Purification and characterization of salicylate 5-hydroxylase, a three-component monooxygenase from Ralstonia sp. strain U2

Salicylate is an important intermediate in the bacterial degradation of polycyclic aromatic hydrocarbons and salicylate hydroxylases play essential roles in linking the peripheral and ring-cleavage catabolic pathways. Unlike the well-characterized salicylate 1-hydroxylases, the rarely occurred salicylate 5-hydroxylase (S5H) has not been characterized in detail. In this study, the three-component Fe-S protein complex (NagAaGHAb) of S5H from Ralstonia sp. strain U2 was purified, and its biochemical and catalytic properties were characterized. The oxygenase component NagGH exhibited an α₃β₃ heterohexameric structure and contained one Rieske-type [2Fe-2S] cluster and one mononuclear iron per α subunit. NagAa is the ferredoxin-NADP⁺ reductase component containing flavin and plant type [2Fe–2S] cluster. The ferredoxin component NagAb was characterized as a [2Fe-2S] dimer which remains remarkably stable in denaturing gel electrophoresis after being heated at 100 °C for 1 h. Purified NagAa and NagAb, NagGH catalyzed the hydroxylation of salicylate to gentisate with a specific activity of 107.12?±?14.38 U/g and showed an apparent K _m for salicylate of 102.79?±?27.20 μM and a similar K _m value for both NADH and NADPH (59.76?±?7.81 μM versus 56.41?±?12.76 μM). The hydroxylase exhibited different affinities for two hydroxysalicylates (2,4-dihydroxybenzoate K _m of 93.54?±?18.50 μM versus 2,6-dihydroxybenzoate K _m of 939.80?±?199.46 μM). Interestingly, this S5H also showed catalytic activity to the pollutant 2-nitrophenol and exhibited steady-state kinetic data of the same order of magnitude as those for salicylate. This study will allow further comparative studies of structure–function relationships of the ring hydroxylating mono- and di-oxygenase systems.     

Hydrogen peroxide-coupled cis-diol formation catalyzed by naphthalene 1,2-dioxygenase

Naphthalene 1,2-dioxygenase (NDOS) catalyzes the NAD(P)H and O(2)-dependent oxidation of naphthalene to (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. NDOS consists of three protein components: a flavo-[2Fe-2S] reductase (NDR), a ferredoxin electron transfer protein (NDF), and an (alphabeta)(3) oxygenase (NDO) containing a mononuclear iron site and a Rieske-type [2Fe-2S] cluster in each alpha-subunit. The active site is built across a subunit-subunit boundary, and each subunit contributes one type of metal center. Our previous studies have shown that NDO with both metal centers reduced is capable of an O(2)-coupled single turnover to yield the correct cis-diol product in the absence of the NDR and NDF components (Wolfe, M. D., Parales, J. V., Gibson, D. T., and Lipscomb, J. D. (2001) J. Biol. Chem. 276, 1945-1953). It is shown here that addition of H(2)O(2) to NDO allows reaction with naphthalene to rapidly yield the correct product in a "peroxide shunt" reaction that does not require a reduced Rieske cluster. The mononuclear Fe(2+) center is oxidized during turnover, while the Rieske cluster remains in the oxidized state. Peroxide shunt turnover in the presence of (18)O-labeled H(2)O(2), H(2)O, or O(2) shows that both oxygen atoms in the product derive primarily from H(2)O(2). The peroxide shunt halts after one turnover despite the presence of excess H(2)O(2) and naphthalene, but this is not the result of enzyme inactivation. Rather, it appears that the product cannot be released when the mononuclear iron is in the Fe(3+) state, blocking a second turnover. This work supports the hypotheses that the cis-dihydroxylation activity of NDOS requires only the NDO component, that a peroxo intermediate is formed during normal catalysis, and that product release requires an additional reducing equivalent beyond those necessary for the first turnover.     

Catabolic role of a three-component salicylate oxygenase from Sphingomonas yanoikuyae B1 in polycyclic aromatic hydrocarbon degradation

Sphingomonas yanoikuyae B1 possesses several different multicomponent oxygenases involved in metabolizing aromatic compounds. Six different pairs of genes encoding large and small subunits of oxygenase iron-sulfur protein components have previously been identified in a gene cluster involved in the degradation of both monocyclic and polycyclic aromatic hydrocarbons. Insertional inactivation of one of the oxygenase large subunit genes, bphA1c, results in a mutant strain unable to grow on naphthalene, phenanthrene, or salicylate. The knockout mutant accumulates salicylate from naphthalene and 1-hydroxy-2-naphthoic acid from phenanthrene indicating the loss of salicylate oxygenase activity. Complementation experiments verify that the salicylate oxygenase in S. yanoikuyae B1 is a three-component enzyme consisting of an oxygenase encoded by bphA2cA1c, a ferredoxin encoded by the adjacent bphA3, and a ferredoxin reductase encoded by bphA4 located over 25kb away. Expression of bphA3-bphA2c-bphA1c genes in Escherichia coli demonstrated the ability of salicylate oxygenase to convert salicylate to catechol and 3-, 4-, and 5-methylsalicylate to methylcatechols.     

Purification,characterization, and crystallization of the components of a biphenyl dioxygenase system from <Emphasis Type="Italic">Sphingobium yanoikuyae</Emphasis> B1

Sphingobium yanoikuyae B1 initiates the catabolism of biphenyl by adding dioxygen to the aromatic nucleus to form (+)-cis-(2R, 3S)-dihydroxy-1-phenylcyclohexa-4,6-diene. The present study focuses on the biphenyl 2,3-dioxygenase system, which catalyzes the dioxygenation reaction. This enzyme has been shown to have a broad substrate range, catalyzing the dioxygenation of not only biphenyl, but also three- and four-ring polycyclic aromatic hydrocarbons. Extracts prepared from biphenyl-grown B1 cells contained three protein components that were required for the oxidation of biphenyl. The genes encoding the three components (bphA4, bphA3 and bphA1f,A2f) were expressed in Escherichia coli. Biotransformations of biphenyl, naphthalene, phenanthrene, and benzo[a]pyrene as substrates using the recombinant E. coli strain resulted in the formation of the expected cis-dihydrodiol products previously shown to be produced by biphenyl-induced strain B1. The three protein components were purified to apparent homogeneity and characterized in detail. The reductase component (bphA4), designated reductase(BPH-B1), was a 43 kD monomer containing one mol FAD/mol reductase(BPH-B1). The ferredoxin component (bphA3), designated ferredoxin(BPH-B1), was a 12 kD monomer containing approximately 2 g-atoms each of iron and acid-labile sulfur. The oxygenase component (bphA1f,A2f), designated oxygenase(BPH-B1), was a 217 kD heterotrimer consisting of alpha and beta subunits (approximately 51 and 21 kD, respectively). The iron and acid-labile sulfur contents of oxygenase(BPH-B1) per alphabeta were 2.4 and 1.8 g-atom per mol, respectively. Reduced ferredoxin(BPH-B1) and oxygenase(BPH-B1) each gave EPR signals typical of Rieske [2Fe-2S] proteins. Crystals of reductase(BPH-B1), ferredoxin(BPH-B1) and oxygenase(BPH-B1 )diffracted to 2.5 A, 2.0 A and 1.75 A, respectively. The structures of the three proteins are currently being determined.     

Single turnover chemistry and regulation of O2 activation by the oxygenase component of naphthalene 1,2-dioxygenase

Naphthalene 1,2-dioxygenase (NDOS) is a three-component enzyme that catalyzes cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene formation from naphthalene, O2, and NADH. We have determined the conditions for a single turnover of NDOS for the first time and studied the regulation of catalysis. As isolated, the alpha3beta3 oxygenase component (NDO) has up to three catalytic pairs of metal centers (one mononuclear Fe2+ and one diferric Rieske iron-sulfur cluster). This form of NDO is unreactive with O2. However, upon reduction of the Rieske cluster and exposure to naphthalene and O2, approximately 0.85 cis-diol product per occupied mononuclear iron site rapidly forms. Substrate binding is required for oxygen reactivity. Stopped-flow and chemical quench analyses indicate that the rate constant of the single turnover product-forming reaction significantly exceeds the NDOS turnover number. UV-visible and electron paramagnetic resonance spectroscopies show that during catalysis, one mononuclear iron and one Rieske cluster are oxidized per product formed, satisfying the two-electron reaction stoichiometry. The addition of oxidized or reduced NDOS ferredoxin component (NDF) increases both the product yield and rate of oxidation of formerly unreactive Rieske clusters. The results show that NDO alone catalyzes dioxygenase chemistry, whereas NDF appears to serve only an electron transport role, in this case redistributing electrons to competent active sites.     

A Gene Cluster Encoding Steps in Conversion of Naphthalene to Gentisate in Pseudomonas sp. Strain U2

Pseudomonas sp. strain U2 was isolated from oil-contaminated soil in Venezuela by selective enrichment on naphthalene as the sole carbon source. The genes for naphthalene dioxygenase were cloned from the plasmid DNA of strain U2 on an 8.3-kb BamHI fragment. The genes for the naphthalene dioxygenase genes nagAa (for ferredoxin reductase), nagAb (for ferredoxin), and nagAc and nagAd (for the large and small subunits of dioxygenase, respectively) were located by Southern hybridizations and by nucleotide sequencing. The genes for nagB (for naphthalene cis-dihydrodiol dehydrogenase) and nagF (for salicylaldehyde dehydrogenase) were inferred from subclones by their biochemical activities. Between nagAa and nagAb were two open reading frames, homologs of which have also been identified in similar locations in two nitrotoluene-using strains (J. V. Parales, A. Kumar, R. E. Parales, and D. T. Gibson, Gene 181:57–61, 1996; W.-C. Suen, B. Haigler, and J. C. Spain, J. Bacteriol. 178:4926–4934, 1996) and a naphthalene-using strain (G. J. Zylstra, E. Kim, and A. K. Goyal, Genet. Eng. 19:257–269, 1997). Recombinant Escherichia coli strains with plasmids carrying this region were able to convert salicylate to gentisate, which was identified by a combination of gas chromatography-mass spectrometry and nuclear magnetic resonance. The first open reading frame, designated nagG, encodes a protein with characteristics of a Rieske-type iron-sulfur center homologous to the large subunits of dihydroxylating dioxygenases, and the second open reading frame, designated nagH, encodes a protein with limited homology to the small subunits of the same dioxygenases. Cloned together in E. coli, nagG, nagH, and nagAb, were able to convert salicylate (2-hydroxybenzoate) into gentisate (2,5-dihydroxybenzoate) and therefore encode a salicylate 5-hydroxylase activity. Single-gene knockouts of nagG, nagH, and nagAb demonstrated their functional roles in the formation of gentisate. It is proposed that NagG and NagH are structural subunits of salicylate 5-hydroxylase linked to an electron transport chain consisting of NagAb and NagAa, although E. coli appears to be able to partially substitute for the latter. This constitutes a novel mechanism for monohydroxylation of the aromatic ring. Salicylate hydroxylase and catechol 2,3-dioxygenase in strain U2 could not be detected either by enzyme assay or by Southern hybridization. However growth on both naphthalene and salicylate caused induction of gentisate 1,2-dioxygenase, confirming this route for salicylate catabolism in strain U2. Sequence comparisons suggest that the novel gene order nagAa-nagG-nagH-nagAb-nagAc-nagAd-nagB-nagF represents the archetype for naphthalene strains which use the gentisate pathway rather than the meta cleavage pathway of catechol.     

A three-component dicamba O-demethylase from Pseudomonas maltophilia, strain DI-6: gene isolation, characterization, and heterologous expression

Dicamba O-demethylase is a multicomponent enzyme from Pseudomonas maltophilia, strain DI-6, that catalyzes the conversion of the widely used herbicide dicamba (2-methoxy-3,6-dichlorobenzoic acid) to DCSA (3,6-dichlorosalicylic acid). We recently described the biochemical characteristics of the three components of this enzyme (i.e. reductase(DIC), ferredoxin(DIC), and oxygenase(DIC)) and classified the oxygenase component of dicamba O-demethylase as a member of the Rieske non-heme iron family of oxygenases. In the current study, we used N-terminal and internal amino acid sequence information from the purified proteins to clone the genes that encode dicamba O-demethylase. Two reductase genes (ddmA1 and ddmA2) with predicted amino acid sequences of 408 and 409 residues were identified. The open reading frames encode 43.7- and 43.9-kDa proteins that are 99.3% identical to each other and homologous to members of the FAD-dependent pyridine nucleotide reductase family. The ferredoxin coding sequence (ddmB) specifies an 11.4-kDa protein composed of 105 residues with similarity to the adrenodoxin family of [2Fe-2S] bacterial ferredoxins. The oxygenase gene (ddmC) encodes a 37.3-kDa protein composed of 339 amino acids that is homologous to members of the Phthalate family of Rieske non-heme iron oxygenases that function as monooxygenases. Southern analysis localized the oxygenase gene to a megaplasmid in cells of P. maltophilia. Mixtures of the three highly purified recombinant dicamba O-demethylase components overexpressed in Escherichia coli converted dicamba to DCSA with an efficiency similar to that of the native enzyme, suggesting that all of the components required for optimal enzymatic activity have been identified. Computer modeling suggests that oxygenase(DIC) has strong similarities with the core alphasubunits of naphthalene 1,2-dioxygenase. Nonetheless, the present studies point to dicamba O-demethylase as an enzyme system with its own unique combination of characteristics.     

The Trichoderma atroviride seb1 (stress response element binding) gene encodes an AGGGG-binding protein which is involved in the response to high osmolarity stress

The chitinase genes of Trichoderma spp. (ech42, chit33, nag1) contain one or more copies of a pentanucleotide element (5'-AGGGG-3') in their 5'-noncoding regions. In Saccharomyces cerevisiae, this motif is recognized and bound by the stress response regulator proteins Msn2p/Msn4p. To test whether this motif in the chitinase promoters is bound by a Trichoderma Msn2/4p homolog, we have cloned a gene (seb1) from T. atroviride which encodes a C2H2 zinc-finger protein that is 62 (64)% identical to S. cerevisiae Msn2p (Msn4p) in the zinc-finger region, and almost identical to the G-box binding protein from Haematonectria haematococca and to polypeptides encoded by uncharacterized ORFs from Neurospora crassa and Aspergillus nidulans. Its zinc-finger domain specifically recognizes the AGGGG sequence of the ech42 and nag1 promoter in band-shift assays. However, a cDNA clone of seb1, when overexpressed in S. cerevisiae, was unable to complement a Delta msn2/4 mutant of S. cerevisiae. Levels of seb1 mRNA increased under conditions of osmotic stress (sorbitol, NaCl) but not under other stress conditions (cadmium sulfate, pH, membrane perturbance). A T. atroviride Delta seb1 strain, produced by transformation with a seb1 copy disrupted by insertion of the A. nidulans amdS gene, showed strongly reduced growth on solid medium, but grew normally in liquid medium. In liquid medium, growth of the disruption strain was significantly more inhibited by the presence of 1 M sorbitol and 1 M NaCl than was that of the wild-type strain. Despite the presence of AGGGG elements in the promoter of the chitinase gene nag1, no differences in its expression were found between the parent and the disruption strain. EMSA analyses with cell-free extracts obtained from the seb1 disruption strain showed the presence of proteins that could bind to the AGGGG-element in nag1 and ech42. We therefore conclude that seb1 encodes a protein that is involved in the osmotic stress response, but not in chitinase gene expression, in T. atroviride.     

nag genes of Ralstonia (formerly Pseudomonas) sp. strain U2 encoding enzymes for gentisate catabolism

Ralstonia sp. strain U2 metabolizes naphthalene via gentisate to central metabolites. We have cloned and sequenced a 21.6-kb region spanning the nag genes. Upstream of the pathway genes are nagY, homologous to chemotaxis proteins, and nagR, a regulatory gene of the LysR family. Divergently transcribed from nagR are the genes for conversion of naphthalene to gentisate (nagAaGHAbAcAdBFCQED) (S. L. Fuenmayor, M. Wild, A. L. Boyes, and P. A. Williams, J. Bacteriol. 180:2522-2530, 1998), which except for the insertion of nagGH, encoding the salicylate 5-hydroxylase, are homologous to and in the same order as the genes in the classical upper pathway operon described for conversion of naphthalene to salicylate found in the NAH7 plasmid of Pseudomonas putida PpG7. Downstream of nahD is a cluster of genes (nagJIKLMN) which are probably cotranscribed with nagAaGHAbAcAdBFCQED as a single large operon. By cloning into expression vectors and by biochemical assays, three of these genes (nagIKL) have been shown to encode the enzymes involved in the further catabolism of gentisate to fumarate and pyruvate. NagI is a gentisate 1,2-dioxygenase which converts gentisate to maleylpyruvate and is also able to catalyze the oxidation of some substituted gentisates. NagL is a reduced glutathione-dependent maleylpyruvate isomerase catalyzing the isomerization of maleylpyruvate to fumarylpyruvate. NagK is a fumarylpyruvate hydrolase which hydrolyzes fumarylpyruvate to fumarate and pyruvate. The three other genes (nagJMN) have also been cloned and overexpressed, but no biochemical activities have been attributed to them. NagJ is homologous to a glutathione S-transferase, and NagM and NagN are proteins homologous to each other and to other proteins of unknown function. Downstream of the operon is a partial sequence with homology to a transposase.     

X-ray crystal structure of benzoate 1,2-dioxygenase reductase from Acinetobacter sp. strain ADP1

One of the major processes for aerobic biodegradation of aromatic compounds is initiated by Rieske dioxygenases. Benzoate dioxygenase contains a reductase component, BenC, that is responsible for the two-electron transfer from NADH via FAD and an iron-sulfur cluster to the terminal oxygenase component. Here, we present the structure of BenC from Acinetobacter sp. strain ADP1 at 1.5 A resolution. BenC contains three domains, each binding a redox cofactor: iron-sulfur, FAD and NADH, respectively. The [2Fe-2S] domain is similar to that of plant ferredoxins, and the FAD and NADH domains are similar to members of the ferredoxin:NADPH reductase superfamily. In phthalate dioxygenase reductase, the only other Rieske dioxygenase reductase for which a crystal structure is available, the ferredoxin-like and flavin binding domains are sequentially reversed compared to BenC. The BenC structure shows significant differences in the location of the ferredoxin domain relative to the other domains, compared to phthalate dioxygenase reductase and other known systems containing these three domains. In BenC, the ferredoxin domain interacts with both the flavin and NAD(P)H domains. The iron-sulfur center and the flavin are about 9 A apart, which allows a fast electron transfer. The BenC structure is the first determined for a reductase from the class IB Rieske dioxygenases, whose reductases transfer electrons directly to their oxygenase components. Based on sequence similarities, a very similar structure was modeled for the class III naphthalene dioxygenase reductase, which transfers electrons to an intermediary ferredoxin, rather than the oxygenase component.     

Substrate specificity of naphthalene dioxygenase: effect of specific amino acids at the active site of the enzyme

The three-component naphthalene dioxygenase (NDO) enzyme system carries out the first step in the aerobic degradation of naphthalene by Pseudomonas sp. strain NCIB 9816-4. The three-dimensional structure of NDO revealed that several of the amino acids at the active site of the oxygenase are hydrophobic, which is consistent with the enzyme's preference for aromatic hydrocarbon substrates. Although NDO catalyzes cis-dihydroxylation of a wide range of substrates, it is highly regio- and enantioselective. Site-directed mutagenesis was used to determine the contributions of several active-site residues to these aspects of catalysis. Amino acid substitutions at Asn-201, Phe-202, Val-260, Trp-316, Thr-351, Trp-358, and Met-366 had little or no effect on product formation with naphthalene or biphenyl as substrates and had slight but significant effects on product formation from phenanthrene. Amino acid substitutions at Phe-352 resulted in the formation of cis-naphthalene dihydrodiol with altered stereochemistry [92 to 96% (+)-1R,2S], compared to the enantiomerically pure [>99% (+)-1R,2S] product formed by the wild-type enzyme. Substitutions at position 352 changed the site of oxidation of biphenyl and phenanthrene. Substitution of alanine for Asp-362, a ligand to the active-site iron, resulted in a completely inactive enzyme.     

Regioselectivity and enantioselectivity of naphthalene dioxygenase during arene cis-dihydroxylation: control by phenylalanine 352 in the alpha subunit

The naphthalene dioxygenase (NDO) system catalyzes the first step in the degradation of naphthalene by Pseudomonas sp. strain NCIB 9816-4. The enzyme has a broad substrate range and catalyzes several types of reactions including cis-dihydroxylation, monooxygenation, and desaturation. Substitution of valine or leucine at Phe-352 near the active site iron in the alpha subunit of NDO altered the stereochemistry of naphthalene cis-dihydrodiol formed from naphthalene and also changed the region of oxidation of biphenyl and phenanthrene. In this study, we replaced Phe-352 with glycine, alanine, isoleucine, threonine, tryptophan, and tyrosine and determined the activity with naphthalene, biphenyl, and phenanthrene as substrates. NDO variants F352W and F352Y were marginally active with all substrates tested. F352G and F352A had reduced but significant activity, and F352I, F352T, F352V, and F352L had nearly wild-type activities with respect to naphthalene oxidation. All active enzymes had altered regioselectivity with biphenyl and phenanthrene. In addition, the F352V and F352T variants formed the opposite enantiomer of biphenyl cis-3,4-dihydrodiol [77 and 60% (-)-(3S,4R), respectively] to that formed by wild-type NDO [>98% (+)-(3R,4S)]. The F352V mutant enzyme also formed the opposite enantiomer of phenanthrene cis-1,2-dihydrodiol from phenanthrene to that formed by biphenyl dioxygenase from Sphingomonas yanoikuyae B8/36. A recombinant Escherichia coli strain expressing the F352V variant of NDO and the enantioselective toluene cis-dihydrodiol dehydrogenase from Pseudomonas putida F1 was used to produce enantiomerically pure (-)-biphenyl cis-(3S,4R)-dihydrodiol and (-)-phenanthrene cis-(1S,2R)-dihydrodiol from biphenyl and phenanthrene, respectively.     

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.     

A novel aromatic-ring-hydroxylating dioxygenase from the diterpenoid-degrading bacterium Pseudomonas abietaniphila BKME-9

Pseudomonas abietaniphila BKME-9 is able to degrade dehydroabietic acid (DhA) via ring hydroxylation by a novel dioxygenase. The ditA1, ditA2, and ditA3 genes, which encode the alpha and beta subunits of the oxygenase and the ferredoxin of the diterpenoid dioxygenase, respectively, were isolated and sequenced. The ferredoxin gene is 9. 2 kb upstream of the oxygenase genes and 872 bp upstream of a putative meta ring cleavage dioxygenase gene, ditC. A Tn5 insertion in the alpha subunit gene, ditA1, resulted in the accumulation by the mutant strain BKME-941 of the pathway intermediate, 7-oxoDhA. Disruption of the ferredoxin gene, ditA3, in wild-type BKME-9 by mutant-allele exchange resulted in a strain (BKME-91) with a phenotype identical to that of the mutant strain BKME-941. Sequence analysis of the putative ferredoxin indicated that it is likely to be a [4Fe-4S]- or [3Fe-4S]-type ferredoxin and not a [2Fe-2S]-type ferredoxin, as found in all previously described ring-hydroxylating dioxygenases. Expression in Escherichia coli of ditA1A2A3, encoding the diterpenoid dioxygenase without its putative reductase component, resulted in a functional enzyme. The diterpenoid dioxygenase attacks 7-oxoDhA, and not DhA, at C-11 and C-12, producing 7-oxo-11, 12-dihydroxy-8,13-abietadien acid, which was identified by 1H nuclear magnetic resonance, UV-visible light, and high-resolution mass spectrometry. The organization of the genes encoding the various components of the diterpenoid dioxygenase, the phylogenetic distinctiveness of both the alpha subunit and the ferredoxin component, and the unusual Fe-S cluster of the ferredoxin all suggest that this enzyme belongs to a new class of aromatic ring-hydroxylating dioxygenases.     

Diverse reactions catalyzed by naphthalene dioxygenase fromPseudomonas sp strain NCIB 9816

Naphthalene dioxygenase (NDO) fromPseudomonas sp strain NCIB 9816 is a multicomponent enzyme system which initiates naphthalene catabolism by catalyzing the addition of both atoms of molecular oxygen and two hydrogen atoms to the substrate to yield enantiomerically pure (+)-cis-(1R,2S)-dihydroxy-1,2-dihydronaphthalene. NDO has a relaxed substrate specificity and catalyzes the dioxygenation of many related 2- and 3-ring aromatic and hydroaromatic (benzocyclic) compounds to their respectivecis-diols. Biotransformations with a diol-accumulating mutant, recombinant strains and purified enzyme components have established that in addition tocis-dihydroxylation, NDO also catalyzes a variety of other oxidations which include monohydroxylation, desaturation (dehydrogenation),O-andN-dealkylation and sulfoxidation reactions. In several cases, the absolute stereochemistry of the oxidation products formed by NDO are opposite to those formed by toluene dioxygenase (TDO). The reactions catalyzed by NDO and other microbial dioxygenases can yield specific hydroxylated compounds which can serve as chiral synthons in the preparation of a variety of compounds of interest to pharmaceutical and specialty chemical industries. We present here recent work documenting the diverse array of oxidation reactions catalyzed by NDO. The trends observed in the oxidation of a series of benzocyclic aromatic compounds are compared to those observed with TDO and provide the basis for prediction of regio- and stereospecificity in the oxidation of related substrates. Based on the types of reactions catalyzed and the biochemical characteristics of NDO, a mechanism for oxygen activation by NDO is proposed.     

Purification, characterization, and crystallization of the components of the nitrobenzene and 2-nitrotoluene dioxygenase enzyme systems

The protein components of the 2-nitrotoluene (2NT) and nitrobenzene dioxygenase enzyme systems from Acidovorax sp. strain JS42 and Comamonas sp. strain JS765, respectively, were purified and characterized. These enzymes catalyze the initial step in the degradation of 2-nitrotoluene and nitrobenzene. The identical shared reductase and ferredoxin components were monomers of 35 and 11.5 kDa, respectively. The reductase component contained 1.86 g-atoms iron, 2.01 g-atoms sulfur, and one molecule of flavin adenine dinucleotide per monomer. Spectral properties of the reductase indicated the presence of a plant-type [2Fe-2S] center and a flavin. The reductase catalyzed the reduction of cytochrome c, ferricyanide, and 2,6-dichlorophenol indophenol. The ferredoxin contained 2.20 g-atoms iron and 1.99 g-atoms sulfur per monomer and had spectral properties indicative of a Rieske [2Fe-2S] center. The ferredoxin component could be effectively replaced by the ferredoxin from the Pseudomonas sp. strain NCIB 9816-4 naphthalene dioxygenase system but not by that from the Burkholderia sp. strain LB400 biphenyl or Pseudomonas putida F1 toluene dioxygenase system. The oxygenases from the 2-nitrotoluene and nitrobenzene dioxygenase systems each had spectral properties indicating the presence of a Rieske [2Fe-2S] center, and the subunit composition of each oxygenase was an alpha(3)beta(3) hexamer. The apparent K(m) of 2-nitrotoluene dioxygenase for 2NT was 20 muM, and that for naphthalene was 121 muM. The specificity constants were 7.0 muM(-1) min(-1) for 2NT and 1.2 muM(-1) min(-1) for naphthalene, indicating that the enzyme is more efficient with 2NT as a substrate. Diffraction-quality crystals of the two oxygenases were obtained.     

Purification and molecular characterization of the electron transfer protein of methanesulfonic acid monooxygenase.

A novel serine pathway methylotroph, strain M2, capable of utilizing methanesulfonic acid (MSA) as a sole source of carbon and energy was investigated. The initial step in the biodegradative pathway of MSA in strain M2 involved an inducible NADH-specific monooxygenase enzyme (MSAMO). Fractionation of MSAMO active cell extracts by ion-exchange chromatography led to the loss of MSAMO activity. Activity was restored by mixing three distinct protein fractions, designated A, B, and C. Further purification to homogeneity of component C indicated that the polypeptide was acidic, with a pI of 3.9, and contained an iron-sulfur center with spectral characteristics similar to those of other proteins containing Rieske [2Fe-2S] centers. The size of the protein subunit and the similarity of the N-terminal sequence to those of ferredoxin components of other oxygenase enzymes have suggested that component C is a specific electron transfer protein of the MSAMO which contains a Rieske [2Fe-2S] cluster. The gene encoding component C of MSAMO was cloned and sequenced, and the predicted protein sequence was compared with those of other Rieske [2Fe-2S]-center-containing ferredoxins. MSAMO appears to be a novel combination of oxygenase elements in which an enzyme related to aromatic-ring dioxygenases attacks a one-carbon (C1) compound via monooxygenation.     

Stereospecific oxidation of (R)- and (S)-1-indanol by naphthalene dioxygenase from Pseudomonas sp. strain NCIB 9816-4.

A recombinant Escherichia coli strain which expresses naphthalene dioxygenase (NDO) from Pseudomonas sp. strain NCIB 9816-4 oxidized (S)-1-indanol to trans-(1S,3S)-indan-1,3-diol (95.5%) and (R)-3-hydroxy-1-indanone (4.5%). The same cells oxidized (R)-1-indanol to cis-1,3-indandiol (71%), (R)-3-hydroxy-1-indanone (18.2%), and cis-1,2,3-indantriol (10.8%). Purified NDO oxidized (S)-1-indenol to both syn- and anti-2,3-dihydroxy-1-indanol.     

Substrate Specificities of Hybrid Naphthalene and 2,4-Dinitrotoluene Dioxygenase Enzyme Systems

Bacterial three-component dioxygenase systems consist of reductase and ferredoxin components which transfer electrons from NAD(P)H to a terminal oxygenase. In most cases, the oxygenase consists of two different subunits (α and β). To assess the contributions of the α and β subunits of the oxygenase to substrate specificity, hybrid dioxygenase enzymes were formed by coexpressing genes from two compatible plasmids in Escherichia coli. The activities of hybrid naphthalene and 2,4-dinitrotoluene dioxygenases containing four different β subunits were tested with four substrates (indole, naphthalene, 2,4-dinitrotoluene, and 2-nitrotoluene). In the active hybrids, replacement of small subunits affected the rate of product formation but had no effect on the substrate range, regiospecificity, or enantiomeric purity of oxidation products with the substrates tested. These studies indicate that the small subunit of the oxygenase is essential for activity but does not play a major role in determining the specificity of these enzymes.     

The effect of ferredoxin(BED) overexpression on benzene dioxygenase activity in Pseudomonas putida ML2.

The benzene dioxygenase from Pseudomonas putida ML2 is a multicomponent complex comprising a flavoprotein reductase, a ferredoxin, and a terminal iron-sulfur protein (ISP). The catalytic activity of the isolated complex shows a nonlinear relationship with protein concentration in cell extracts, with the limiting factor for activity in vitro being ferredoxin(BED). The relative levels of the three components were analyzed by using 125I-labelled antibodies, and the functional molar ratio of ISP(BED), ferredoxin(BED), and reductase(BED) was shown to be 1:0.9:0.8, respectively. The concentration of ferredoxin(BED) was confirmed by quantitative electron paramagnetic resonance spectroscopy of the 2Fe-2S centers in ferredoxin(BED) and ISP(BED) of whole cells. These results demonstrate that the ferredoxin(BED) component is a limiting factor in dioxygenase activity in vitro. To determine if it is a limiting factor in vivo, a plasmid (pJRM606) overproducing ferredoxin(BED) was introduced into P. putida ML2. The benzene dioxygenase activity of this strain, measured in cell extracts, was fivefold greater than in the wild type, and the activity was linear with protein concentration in cell extracts above 2 mg/ml. Western blotting (immunoblotting) and electron paramagnetic resonance spectroscopic analysis confirmed an elevated level of ferredoxin(BED) protein and active redox centers in the recombinant strain. However, in these cells, the increased level of ferredoxin(BED) had no effect on the overall rate of benzene oxidation by whole cells. Thus, we conclude that ferredoxin(BED) is not limiting at the high intracellular concentration (0.48 mM) found in cells.