Cloning, Sequencing, and Characterization of a Gene Cluster Involved in EDTA Degradation from the Bacterium BNC1

EDTA is a chelating agent, widely used in many industries. Because of its ability to mobilize heavy metals and radionuclides, it can be an environmental pollutant. The EDTA monooxygenases that initiate EDTA degradation have been purified and characterized in bacterial strains BNC1 and DSM 9103. However, the genes encoding the enzymes have not been reported. The EDTA monooxygenase gene was cloned by probing a genomic library of strain BNC1 with a probe generated from the N-terminal amino acid sequence of the monooxygenase. Sequencing of the cloned DNA fragment revealed a gene cluster containing eight genes. Two of the genes, emoA and emoB, were expressed in Escherichia coli, and the gene products, EmoA and EmoB, were purified and characterized. Both experimental data and sequence analysis showed that EmoA is a reduced flavin mononucleotide-utilizing monooxygenase and that EmoB is an NADH:flavin mononucleotide oxidoreductase. The two-enzyme system oxidized EDTA to ethylenediaminediacetate (EDDA) and nitrilotriacetate (NTA) to iminodiacetate (IDA) with the production of glyoxylate. The emoA and emoB genes were cotranscribed when BNC1 cells were grown on EDTA. Other genes in the cluster encoded a hypothetical transport system, a putative regulatory protein, and IDA oxidase that oxidizes IDA and EDDA. We concluded that this gene cluster is responsible for the initial steps of EDTA and NTA degradation.     

Identification, purification, and characterization of iminodiacetate oxidase from the EDTA-degrading bacterium BNC1

Microbial degradation of synthetic chelating agents, such as EDTA and nitrilotriacetate (NTA), may help immobilizing radionuclides and heavy metals in the environment. The EDTA- and NTA-degrading bacterium BNC1 uses EDTA monooxygenase to oxidize NTA to iminodiacetate (IDA) and EDTA to ethylenediaminediacetate (EDDA). IDA- and EDDA-degrading enzymes have not been purified and characterized to date. In this report, an IDA oxidase was purified to apparent homogeneity from strain BNC1 by using a combination of eight purification steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein band of 40 kDa, and by using size exclusion chromatography, we estimated the native enzyme to be a homodimer. Flavin adenine dinucleotide was determined as its prosthetic group. The purified enzyme oxidized IDA to glycine and glyoxylate with the consumption of O2. The temperature and pH optima for IDA oxidation were 35 degrees C and 8, respectively. The apparent Km for IDA was 4.0 mM with a kcat of 5.3 s(-1). When the N-terminal amino acid sequence was determined, it matched exactly with that encoded by a previously sequenced hypothetical oxidase gene of BNC1. The gene was expressed in Escherichia coli, and the gene product as a C-terminal fusion with a His tag was purified by a one-step nickel affinity chromatography. The purified fusion protein had essentially the same enzymatic activity and properties as the native IDA oxidase. IDA oxidase also oxidized EDDA to ethylenediamine and glyoxylate. Thus, IDA oxidase is likely the second enzyme in both NTA and EDTA degradation pathways in strain BNC1.     

Identification, Purification, and Characterization of Iminodiacetate Oxidase from the EDTA-Degrading Bacterium BNC1

Microbial degradation of synthetic chelating agents, such as EDTA and nitrilotriacetate (NTA), may help immobilizing radionuclides and heavy metals in the environment. The EDTA- and NTA-degrading bacterium BNC1 uses EDTA monooxygenase to oxidize NTA to iminodiacetate (IDA) and EDTA to ethylenediaminediacetate (EDDA). IDA- and EDDA-degrading enzymes have not been purified and characterized to date. In this report, an IDA oxidase was purified to apparent homogeneity from strain BNC1 by using a combination of eight purification steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein band of 40 kDa, and by using size exclusion chromatography, we estimated the native enzyme to be a homodimer. Flavin adenine dinucleotide was determined as its prosthetic group. The purified enzyme oxidized IDA to glycine and glyoxylate with the consumption of O₂. The temperature and pH optima for IDA oxidation were 35°C and 8, respectively. The apparent K_m for IDA was 4.0 mM with a k_cat of 5.3 s⁻¹. When the N-terminal amino acid sequence was determined, it matched exactly with that encoded by a previously sequenced hypothetical oxidase gene of BNC1. The gene was expressed in Escherichia coli, and the gene product as a C-terminal fusion with a His tag was purified by a one-step nickel affinity chromatography. The purified fusion protein had essentially the same enzymatic activity and properties as the native IDA oxidase. IDA oxidase also oxidized EDDA to ethylenediamine and glyoxylate. Thus, IDA oxidase is likely the second enzyme in both NTA and EDTA degradation pathways in strain BNC1.     

Structural and biochemical characterization of EDTA monooxygenase and its physical interaction with a partner flavin reductase

Ethylenediaminetetraacetate (EDTA) is currently the most abundant organic pollutant due to its recalcitrance and extensive use. Only a few bacteria can degrade it, using EDTA monooxygenase (EmoA) to initiate the degradation. EmoA is an FMNH₂‐dependent monooxygenase that requires an NADH:FMN oxidoreductase (EmoB) to provide FMNH₂ as a cosubstrate. Although EmoA has been identified from Chelativorans (ex. Mesorhizobium) sp. BNC1, its catalytic mechanism is unknown. Crystal structures of EmoA revealed a domain‐like insertion into a TIM‐barrel, which might serve as a flexible lid for the active site. Docking of MgEDTA²⁻ into EmoA identified an intricate hydrogen bond network connected to Tyr⁷¹, which should potentially lower its pKa. Tyr⁷¹, along with nearby Glu⁷⁰ and a peroxy flavin, facilitates a keto‐enol transition of the leaving acetyl group of EDTA. Further, for the first time, the physical interaction between EmoA and EmoB was observed by ITC, molecular docking and enzyme kinetic assay, which enhanced both EmoA and EmoB activities probably through coupled channelling of FMNH₂.     

Crystal structures of NADH:FMN oxidoreductase (EmoB) at different stages of catalysis

EDTA has become a major organic pollutant in the environment because of its extreme usage and resistance to biodegradation. Recently, two critical enzymes, EDTA monooxygenase (EmoA) and NADH:FMN oxidoreductase (EmoB), belonging to the newly established two-component flavin-diffusible monooxygenase family, were identified in the EDTA degradation pathway in Mesorhizobium sp. BNC1. EmoA is an FMNH2-dependent enzyme that requires EmoB to provide FMNH2 for the conversion of EDTA to ethylenediaminediacetate. To understand the molecular basis of this FMN-mediated reaction, the crystal structures of the apo-form, FMN.FMN complex, and FMN.NADH complex of EmoB were determined at 2.5 angstroms resolution. The structure of EmoB is a homotetramer consisting of four alpha/beta-single-domain monomers of five parallel beta-strands flanked by five alpha-helices, which is quite different from those of other known two-component flavin-diffusible monooxygenase family members, such as PheA2 and HpaC, in terms of both tertiary and quaternary structures. For the first time, the crystal structures of both the FMN.FMN and FMN.NADH complexes of an NADH:FMN oxidoreductase were determined. Two stacked isoalloxazine rings and nicotinamide/isoalloxazine rings were at a proper distance for hydride transfer. The structures indicated a ping-pong reaction mechanism, which was confirmed by activity assays. Thus, the structural data offer detailed mechanistic information for hydride transfer between NADH to an enzyme-bound FMN and between the bound FMNH2 and a diffusible FMN.     

Cloning, sequencing, and analysis of a gene cluster from Chelatobacter heintzii ATCC 29600 encoding nitrilotriacetate monooxygenase and NADH:flavin mononucleotide oxidoreductase.

Nitrilotriacetate (NTA) is an important chelating agent in detergents and has also been used extensively in processing radionuclides. In Chelatobacter heintzii ATCC 29600, biodegradation of NTA is initiated by NTA monooxygenase that oxidizes NTA to iminodiacetate and glyoxylate. The NTA monooxygenase activity requires two component proteins, component A and component B, but the function of each component is unclear. We have cloned and sequenced a gene cluster encoding components A and B (nmoA and nmoB) and two additional open reading frames, nmoR and nmoT, downstream of nmoA. Based on sequence similarities, nmoR and nmoT probably encode a regulatory protein and a transposase, respectively. The NmoA sequence was similar to a monooxygenase that uses reduced flavin mononucleotide (FMNH2) as reductant; NmoB was similar to an NADH:flavin mononucleotide (FMN) oxidoreductase. On the basis of this information, we tested the function of each component. Purified component B was shown to be an NADH:FMN oxidoreductase, and its activity could be separated from that of component A. When the Photobacterium fischeri NADH:FMN oxidoreductase was substituted for component B in the complete reaction, NTA was oxidized, showing that the substrate specificity of the reaction resides in component A. Component A is therefore an NTA monooxygenase that uses FMNH2 and O2 to oxidize NTA, and component B is an NADH:FMN oxidoreductase that provides FMNH2 for NTA oxidation.     

Oligonucleotide primers for detection and amplification of the emoA gene encoding bacterial ethylenediaminetetraacetate monooxygenase

A system of primers was designed on the basis of analysis of nucleotide sequences of the emoA gene encoding ethylenediaminetetraacetate (EDTA) monooxygenase, which are deposited in GenBank. This system of primers makes it possible to obtain emoA gene fragments approximately 750 bp long for bacterial destructors of EDTA. Polymerase chain reaction (PCR) of total DNA isolated from enrichment and pure cultures showed that this system can be effectively used for detecting the emoA gene in representatives of Alpha- and Gammaproteobacteria. Partial sequences of emoA genes of bacteria of the genera Chelativorans and Stenotrophomonas, which are able to degrade this pollutant, have been sequenced and deposited in GenBank.     

Pathway of degradation of nitrilotriacetate by a Pseudomonas species.

The pathway of degradation of nitrilotriacetate (NTA) was determined by using cell-free extracts and a 35-fold purification of NTA monooxygenase. The first step in the breakdown was an oxidative cleavage of the tertiary amine by the monooxygenase to form the aldo acid, glyoxylate, and the secondary amine, iminodiacetate (IDA). NTA N-oxide acted as a substrate analog for induction of the monooxygenase and was slowly metabolized by the enzyme, but was not an intermediate in the pathway. No intermediate before IDA was found, but an unstable alpha-hydroxy-NTA intermediate was postulated. IDA did undergo cleavage in the presence of the purified monooxygenase to give glyoxylate and glycine, but was not metabolized in cell-free extracts. Glyoxylate was further metabolized by cell-free extracts to yield CO2 and glycerate or glycine, products also found from NTA metabolism. Of the three bacterial isolates in which the NTA pathway has been studied, two strains, one isolated from a British soil and ours from a Michigan soil, appear to be almost identical.     

Evidence that bacterial ABC-type transporter imports free EDTA for metabolism

EDTA, a common chelating agent, is becoming a major organic pollutant in the form of metal-EDTA complexes in surface waters, partly due to its recalcitrance to biodegradation. Even an EDTA-degrading bacterium, BNC1, does not degrade stable metal-EDTA complexes. In the present study, an ABC-type transporter was identified for possible uptake of EDTA because the transporter genes and the EDTA monooxygenase gene were expressed from a single operon in BNC1. The ABC-type transporter had a periplasmic-binding protein (EppA) that should confer the substrate specificity for the transporter; therefore, EppA was produced in Escherichia coli, purified, and characterized. EppA was shown to bind free EDTA with a dissociation constant as low as 25 nM by using isothermal titration calorimetry. When unstable metal-EDTA complexes, e.g., (Mg-EDTA)²⁻, were added to the EppA solution, binding was also observed. However, experimental data and theoretical analysis supported EppA binding only of free EDTA. When stable metal-EDTA complexes, e.g., (Cu-EDTA)²⁻, were titrated into the EppA solution, no binding was observed. Since EDTA monooxygenase in the cytoplasm uses some of the stable metal-EDTA complexes as substrates, we suggest that the lack of EppA binding and EDTA uptake are responsible for the failure of BNC1 cells to degrade the stable complexes.     

Purification and sequence analysis of 4-methyl-5-nitrocatechol oxygenase from Burkholderia sp. strain DNT.

4-Methyl-5-nitrocatechol (MNC) is an intermediate in the degradation of 2,4-dinitrotoluene by Burkholderia sp. strain DNT. In the presence of NADPH and oxygen, MNC monooxygenase catalyzes the removal of the nitro group from MNC to form 2-hydroxy-5-methylquinone. The gene (dntB) encoding MNC monooxygenase has been previously cloned and characterized. In order to examine the properties of MNC monooxygenase and to compare it with other enzymes, we sequenced the gene encoding the MNC monooxygenase and purified the enzyme from strain DNT. dntB was localized within a 2.2-kb ApaI DNA fragment. Sequence analysis of this fragment revealed an open reading frame of 1,644 bp with an N-terminal amino acid sequence identical to that of purified MNC monooxygenase from strain DNT. Comparison of the derived amino acid sequences with those of other genes showed that DntB contains the highly conserved ADP and flavin adenine dinucleotide (FAD) binding motifs characteristic of flavoprotein hydroxylases. MNC monooxygenase was purified to homogeneity from strain DNT by anion exchange and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein with a molecular weight of 60,200, which is consistent with the size determined from the gene sequence. The native molecular weight determined by gel filtration was 65,000, which indicates that the native enzyme is a monomer. It used either NADH or NADPH as electron donors, and NADPH was the preferred cofactor. The purified enzyme contained 1 mol of FAD per mol of protein, which is also consistent with the detection of an FAD binding motif in the amino acid sequence of DntB. MNC monooxygenase has a narrow substrate specificity. MNC and 4-nitrocatechol are good substrates whereas 3-methyl-4-nitrophenol, 3-methyl-4-nitrocatechol, 4-nitrophenol, 3-nitrophenol, and 4-chlorocatechol were not. These studies suggest that MNC monooxygenase is a flavoprotein that shares some properties with previously studied nitrophenol oxygenases.     

Purification and Characterization of EDTA Monooxygenase from the EDTA-Degrading Bacterium BNC1

The synthetic chelating agent EDTA can mobilize radionuclides and heavy metals in the environment. Biodegradation of EDTA should reduce this mobilization. Although several bacteria have been reported to mineralize EDTA, little is known about the biochemistry of EDTA degradation. Understanding the biochemistry will facilitate the removal of EDTA from the environment. EDTA-degrading activities were detected in cell extracts of bacterium BNC1 when flavin mononucleotide (FMN), NADH, and O₂ were present. The degradative enzyme system was separated into two different enzymes, EDTA monooxygenase and an FMN reductase. EDTA monooxygenase oxidized EDTA to glyoxylate and ethylenediaminetriacetate (ED3A), with the coconsumption of FMNH₂ and O₂. The FMN reductase provided EDTA monooxygenase with FMNH₂ by reducing FMN with NADH. The FMN reductase was successfully substituted in the assay mixture by other FMN reductases. EDTA monooxygenase was purified to greater than 95% homogeneity and had a single polypeptide with a molecular weight of 45,000. The enzyme oxidized both EDTA complexed with various metal ions and uncomplexed EDTA. The optimal conditions for activity were pH 7.8 and 35°C. K_ms were 34.1 μM for uncomplexed EDTA and 8.5 μM for MgEDTA²⁻; this difference in K_m indicates that the enzyme has greater affinity for MgEDTA²⁻. The enzyme also catalyzed the release of glyoxylate from nitrilotriacetate and diethylenetriaminepentaacetate. EDTA monooxygenase belongs to a small group of FMNH₂-utilizing monooxygenases that attack carbon-nitrogen, carbon-sulfur, and carbon-carbon double bonds.     

Bacterial growth yields on EDTA,NTA, and their biodegradation intermediates

Ethylenediaminetetraacetic acid (EDTA) and nitrilotriacetic acid (NTA) are widely used anthropogenic chelating agents for control of metal speciation and are ubiquitous in natural waters and wastewaters. This is the first report of systematic measurement of the growth yields of a mixed culture (BNC1-BNC2) on EDTA and its biodegradation intermediates, and of Aminobacter aminovorans (aka Chelatobacter heintzii) ATCC 29600 on NTA and its biodegradation intermediates. The yields measured for BNC1-BNC2 co-culture were 75.0 g of cell dry weight (CDW) (mole of EDTA)⁻¹, 68.6 g of CDW (mole of ED3 A)⁻¹, 51.2 g of CDW (mole of N,N′-EDDA)⁻¹, 34.5 g of CDW (mole of ED)⁻¹, 26.3 g of CDW (mole of IDA)⁻¹, 12.2 g of CDW (mole of glycine)⁻¹, and 9.7 g of CDW (mole of glyoxylate)⁻¹. The yields measured for A. aminovorans were 44.3 g of CDW (mole of NTA)⁻¹, 37.9 g of CDW (mole of IDA)⁻¹, 15.2 g of CDW (mole of glycine)⁻¹, and 10.4 g of CDW (mole of glyoxylate)⁻¹. The biodegradation pathways of EDTA, NTA, and several of their metabolic intermediates include reactions catalyzed by oxygenase enzymes, which may reduce energy available for cell synthesis. Comparison of measured yields with predicted yields indicates that the effect of oxygenase reaction on cell yield can be quantified experimentally as well as modeled based on thermodynamics.     

2-萘酸加单氧酶基因及NADH:黄素还原酶基因的克隆与分析

伯克霍尔德氏菌(Burkholderiasp.)JT1500对2-萘酸(2-naphthoate)生物降解的关键步骤之一是通过2-萘酸加单氧酶羟化2-萘酸生成1-羟基-2-萘酸(1-hydroxy-2-naphthoate)。在已确定2-萘酸加单氧酶基因及其功能的基础上对含有该基因的一个4.8kb长度的基因簇进行了克隆测序。该序列上含有4个可能的阅读框orfB、orfC、orfD、orfA。序列比对发现,orfA序列与JaponicumUSDA110和RalstoniaeutrophaHF39中的加单氧酶基因同源性较高,orfB序列与BordetllapertussisTohamaI、RalstoniasolanacearumGMI1000和BordetellabronchisepticaRB50等菌中的黄素还原酶基因有一定的同源性。酶活分析发现只含基因orfA的重组大肠杆菌SA细胞提取液有很低的加氧活性,含基因orfB的重组子SB细胞提取液没有加氧活性,但在反应体系中同时加入SA和SB的细胞提取液后,其加氧活性显著增强,包含片段orfB orfA的重组子SB A在黄素(FMN、FAD)存在的情况下也表现出很强的加氧活性;在厌氧条件下,能检测出SB细胞提取液的黄素还原活性。基于以上信息,认为2-萘酸加单氧酶基因簇含有两个重要的组分黄素还原酶基因(nmoB)和加单氧酶基因(nmoA)。2-萘酸加单氧酶Nmo羟化2-萘酸的过程为先由黄素还原酶(NmoB)在NADH存在的条件下将黄素(FMN、FAD)还原为还原型黄素(FMNH2、FADH2),然后加单氧酶(NmoA)利用还原型黄素和O2羟化底物2-萘酸,生成1-羟基-2-萘酸。NmoB是NmoA的偶联蛋白。     

Structural and biochemical characterization of iminodiacetate oxidase from Chelativorans sp. BNC1

Ethylenediaminetetraacetate (EDTA) is the most abundant organic pollutant in surface water because of its extensive usage and the recalcitrance of stable metal‐EDTA complexes. A few bacteria including Chelativorans sp. BNC1 can degrade EDTA with a monooxygenase to ethylenediaminediacetate (EDDA) and then use iminodiacetate oxidase (IdaA) to further degrade EDDA into ethylenediamine in a two‐step oxidation. To alleviate EDTA pollution into the environment, deciphering the mechanisms of the metabolizing enzymes is an imperative prerequisite for informed EDTA bioremediation. Although IdaA cannot oxidize glycine, the crystal structure of IdaA shows its tertiary and quaternary structures similar to those of glycine oxidases. All confirmed substrates, EDDA, ethylenediaminemonoacetate, iminodiacetate and sarcosine are secondary amines with at least one N‐acetyl group. Each substrate was bound at the re‐side face of the isoalloxazine ring in a solvent‐connected cavity. The carboxyl group of the substrate was bound by Arg²⁶⁵ and Arg³⁰⁷. The catalytic residue, Tyr²⁵⁰, is under the hydrogen bond network to facilitate its deprotonation acting as a general base, removing an acetate group of secondary amines as glyoxylate. Thus, IdaA is a secondary amine oxidase, and our findings improve understanding of molecular mechanism involved in the bioremediation of EDTA and the metabolism of secondary amines.     

Characterization of an inducible,membrane-bound iminodiacetate dehydrogenase from Chelatobacter heintzii ATCC 29600

Iminodiacetate (IDA) is a xenobiotic intermediate common to both aerobic and anaerobic metabolism of nitrilotriacetate (NTA). It is formed by either NTA monooxygenase or NTA dehydrogenase. In this paper the detection and characterization of a membrane-bound iminodiacete dehydrogenase (IDA-DH) from Chelatobacter heintzii ATCC 29600 is reported, which oxidizes IDA to glycine and glyoxylate. Out of 15 compounds tested, IDA was the only substrate for the enzyme. Optimum activity of IDA-DH was found at pH 8.5 and 25°C, respectively, and the K_m for IDA was found to be 8mM. Activity of the membrane-bound enzyme was inhibited by KCN, antimycine and dibromomethylisopropyl-benzoquinone. When inhibited by KCN IDA-DH was able to reduce the artificial electron acceptor iodonitrotetrazolium (INT). It was possible to extract IDA-DH from the membranes with 2% cholate, to reconstitute the enzyme into soybean phospholipid vesicles and to obtain IDA-DH activity (more than 50% recovery) using ubiquinone Q₁ as the intermediate electron carrier and INT as the final electron acceptor. Growth experiments with different substrates revealed that in all NTA-degrading strains tested both NTA monooxygenase and IDA-DH were only expressed when the cells were grown on NTA or IDA. Furthermore, in Cb. heintzii ATCC 29600 growing exponentially on succinate and ammonia, addition of 0.4 g l^-1 NTA led to the induction of the two enzymes within an hour and NTA was utilized simultaneously with succinate. The presence of IDA-DH was confirmed in ten different NTA-degrading strains belonging to three different genera.Abbreviations cA component A - cB component B - DBMIB dibromomethylisopropyl-benzoquinone - HEPES hydroxyethylpiperazinethanesulfonic acid - IDA iminodiacetate, HN(CH₂COOH)₂ - IDA-DH iminodiacetate dehydrogenase - INT iodonitrotetrazolium chloride - NTA nitrilotriacetate, N(CH₂COOH)₃ - NTA-MO nitrilotriacetate monooxygenase - PMS phenazine methosulphate - SDS-PAGE sodium dodecylsulfate polyacrylamide gel electrophoresis - Suc-DH succinate dehydrogenase     

Cloning and characterization of a gene cluster for cyclododecanone oxidation in Rhodococcus ruber SC1

Biological oxidation of cyclic ketones normally results in formation of the corresponding dicarboxylic acids, which are further metabolized in the cell. Rhodococcus ruber strain SC1 was isolated from an industrial wastewater bioreactor that was able to utilize cyclododecanone as the sole carbon source. A reverse genetic approach was used to isolate a 10-kb gene cluster containing all genes required for oxidative conversion of cyclododecanone to 1,12-dodecanedioic acid (DDDA). The genes required for cyclododecanone oxidation were only marginally similar to the analogous genes for cyclohexanone oxidation. The biochemical function of the enzymes encoded on the 10-kb gene cluster, the flavin monooxygenase, the lactone hydrolase, the alcohol dehydrogenase, and the aldehyde dehydrogenase, was determined in Escherichia coli based on the ability to convert cyclododecanone. Recombinant E. coli strains grown in the presence of cyclododecanone accumulated lauryl lactone, 12-hydroxylauric acid, and/or DDDA depending on the genes cloned. The cyclododecanone monooxygenase is a type 1 Baeyer-Villiger flavin monooxygenase (FAD as cofactor) and exhibited substrate specificity towards long-chain cyclic ketones (C₁₁ to C₁₅), which is different from the specificity of cyclohexanone monooxygenase favoring short-chain cyclic compounds (C₅ to C₇).     

Purification, characterization, and overexpression of flavin reductase involved in dibenzothiophene desulfurization by Rhodococcus erythropolis D-1

The dibenzothiophene (DBT)-desulfurizing bacterium, Rhodococcus erythropolis D-1, removes sulfur from DBT to form 2-hydroxybiphenyl using four enzymes, DszC, DszA, DszB, and flavin reductase. In this study, we purified and characterized the flavin reductase from R. erythropolis D-1 grown in a medium containing DBT as the sole source of sulfur. It is conceivable that the enzyme is essential for two monooxygenase (DszC and DszA) reactions in vivo. The purified flavin reductase contains no chromogenic cofactors and was found to have a molecular mass of 86 kDa and four identical 22-kDa subunits. The enzyme catalyzed NADH-dependent reduction of flavin mononucleotide (FMN), and the K(m) values for NADH and FMN were 208 and 10.8 microM, respectively. Flavin adenine dinucleotide was a poor substrate, and NADPH was inert. The enzyme did not catalyze reduction of any nitroaromatic compound. The optimal temperature and optimal pH for enzyme activity were 35 degrees C and 6.0, respectively, and the enzyme retained 30% of its activity after heat treatment at 80 degrees C for 30 min. The N-terminal amino acid sequence of the purified flavin reductase was identical to that of DszD of R. erythropolis IGTS8 (K. A. Gray, O. S. Pogrebinsky, G. T. Mrachko, L. Xi, D. J. Monticello, and C. H. Squires, Nat. Biotechnol. 14:1705-1709, 1996). The flavin reductase gene was amplified with primers designed by using dszD of R. erythropolis IGTS8, and the enzyme was overexpressed in Escherichia coli. The specific activity in crude extracts of the overexpressed strain was about 275-fold that of the wild-type strain.     

Characterization of the baiH gene encoding a bile acid-inducible NADH:flavin oxidoreductase from Eubacterium sp. strain VPI 12708.

A cholate-inducible, NADH-dependent flavin oxidoreductase from the intestinal bacterium Eubacterium sp. strain VPI 12708 was purified 372-fold to apparent electrophoretic homogeneity. The subunit and native molecular weights were estimated to be 72,000 and 210,000, respectively, suggesting a homotrimeric organization. Three peaks of NADH:flavin oxidoreductase activity (forms I, II, and III) eluted from a DEAE-high-performance liquid chromatography column. Absorption spectra revealed that purified form III, but not form I, contained bound flavin, which dissociated during purification to generate form I. Enzyme activity was inhibited by sulfhydryl-reactive compounds, acriflavine, o-phenanthroline, and EDTA. Activity assays and Western blot (immunoblot) analysis confirmed that expression of the enzyme was cholate inducible. The first 25 N-terminal amino acid residues of purified NADH:flavin oxidoreductase were determined, and a corresponding oligonucleotide probe was synthesized for use in cloning of the associated gene, baiH. Restriction mapping, sequence data, and RNA blot analysis suggested that the baiH gene was located on a previously described, cholate-inducible operon > or = 10 kb long. The baiH gene encoded a 72,006-Da polypeptide containing 661 amino acids. The deduced amino acid sequence of the baiH gene was homologous to that of NADH oxidase from Thermoanaerobium brockii, trimethylamine dehydrogenase from methylotrophic bacterium W3A1, Old Yellow Enzyme from Saccharomyces carlsbergensis, and the product of the baiC gene of Eubacterium sp. strain VPI 12708, located upstream from the baiH gene in the cholate-inducible operon. Alignment of these five sequences revealed potential ligands for an iron-sulfur cluster, a putative flavin adenine dinucleotide-binding domain, and two other well-conserved domains of unknown function.     

Cloning, purification and characterization of two components of phenol hydroxylase from Rhodococcus erythropolis UPV-1

Phenol hydroxylase that catalyzes the conversion of phenol to catechol in Rhodococcus erythropolis UPV-1 was identified as a two-component flavin-dependent monooxygenase. The two proteins are encoded by the genes pheA1 and pheA2, located very closely in the genome. The sequenced pheA1 gene was composed of 1,629 bp encoding a protein of 542 amino acids, whereas the pheA2 gene consisted of 570 bp encoding a protein of 189 amino acids. The deduced amino acid sequences of both genes showed high homology with several two-component aromatic hydroxylases. The genes were cloned separately in cells of Escherichia coli M15 as hexahistidine-tagged proteins, and the recombinant proteins His₆PheA1 and His₆PheA2 were purified and its catalytic activity characterized. His₆PheA1 exists as a homotetramer of four identical subunits of 62 kDa that has no phenol hydroxylase activity on its own. His₆PheA2 is a homodimeric flavin reductase, consisting of two identical subunits of 22 kDa, that uses NAD(P)H in order to reduce flavin adenine dinucleotide (FAD), according to a random sequential kinetic mechanism. The reductase activity was strongly inhibited by thiol-blocking reagents. The hydroxylation of phenol in vitro requires the presence of both His₆PheA1 and His₆PheA2 components, in addition to NADH and FAD, but the physical interaction between the proteins is not necessary for the reaction.