ATP‐dependent/‐independent enzymatic ring reductions involved in the anaerobic catabolism of naphthalene

Polycyclic aromatic hydrocarbons are among the most hazardous environmental pollutants. However, in contrast to aerobic degradation, the respective degradation pathways in anaerobes are greatly unknown which has so far prohibited many environmental investigations. In this work, we studied the enzymatic dearomatization reactions involved in the degradation of the PAH model compounds naphthalene and 2‐methylnaphthalene in the sulfate‐reducing enrichment culture N47. Cell extracts of N47 grown on naphthalene catalysed the sodium dithionite‐dependent four‐electron reduction of the key intermediate 2‐naphthoyl‐coenzyme A (NCoA) to 5,6,7,8‐tetrahydro‐2‐naphthoyl‐CoA (THNCoA). The NCoA reductase activity was independent of ATP and was, surprisingly, not sensitive to oxygen. In cell extracts in the presence of various electron donors the product THNCoA was further reduced by a two‐electron reaction to most likely a conjugated hexahydro‐2‐naphthoyl‐CoA isomer (HHNCoA). The reaction assigned to THNCoA reductase strictly depended on ATP and was oxygen‐sensitive with a half‐life time between 30 s and 1 min when exposed to air. The rate was highest with NADH as electron donor. The results indicate that two novel and completely different dearomatizing ring reductases are involved in anaerobic naphthalene degradation. While the THNCoA reducing activity shows some properties of ATP‐dependent class I benzoyl‐CoA reductases, NCoA reduction appears to be catalysed by a previously unknown class of dearomatizing aryl‐carboxyl‐CoA reductases.     

Of mothballs and old yellow enzymes

Polycyclic aromatic hydrocarbons (PAHs) are persistent and toxic environmental pollutants that accumulate in anoxic habitats. With the exception of naphthalene, nothing is known about the microbial degradation of PAH in these environments. The challenge that must be met in anaerobic PAH degradation is the destabilization of the resonance energy of the aromatic ring system, which requires electrons with very negative redox potentials. Estelmann et al. (2014) identify two enzymes from sulphate‐reducing bacteria that perform successive 2‐electron reductions of a coenzyme A thioester derivative of naphthalene. The first reduces 2‐naphthoyl‐CoA to 5,6‐dihydro‐2‐naphthoyl‐CoA and the second generates 5,6,7,8‐tetrahydro‐2‐naphthoyl‐CoA. Surprisingly, both enzymes are members of the ‘old yellow enzyme’ (OYE) family of flavoproteins. Neither uses adenosine triphosphate to achieve reduction of the aromatic ring. Typically, OYEs have flavin mononucleotide as cofactor and use nicotinamide adenine dinucleotide (phosphate) as reductant. Both ring reductases have flavin adenine dinucleotide and an iron‐sulphur cluster as additional cofactors. Evidence also suggests that in the sulphate‐reducing bacteria, these enzymes form a complex, allowing substrate channeling. The findings of this superb study represent unprecedented biochemistry. This work sheds light on how microbes meet the thermodynamic challenges of life at the redox limit.     

Identification and characterization of 2‐naphthoyl‐coenzyme A reductase,the prototype of a novel class of dearomatizing reductases

The enzymatic dearomatization of aromatic ring systems by reduction represents a highly challenging redox reaction in biology and plays a key role in the degradation of aromatic compounds under anoxic conditions. In anaerobic bacteria, most monocyclic aromatic growth substrates are converted to benzoyl‐coenzyme A (CoA), which is then dearomatized to a conjugated dienoyl‐CoA by ATP‐dependent or ‐independent benzoyl‐CoA reductases. It was unresolved whether or not related enzymes are involved in the anaerobic degradation of environmentally relevant polycyclic aromatic hydrocarbons (PAHs). In this work, a previously unknown dearomatizing 2‐naphthoyl‐CoA reductase was purified from extracts of the naphthalene‐degrading, sulphidogenic enrichment culture N47. The oxygen‐tolerant enzyme dearomatized the non‐activated ring of 2‐naphthoyl‐CoA by a four‐electron reduction to 5,6,7,8‐tetrahydro‐2‐naphthoyl‐CoA. The dimeric 150 kDa enzyme complex was composed of a 72 kDa subunit showing sequence similarity to members of the flavin‐containing ‘old yellow enzyme’ family. NCR contained FAD, FMN, and an iron‐sulphur cluster as cofactors. Extracts of Escherichia coli expressing the encoding gene catalysed 2‐naphthoyl‐CoA reduction. The identified NCR is a prototypical enzyme of a previously unknown class of dearomatizing arylcarboxyl‐CoA reductases that are involved in anaerobic PAH degradation; it fundamentally differs from known benzoyl‐CoA reductases.     

Conversion of cis‐2‐carboxycyclohexylacetyl‐CoA in the downstream pathway of anaerobic naphthalene degradation

The cyclohexane derivative cis‐2‐(carboxymethyl)cyclohexane‐1‐carboxylic acid [(1R,2R)‐/(1S,2S)‐2‐(carboxymethyl)cyclohexane‐1‐carboxylic acid] has previously been identified as metabolite in the pathway of anaerobic degradation of naphthalene by sulfate‐reducing bacteria. We tested the corresponding CoA esters of isomers and analogues of this compound for conversion in cell free extracts of the anaerobic naphthalene degraders Desulfobacterium strain N47 and Deltaproteobacterium strain NaphS2. Conversion was only observed for the cis‐isomer, verifying that this is a true intermediate and not a dead‐end product. Mass‐spectrometric analyses confirmed that conversion is performed by an acyl‐CoA dehydrogenase and a subsequent hydratase yielding an intermediate with a tertiary hydroxyl‐group. We propose that a novel kind of ring‐opening lyase is involved in the further catabolic pathway proceeding via pimeloyl‐CoA. In contrast to degradation pathways of monocyclic aromatic compounds where ring‐cleavage is achieved via hydratases, this lyase might represent a new ring‐opening strategy for the degradation of polycyclic compounds. Conversion of the potential downstream metabolites pimeloyl‐CoA and glutaryl‐CoA was proved in cell free extracts, yielding 2,3‐dehydropimeloyl‐CoA, 3‐hydroxypimeloyl‐CoA, 3‐oxopimeloyl‐CoA, glutaconyl‐CoA, crotonyl‐CoA, 3‐hydroxybutyryl‐CoA and acetyl‐CoA as observable intermediates. This indicates a link to central metabolism via β‐oxidation, a non‐decarboxylating glutaryl‐CoA dehydrogenase and a subsequent glutaconyl‐CoA decarboxylase.     

Enzymes involved in phthalate degradation in sulphate-reducing bacteria

The complete degradation of the xenobiotic and environmentally harmful phthalate esters is initiated by hydrolysis to alcohols and o-phthalate (phthalate) by esterases. While further catabolism of phthalate has been studied in aerobic and denitrifying microorganisms, the degradation in obligately anaerobic bacteria has remained obscure. Here, we demonstrate a previously overseen growth of the δ-proteobacterium Desulfosarcina cetonica with phthalate/sulphate as only carbon and energy sources. Differential proteome and CoA ester pool analyses together with in vitro enzyme assays identified the genes, enzymes and metabolites involved in phthalate uptake and degradation in D. cetonica. Phthalate is initially activated to the short-lived phthaloyl-CoA by an ATP-dependent phthalate CoA ligase (PCL) followed by decarboxylation to the central intermediate benzoyl-CoA by an UbiD-like phthaloyl-CoA decarboxylase (PCD) containing a prenylated flavin cofactor. Genome/metagenome analyses predicted phthalate degradation capacity also in the sulphate-reducing Desulfobacula toluolica, strain NaphS2, and other δ-proteobacteria. Our results suggest that phthalate degradation proceeds in all anaerobic bacteria via the labile phthaloyl-CoA that is captured and decarboxylated by highly abundant PCDs. In contrast, two alternative strategies have been established for the formation of phthaloyl-CoA, the possibly most unstable CoA ester in biology.     

Effect of energy deprivation on metabolite release by anaerobic marine naphthalene-degrading sulfate-reducing bacteria

The aromatic hydrocarbon naphthalene, which occurs in coal and oil, can be degraded by aerobic or anaerobic microorganisms. A wide-spread electron acceptor for the latter is sulfate. Evidence for in situ naphthalene degradation stems in particular from the detection of 2-naphthoate and [5,6,7,8]-tetrahydro-2-naphthoate in oil field samples. Because such intermediates are usually not detected in laboratory cultures with high sulfate concentrations, one may suppose that conditions in reservoirs, such as sulfate limitation, trigger metabolite release. Indeed, if naphthalene-grown cells of marine sulfate-reducing Deltaproteobacteria (strains NaphS2, NaphS3 and NaphS6) were transferred to sulfate-free medium, they released 2-naphthoate and [5,6,7,8]-tetrahydro-2-naphthoate while still consuming naphthalene. With 2-naphthoate as initial substrate, cells produced [5,6,7,8]-tetrahydro-2-naphthoate and the hydrocarbon, naphthalene, indicating reversibility of the initial naphthalene-metabolizing reaction. The reactions in the absence of sulfate were not coupled to observable growth. Excretion of naphthalene-derived metabolites was also achieved in sulfate-rich medium upon addition of the protonophore carbonyl cyanide4-(trifluoromethoxy)phenylhydrazone or the ATPase inhibitor N,N′-dicyclohexylcarbodiimide. In conclusion, obstruction of electron flow and energy gain by sulfate limitation offers an explanation for the occurrence of naphthalene-derived metabolites in oil reservoirs, and provides a simple experimental tool for gaining insights into the anaerobic naphthalene oxidation pathway from an energetic perspective.     

Purification and properties of NADH-ferredoxinNAP reductase, a component of naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816.

Cells of Pseudomonas sp. strain NCIB 9816, after growth with naphthalene or salicylate, contain a multicomponent enzyme system that oxidizes naphthalene to cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. We purified one of these components to homogeneity and found it to be an iron-sulfur flavoprotein that loses the flavin cofactor during purification. Dialysis against flavin adenine dinucleotide (FAD) showed that the enzyme bound 1 mol of FAD per mol of enzyme protein. The enzyme consisted of a single polypeptide with an apparent molecular weight of 36,300. The purified protein contained 1.8 g-atoms of iron and 2.0 g-atoms of acid-labile sulfur and showed absorption maxima at 278, 340, 420, and 460 nm, with a broad shoulder at 540 nm. The purified enzyme catalyzed the reduction of cytochrome c, dichlorophenolindophenol, Nitro Blue Tetrazolium, and ferricyanide. These activities were enhanced in the presence of added FAD. The ability of the enzyme to catalyze the reduction of the ferredoxin involved in naphthalene reduction and other electron acceptors indicates that it functions as an NAD(P)H-oxidoreductase in the naphthalene dioxygenase system. The results suggest that naphthalene dioxygenase requires two proteins with three redox groups to transfer electrons from NADH to the terminal oxygenase.     

Cyclin-dependent kinase 5 phosphorylates mammalian HMGB1 protein only if acetylated

Old yellow enzyme (OYE) is an NADPH oxidoreductase capable of reducing a variety of compounds. It contains flavin mononucleotide (FMN) as a prosthetic group. A ternary complex structure of OYE from Trypanosoma cruzi (TcOYE) with FMN and one of the substrates, p-hydroxybenzaldehyde, shows a striking movement around the active site upon binding of the substrate. From a structural comparison of other OYE complexed with 12-oxophytodienoate, we have constructed a complex structure with another substrate, prostaglandin H(2) (PGH(2)), to provide a proposed stereoselective reaction mechanism for the reduction of PGH(2) to prostaglandin F(2α) by TcOYE.     

The 1.3 A crystal structure of the flavoprotein YqjM reveals a novel class of Old Yellow Enzymes

Here we report the crystal structure of YqjM, a homolog of Old Yellow Enzyme (OYE) that is involved in the oxidative stress response of Bacillus subtilis. In addition to the oxidized and reduced enzyme form, the structures of complexes with p-hydroxybenzaldehyde and p-nitrophenol, respectively, were solved. As for other OYE family members, YqjM folds into a (alpha/beta)8-barrel and has one molecule of flavin mononucleotide bound non-covalently at the COOH termini of the beta-sheet. Most of the interactions that control the electronic properties of the flavin mononucleotide cofactor are conserved within the OYE family. However, in contrast to all members of the OYE family characterized to date, YqjM exhibits several unique structural features. For example, the enzyme exists as a homotetramer that is assembled as a dimer of catalytically dependent dimers. Moreover, the protein displays a shared active site architecture where an arginine finger (Arg336) at the COOH terminus of one monomer extends into the active site of the adjacent monomer and is directly involved in substrate recognition. Another remarkable difference in the binding of the ligand in YqjM is represented by the contribution of the NH2-terminal Tyr28 instead of a COOH-terminal tyrosine in OYE and its homologs. The structural information led to a specific data base search from which a new class of OYE oxidoreductases was identified that exhibits a strict conservation of active site residues, which are critical for this subfamily, most notably Cys26, Tyr28, Lys109, and Arg336. Therefore, YqjM is the first representative of a new bacterial subfamily of OYE homologs.     

Anaerobic degradation of naphthalene and 2-methylnaphthalene by strains of marine sulfate-reducing bacteria

The anaerobic biodegradation of naphthalene, an aromatic hydrocarbon in tar and petroleum, has been repeatedly observed in environments but scarcely in pure cultures. To further explore the relationships and physiology of anaerobic naphthalene-degrading microorganisms, sulfate-reducing bacteria (SRB) were enriched from a Mediterranean sediment with added naphthalene. Two strains (NaphS3, NaphS6) with oval cells were isolated which showed naphthalene-dependent sulfate reduction. According to 16S rRNA gene sequences, both strains were Deltaproteobacteria and closely related to each other and to a previously described naphthalene-degrading sulfate-reducing strain (NaphS2) from a North Sea habitat. Other close relatives were SRB able to degrade alkylbenzenes, and phylotypes enriched anaerobically with benzene. If in adaptation experiments the three naphthalene-grown strains were exposed to 2-methylnaphthalene, this compound was utilized after a pronounced lag phase, indicating that naphthalene did not induce the capacity for 2-methylnaphthalene degradation. Comparative denaturing gel electrophoresis of cells grown with naphthalene or 2-methylnaphthalene revealed a striking protein band which was only present upon growth with the latter substrate. Peptide sequences from this band perfectly matched those of a protein predicted from genomic libraries of the strains. Sequence similarity (50% identity) of the predicted protein to the large subunit of the toluene-activating enzyme (benzylsuccinate synthase) from other anaerobic bacteria indicated that the detected protein is part of an analogous 2-methylnaphthalene-activating enzyme. The absence of this protein in naphthalene-grown cells together with the adaptation experiments as well as isotopic metabolite differentiation upon growth with a mixture of d(8)-naphthalene and unlabelled 2-methylnaphthalene suggest that the marine strains do not metabolize naphthalene by initial methylation via 2-methylnaphthalene, a previously suggested mechanism. The inability to utilize 1-naphthol or 2-naphthol also excludes these compounds as free intermediates. Results leave open the possibility of naphthalene carboxylation, another previously suggested activation mechanism.     

Specific 16S rDNA Sequences Associated with Naphthalene Degradation under Sulfate-Reducing Conditions in Harbor Sediments

Previous studies have demonstrated that naphthalene and other polycyclic aromatic hydrocarbons (PAHs) can be anaerobically oxidized with the reduction of sulfate in PAH-contaminated marine harbor sediments, including those in San Diego Bay. In order to learn more about the microorganisms that might be involved in anaerobic naphthalene degradation, the microorganisms associated with naphthalene degradation in San Diego Bay sediments were evaluated. A dilution-to-extinction enrichment culture strategy, designed to recover the most numerous culturable naphthalene-degrading sulfate reducers, resulted in the enrichment of microorganisms with 16S rDNA sequences in the d-Proteobacteria, which were closely related to a previously described pure culture of a naphthalene-degrading sulfate reducer, NaphS2, isolated from sediments in Germany. A more traditional enrichment culture approach, expected to enrich for the fastest-growing naphthalene-degrading sulfate reducers, yielded 16S rDNA sequences closely related to those found in the dilution-to-extinction enrichments and NaphS2. Analysis of 16S rDNA sequences in sediments from two sites in San Diego Bay that had been adapted for rapid naphthalene degradation by continual amendment with low levels of naphthalene suggested that the microbial community composition in the amended sediments differed from that present in the unamended sediments from the same sites. Most significantly, 6-8% of the sequences recovered from 100 clones of each of the naphthalene-amended sediments were closely related to the 16S rDNA sequences in the enrichment cultures as well as the sequence of the pure culture, NaphS2. No sequences in this NaphS2 phylotype were recovered from the sediments that were not continually exposed to naphthalene. A PCR primer, which was designed based on these phylotype sequences, was used to amplify additional 16S rDNA sequences belonging to the NaphS2 phylotype from PAH-degrading sediments from Island End River (Boston), MA, and Liepaja Harbor, Latvia. Closely related sequences were also recovered from highly contaminated sediment from Tampa Bay, FL. These results suggest that microorganisms closely related to NaphS2 might be involved in naphthalene degradation in harbor sediments. This finding contrasts with the frequent observation that the environmentally relevant microorganisms cannot be readily recovered in pure culture and suggests that further study of the physiology of NaphS2 may provide insights into factors controlling the rate and extent of naphthalene degradation in marine harbor sediments.     

The Bacterial signal transduction protein GlnB regulates the committed step in fatty acid biosynthesis by acting as a dissociable regulatory subunit of acetyl‐CoA carboxylase

Biosynthesis of fatty acids is one of the most fundamental biochemical pathways in nature. In bacteria and plant chloroplasts, the committed and rate‐limiting step in fatty acid biosynthesis is catalyzed by a multi‐subunit form of the acetyl‐CoA carboxylase enzyme (ACC). This enzyme carboxylates acetyl‐CoA to produce malonyl‐CoA, which in turn acts as the building block for fatty acid elongation. In Escherichia coli, ACC is comprised of three functional modules: the biotin carboxylase (BC), the biotin carboxyl carrier protein (BCCP) and the carboxyl transferase (CT). Previous data showed that both bacterial and plant BCCP interact with signal transduction proteins belonging to the P_II family. Here we show that the GlnB paralogues of the P_II proteins from E. coli and Azospirillum brasiliense, but not the GlnK paralogues, can specifically form a ternary complex with the BC‐BCCP components of ACC. This interaction results in ACC inhibition by decreasing the enzyme turnover number. Both the BC‐BCCP‐GlnB interaction and ACC inhibition were relieved by 2‐oxoglutarate and by GlnB uridylylation. We propose that the GlnB protein acts as a 2‐oxoglutarate‐sensitive dissociable regulatory subunit of ACC in Bacteria.     

Reaction of morphinone reductase with 2-cyclohexen-1-one and 1-nitrocyclohexene: proton donation, ligand binding, and the role of residues Histidine 186 and Asparagine 189

Morphinone reductase (MR) catalyzes the NADH-dependent reduction of alpha/beta unsaturated carbonyl compounds in a reaction similar to that catalyzed by Old Yellow Enzyme (OYE1). The two enzymes are related at the sequence and structural levels, but key differences in active site architecture exist which have major implications for the reaction mechanism. We report detailed kinetic and solution NMR data for wild-type MR and two mutant forms in which residues His-186 and Asn-189 have been exchanged for alanine residues. We show that both residues are involved in the binding of the reducing nicotinamide coenzyme NADH and also the binding of the oxidizing substrates 2-cyclohexen-1-one and 1-nitrocyclohexene. Reduction of 2-cyclohexen-1-one by FMNH(2) is concerted with proton transfer from an unknown proton donor in the active site. NMR spectroscopy and flavin reoxidation studies with 2-cyclohexen-1-one are consistent with His-186 being unprotonated in oxidized, reduced, and ligand-bound MR, suggesting that His-186 is not the key proton donor required for the reduction of 2-cyclohexen-1-one. Hydride transfer is decoupled from proton transfer with 1-nitrocyclohexene as oxidizing substrate, and unlike with OYE1 the intermediate nitronate species produced after hydride transfer from FMNH(2) is not converted to 1-nitrocyclohexane. The work highlights key mechanistic differences in the reactions catalyzed by MR and OYE1 and emphasizes the need for caution in inferring mechanistic similarities in structurally related proteins.     

A spectrophotometric assay for measuring acetyl–coenzyme A carboxylase

Acetyl–coenzyme A (CoA) carboxylase catalyzes the first step in the biosynthesis of fatty acids in bacteria and eukaryota. This enzyme is the target of drug design for treatment of human metabolic diseases and of herbicides acting specifically on the eukaryotic form of the enzyme in grasses. Acetyl–CoA carboxylase activity screening in drug and herbicide design depends mostly on a time-consuming enzyme assay that is based on the incorporation of radiolabeled bicarbonate into the product malonyl–CoA. Here we describe a new simple, continuous, and quick photometric assay avoiding radioactive substrate. It couples the carboxylation of acetyl–CoA to the nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of malonyl–CoA, which is catalyzed by recombinant malonyl–CoA reductase of Chloroflexus aurantiacus. This assay can be adapted for high-throughput screening.     

X-ray structure of 12-oxophytodienoate reductase 1 provides structural insight into substrate binding and specificity within the family of OYE

BACKGROUND: 12-Oxophytodienoate reductase (OPR) is a flavin mononucleotide (FMN)-dependent oxidoreductase in plants that belongs to the family of Old Yellow Enzyme (OYE). It was initially characterized as an enzyme involved in the biosynthesis of the plant hormone jasmonic acid, where it catalyzes the reduction of the cyclic fatty acid derivative 9S,13S-12-oxophytodienoate (9S,13S-OPDA) to 1S,2S-3-oxo-2(2'[Z]-pentenyl)-cyclopentane-1-octanoate. Several isozymes of OPR are now known that show different stereoselectivities with regard to the four stereoisomers of OPDA. RESULTS: Here, we report the high-resolution crystal structure of OPR1 from Lycopersicon esculentum and its complex structures with the substrate 9R,13R-OPDA and with polyethylene glycol 400. OPR1 crystallizes as a monomer and folds into a (betaalpha)(8) barrel with an overall structure similar to OYE. The cyclopentenone ring of 9R,13R-OPDA is stacked above the flavin and activated by two hydrogen bonds to His187 and His190. The olefinic bond is properly positioned for hydride transfer from the FMN N(5) and proton transfer from Tyr192 to Cbeta and Calpha, respectively. Comparison of the OPR1 and OYE structures reveals striking differences in the loops responsible for binding 9R,13R-OPDA in OPR1. CONCLUSIONS: Despite extensive biochemical characterization, the physiological function of OYE still remains unknown. The similar catalytic cavity structures and the substrate binding mode in OPR1 strongly support the assumption that alpha,beta-unsaturated carbonyl compounds are physiological substrates of the OYE family. The specific binding of 9R,13R-OPDA by OPR1 explains the experimentally observed stereoselectivity and argues in favor of 9R,13R-OPDA or a structurally related oxylipin as natural substrate of OPR1.     

The SCP2‐thiolase‐like protein (SLP) of Trypanosoma brucei is an enzyme involved in lipid metabolism

Bioinformatics studies have shown that the genomes of trypanosomatid species each encode one SCP2‐thiolase‐like protein (SLP), which is characterized by having the YDCF thiolase sequence fingerprint of the Cβ2‐Cα2 loop. SLPs are only encoded by the genomes of these parasitic protists and not by those of mammals, including human. Deletion of the Trypanosoma brucei SLP gene (TbSLP) increases the doubling time of procyclic T. brucei and causes a 5‐fold reduction of de novo sterol biosynthesis from glucose‐ and acetate‐derived acetyl‐CoA. Fluorescence analyses of EGFP‐tagged TbSLP expressed in the parasite located the TbSLP in the mitochondrion. The crystal structure of TbSLP (refined at 1.75 Å resolution) confirms that TbSLP has the canonical dimeric thiolase fold. In addition, the structures of the TbSLP‐acetoacetyl‐CoA (1.90 Å) and TbSLP‐malonyl‐CoA (2.30 Å) complexes reveal that the two oxyanion holes of the thiolase active site are preserved. TbSLP binds malonyl‐CoA tightly (K_d 90 µM), acetoacetyl‐CoA moderately (K_d 0.9 mM) and acetyl‐CoA and CoA very weakly. TbSLP possesses low malonyl‐CoA decarboxylase activity. Altogether, the data show that TbSLP is a mitochondrial enzyme involved in lipid metabolism. Proteins 2016; 84:1075–1096. © 2016 Wiley Periodicals, Inc.     

Characterization of glycerol trinitrate reductase (NerA) and the catalytic role of active-site residues

Glycerol trinitrate reductase (NerA) from Agrobacterium radiobacter, a member of the old yellow enzyme (OYE) family of oxidoreductases, was expressed in and purified from Escherichia coli. Denaturation of pure enzyme liberated flavin mononucleotide (FMN), and spectra of NerA during reduction and reoxidation confirmed its catalytic involvement. Binding of FMN to apoenzyme to form the holoenzyme occurred with a dissociation constant of ca. 10(-7) M and with restoration of activity. The NerA-dependent reduction of glycerol trinitrate (GTN; nitroglycerin) by NADH followed ping-pong kinetics. A structural model of NerA based on the known coordinates of OYE showed that His-178, Asn-181, and Tyr-183 were close to FMN in the active site. The NerA mutation H178A produced mutant protein with bound FMN but no activity toward GTN. The N181A mutation produced protein that did not bind FMN and was isolated in partly degraded form. The mutation Y183F produced active protein with the same k(cat) as that of wild-type enzyme but with altered K(m) values for GTN and NADH, indicating a role for this residue in substrate binding. Correlation of the ratio of K(m)(GTN) to K(m)(NAD(P)H), with sequence differences for NerA and several other members of the OYE family of oxidoreductases that reduce GTN, indicated that Asn-181 and a second Asn-238 that lies close to Tyr-183 in the NerA model structure may influence substrate specificity.