NADPH) binding regions, we produced truncation mutants within the nNOS reductase domain to investigate the role of these sequences in the ability of nNOS to dimerize and to metabolize L-arginine. The results demonstrated that the deletion of the final 56 amino acids or the NADPH-binding region had no effect on dimerization but produced an inactive enzyme. However, when the FAD-binding site (located between amino acids 920 and 1161) was deleted, both activity and dimerization were abolished. These results implicate sequences within the FAD-binding site as essential for nNOS dimerization but sequences within amino acids 1373 to 1429 as essential for activity.     

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.