Ohgew Kweon

Complete and integrated pyrene degradation pathway in Mycobacterium vanbaalenii PYR-1 based on systems biology

Mycobacterium vanbaalenii PYR-1 was the first bacterium isolated by virtue of its ability to metabolize the high-molecular-weight polycyclic aromatic hydrocarbon (PAH) pyrene. We used metabolic, genomic, and proteomic approaches in this investigation to construct a complete and integrated pyrene degradation pathway for M. vanbaalenii PYR-1. Genome sequence analyses identified genes involved in the pyrene degradation pathway that we have proposed for this bacterium. To identify proteins involved in the degradation, we conducted a proteome analysis of cells exposed to pyrene using one-dimensional gel electrophoresis in combination with liquid chromatography-tandem mass spectrometry. Database searching performed with the M. vanbaalenii PYR-1 genome resulted in identification of 1,028 proteins with a protein false discovery rate of <1%. Based on both genomic and proteomic data, we identified 27 enzymes necessary for constructing a complete pathway for pyrene degradation. Our analyses indicate that this bacterium degrades pyrene to central intermediates through o-phthalate and the beta-ketoadipate pathway. Proteomic analysis also revealed that 18 enzymes in the pathway were upregulated more than twofold, as indicated by peptide counting when the organism was grown with pyrene; three copies of the terminal subunits of ring-hydroxylating oxygenase (NidAB2, MvanDraft_0817/0818, and PhtAaAb), dihydrodiol dehydrogenase (MvanDraft_0815), and ring cleavage dioxygenase (MvanDraft_3242) were detected only in pyrene-grown cells. The results presented here provide a comprehensive picture of pyrene metabolism in M. vanbaalenii PYR-1 and a useful framework for understanding cellular processes involved in PAH degradation.

Molecular Cloning and Expression of Genes Encoding a Novel Dioxygenase Involved in Low- and High-Molecular-Weight Polycyclic Aromatic Hydrocarbon Degradation in Mycobacterium vanbaalenii PYR-1

ABSTRACT Mycobacterium vanbaalenii PYR-1 is able to metabolize a wide range of low- and high-molecular-weight (HMW) polycyclic aromatic hydrocarbons (PAHs). A 20-kDa protein was upregulated in PAH-metabolizing M. vanbaalenii PYR-1 cells compared to control cultures. The differentially expressed protein was identified as a β subunit of the terminal dioxygenase using mass spectrometry. PCR with degenerate primers designed based on de novo sequenced peptides and a series of plaque hybridizations were done to screen the M. vanbaalenii PYR-1 genomic library. The genes, designated nidA3B3, encoding the α and β subunits of terminal dioxygenase, were subsequently cloned and sequenced. The deduced enzyme revealed close similarities to the corresponding PAH ring-hydroxylating dioxygenases from Mycobacterium and Rhodococcus spp. but had the highest similarity, 61.9%, to the α subunit from Nocardioides sp. strain KP7. The α subunit also showed 52% sequence homology with the previously reported NidA from M. vanbaalenii PYR-1. The genes nidA3B3 were subcloned into the expression vector pET-17b, and the enzyme activity in Escherichia coli cells was reconstituted through coexpression with the ferredoxin (PhdC) and ferredoxin reductase (PhdD) genes of the phenanthrene dioxygenase from Nocardioides sp. strain KP7. The recombinant PAH dioxygenase appeared to favor the HMW PAH substrates fluoranthene, pyrene, and phenanthrene. Several other PAHs, including naphthalene, anthracene, and benz[a]anthracene, were also converted to their corresponding cis-dihydrodiols. The recombinant E. coli, however, did not show any dioxygenation activity for phthalate and biphenyl. The upregulation of nidA3B3 in M. vanbaalenii PYR-1 induced by PAHs was confirmed by reverse transcription-PCR analysis.

A new classification system for bacterial Rieske non-heme iron aromatic ring-hydroxylating oxygenases

BackgroundRieske non-heme iron aromatic ring-hydroxylating oxygenases (RHOs) are multi-component enzyme systems that are remarkably diverse in bacteria isolated from diverse habitats. Since the first classification in 1990, there has been a need to devise a new classification scheme for these enzymes because many RHOs have been discovered, which do not belong to any group in the previous classification. Here, we present a scheme for classification of RHOs reflecting new sequence information and interactions between RHO enzyme components.ResultWe have analyzed a total of 130 RHO enzymes in which 25 well-characterized RHO enzymes were used as standards to test our hypothesis for the proposed classification system. From the sequence analysis of electron transport chain (ETC) components of the standard RHOs, we extracted classification keys that reflect not only the phylogenetic affiliation within each component but also relationship among components. Oxygenase components of standard RHOs were phylogenetically classified into 10 groups with the classification keys derived from ETC components. This phylogenetic classification scheme was converted to a new systematic classification consisting of 5 distinct types. The new classification system was statistically examined to justify its stability. Type I represents two-component RHO systems that consist of an oxygenase and an FNRC-type reductase. Type II contains other two-component RHO systems that consist of an oxygenase and an FNRN-type reductase. Type III represents a group of three-component RHO systems that consist of an oxygenase, a [2Fe-2S]-type ferredoxin and an FNRN-type reductase. Type IV represents another three-component systems that consist of oxygenase, [2Fe-2S]-type ferredoxin and GR-type reductase. Type V represents another different three-component systems that consist of an oxygenase, a [3Fe-4S]-type ferredoxin and a GR-type reductase.ConclusionThe new classification system provides the following features. First, the new classification system analyzes RHO enzymes as a whole. RwithSecond, the new classification system is not static but responds dynamically to the growing pool of RHO enzymes. Third, our classification can be applied reliably to the classification of incomplete RHOs. Fourth, the classification has direct applicability to experimental work. Fifth, the system provides new insights into the evolution of RHO systems based on enzyme interaction.

Molecular Characterization of Resistance to Extended-Spectrum Cephalosporins in Clinical Escherichia coli Isolates from Companion Animals in the United States

ABSTRACT Resistance to extended-spectrum cephalosporins (ESC) among members of the family Enterobacteriaceae occurs worldwide; however, little is known about ESC resistance in Escherichia coli strains from companion animals. Clinical isolates of E. coli were collected from veterinary diagnostic laboratories throughout the United States from 2008 to 2009. E. coli isolates (n = 54) with reduced susceptibility to ceftazidime or cefotaxime (MIC ≥ 16 μg/ml) and extended-spectrum-β-lactamase (ESBL) phenotypes were analyzed. PCR and sequencing were used to detect mutations in ESBL-encoding genes and the regulatory region of the chromosomal gene ampC. Conjugation experiments and plasmid identification were conducted to examine the transferability of resistance to ESCs. All isolates carried the blaCTX-M-1-group β-lactamase genes in addition to one or more of the following β-lactamase genes: blaTEM, blaSHV-3, blaCMY-2, blaCTX-M-14-like, and blaOXA-1. Different blaTEM sequence variants were detected in some isolates (n = 40). Three isolates harbored a blaTEM-181 gene with a novel mutation resulting in an Ala184Val substitution. Approximately 78% of the isolates had mutations in promoter/attenuator regions of the chromosomal gene ampC, one of which was a novel insertion of adenine between bases −28 and −29. Plasmids ranging in size from 11 to 233 kbp were detected in the isolates, with a common plasmid size of 93 kbp identified in 60% of isolates. Plasmid-mediated transfer of β-lactamase genes increased the MICs (≥16-fold) of ESCs for transconjugants. Replicon typing among isolates revealed the predominance of IncI and IncFIA plasmids, followed by IncFIB plasmids. This study shows the emergence of conjugative plasmid-borne ESBLs among E. coli strains from companion animals in the United States, which may compromise the effective therapeutic use of ESCs in veterinary medicine.

A Polyomic Approach To Elucidate the Fluoranthene-Degradative Pathway in Mycobacterium vanbaalenii PYR-1

Mycobacterium vanbaalenii PYR-1 is capable of degrading a wide range of high-molecular-weight polycyclic aromatic hydrocarbons (PAHs), including fluoranthene. We used a combination of metabolomic, genomic, and proteomic technologies to investigate fluoranthene degradation in this strain. Thirty-seven fluoranthene metabolites including potential isomers were isolated from the culture medium and analyzed by high-performance liquid chromatography, gas chromatography-mass spectrometry, and UV-visible absorption. Total proteins were separated by one-dimensional gel and analyzed by liquid chromatography-tandem mass spectrometry in conjunction with the M. vanbaalenii PYR-1 genome sequence (http://jgi.doe.gov), which resulted in the identification of 1,122 proteins. Among them, 53 enzymes were determined to be likely involved in fluoranthene degradation. We integrated the metabolic information with the genomic and proteomic results and proposed pathways for the degradation of fluoranthene. According to our hypothesis, the oxidation of fluoranthene is initiated by dioxygenation at the C-1,2, C-2,3, and C-7,8 positions. The C-1,2 and C-2,3 dioxygenation routes degrade fluoranthene via fluorene-type metabolites, whereas the C-7,8 routes oxidize fluoranthene via acenaphthylene-type metabolites. The major site of dioxygenation is the C-2,3 dioxygenation route, which consists of 18 enzymatic steps via 9-fluorenone-1-carboxylic acid and phthalate with the initial ring-hydroxylating oxygenase, NidA3B3, oxidizing fluoranthene to fluoranthene cis-2,3-dihydrodiol. Nonspecific monooxygenation of fluoranthene with subsequent O methylation of dihydroxyfluoranthene also occurs as a detoxification reaction.

Polycyclic Aromatic Hydrocarbon Metabolic Network in Mycobacterium vanbaaleniiPYR-1

This study investigated a metabolic network (MN) from Mycobacterium vanbaalenii PYR-1 for polycyclic aromatic hydrocarbons (PAHs) from the perspective of structure, behavior, and evolution, in which multilayer omics data are integrated. Initially, we utilized a high-throughput proteomic analysis to assess the protein expression response of M. vanbaalenii PYR-1 to seven different aromatic compounds. A total of 3,431 proteins (57.38% of the genome-predicted proteins) were identified, which included 160 proteins that seemed to be involved in the degradation of aromatic hydrocarbons. Based on the proteomic data and the previous metabolic, biochemical, physiological, and genomic information, we reconstructed an experiment-based system-level PAH-MN. The structure of PAH-MN, with 183 metabolic compounds and 224 chemical reactions, has a typical scale-free nature. The behavior and evolution of the PAH-MN reveals a hierarchical modularity with funnel effects in structure/function and intimate association with evolutionary modules of the functional modules, which are the ring cleavage process (RCP), side chain process (SCP), and central aromatic process (CAP). The 189 commonly upregulated proteins in all aromatic hydrocarbon treatments provide insights into the global adaptation to facilitate the PAH metabolism. Taken together, the findings of our study provide the hierarchical viewpoint from genes/proteins/metabolites to the network via functional modules of the PAH-MN equipped with the engineering-driven approaches of modularization and rationalization, which may expand our understanding of the metabolic potential of M. vanbaalenii PYR-1 for bioremediation applications.

Substrate Specificity and Structural Characteristics of the Novel Rieske Nonheme Iron Aromatic Ring-Hydroxylating Oxygenases NidAB and NidA3B3 from Mycobacterium vanbaalenii PYR-1

ABSTRACT The Rieske nonheme iron aromatic ring-hydroxylating oxygenases (RHOs) NidAB and NidA3B3 from Mycobacterium vanbaalenii PYR-1 have been implicated in the initial oxidation of high-molecular-weight (HMW) polycyclic aromatic hydrocarbons (PAHs), forming cis- dihydrodiols. To clarify how these two RHOs are functionally different with respect to the degradation of HMW PAHs, we investigated their substrate specificities to 13 representative aromatic substrates (toluene, m-xylene, phthalate, biphenyl, naphthalene, phenanthrene, anthracene, fluoranthene, pyrene, benz[a]anthracene, benzo[a]pyrene, carbazole, and dibenzothiophene) by enzyme reconstitution studies of Escherichia coli. Both Nid systems were identified to be compatible with type V electron transport chain (ETC) components, consisting of a [3Fe-4S]-type ferredoxin and a glutathione reductase (GR)-type reductase. Metabolite profiles indicated that the Nid systems oxidize a wide range of aromatic hydrocarbon compounds, producing various isomeric dihydrodiol and phenolic compounds. NidAB and NidA3B3 showed the highest conversion rates for pyrene and fluoranthene, respectively, with high product regiospecificity, whereas other aromatic substrates were converted at relatively low regiospecificity. Structural characteristics of the active sites of the Nid systems were investigated and compared to those of other RHOs. The NidAB and NidA3B3 systems showed the largest substrate-binding pockets in the active sites, which satisfies spatial requirements for accepting HMW PAHs. Spatially conserved aromatic amino acids, Phe-Phe-Phe, in the substrate-binding pockets of the Nid systems appeared to play an important role in keeping aromatic substrates within the reactive distance from the iron atom, which allows each oxygen to attack the neighboring carbons. IMPORTANCE Since the discovery of microbial ring-hydroxylating oxygenases (RHOs) in 1970, the sequences, structures, and enzyme biochemistry, including enantiospecific products, of RHOs have been studied and discussed extensively from the perspective of biodegradation, biotransformation, and biocatalysis processes. However, with all that effort to elucidate the enzymatic mechanisms of RHOs, little is known about the biochemistry and enzymology underlying high-molecular-weight (HMW) polycyclic aromatic hydrocarbon (PAH) degradation. We used Mycobacterium vanbaalenii PYR-1 Nid enzymes, the first type V RHO members to display an apparent substrate preference for HMW PAHs. Here, we examine the mechanism of the RHO reaction by integrating structural information of the NidAB and NidA3B3 enzymes with substrate and product data. This study gives us an understanding of how the model RHO systems of M. vanbaalenii PYR-1 metabolize HMW PAHs. The information obtained would also be helpful for successful application of RHO enzymes to the production of industrially and medically important chiral chemicals and the development of PAH bioremediation technologies. Since the discovery of microbial ring-hydroxylating oxygenases (RHOs) in 1970, the sequences, structures, and enzyme biochemistry, including enantiospecific products, of RHOs have been studied and discussed extensively from the perspective of biodegradation, biotransformation, and biocatalysis processes. However, with all that effort to elucidate the enzymatic mechanisms of RHOs, little is known about the biochemistry and enzymology underlying high-molecular-weight (HMW) polycyclic aromatic hydrocarbon (PAH) degradation. We used Mycobacterium vanbaalenii PYR-1 Nid enzymes, the first type V RHO members to display an apparent substrate preference for HMW PAHs. Here, we examine the mechanism of the RHO reaction by integrating structural information of the NidAB and NidA3B3 enzymes with substrate and product data. This study gives us an understanding of how the model RHO systems of M. vanbaalenii PYR-1 metabolize HMW PAHs. The information obtained would also be helpful for successful application of RHO enzymes to the production of industrially and medically important chiral chemicals and the development of PAH bioremediation technologies.

Proteomic Applications to Elucidate Bacterial Aromatic Hydrocarbon Metabolic Pathways

The growing availability of genome sequences and advancement of high-throughput omics and analytical chemistry technologies have reinvigorated the study of biodegradation processes in aromatic hydrocarbon catabolism. In particular, proteomics approaches globally have identified and quantified bacterial enzymes responsible for aromatic hydrocarbon metabolism, which significantly assists in determining strategies for the biodegradation of aromatic hydrocarbons with applications in environmental bioremediation.

Identification and molecular characterization of a novel flavin-free NADPH preferred azoreductase encoded by azoB in Pigmentiphaga kullae K24

BackgroundMicrobial degradation of azo dyes is commonly initiated by the reduction of the azo bond(s) by a group of NADH or NADPH dependant azoreductases with many requiring flavin as a cofactor. In this study, we report the identification of a novel flavin-free NADPH preferred azoreductase encoded by azoB in Pigmentiphaga kullae K24.ResultsThe deduced amino acid sequence of azoB from P. kullae K24 showed 61% identity to a previously studied azoreductase (AzoA) from the same strain. azoB encoded a protein of 203 amino acids and heterologously expressed in Escherichia coli. The purified recombinant enzyme was a monomer with a molecular mass of 22 kDa. Both NADH and NADPH can be used as an electron donor for its activity with 4-(4-hydroxy-1-naphthylazo) benzenesulfonic acid (Orange I) as substrate. The apparent Km values for both NADH and Orange I were 170 and 8.6 μM, respectively. The Km of NADPH for the enzyme is 1.0 μM. When NADPH served as the electron donor, the activity of the enzyme is 63% higher than that when NADH was used. The pH and temperature optima for activity of the enzyme with Orange I as the substrate were at pH 6.0 and between 37 and 45°C. Phylogenetic analysis shows that AzoB belongs to the flavin-free azoreductase group which has a key fingerprint motif GXXGXXG for NAD(P)H binding at the N-terminus of the amino acid sequences. The 3D structure of AzoB was generated by comparative modeling approach. The structural combination of three conserved glycine residues (G7xxG10xxG13) in the pyrophosphate-binding loop with the Arg-32 explains the preference for NADPH of AzoB.ConclusionThe biochemical and structural properties of AzoB from P. kullae K24 revealed its preference for NADPH over NADH and it is a member of the monomeric flavin-free azoreductase group. Our studies show the substrate specificity of AzoB based on structure and cofactor requirement and the phylogenetic relationship among azoreductase groups.

Functional role of Trp-105 of Enterococcus faecalis azoreductase (AzoA) as resolved by structural and mutational analysis.

Enterococcus faecalis azoreductase (AzoA) is a very active enzyme with a broad spectrum of substrate specificity and is capable of degrading various azo dyes. The enzyme has an absolute requirement for reduced FMN, which delivers a total of four electrons from NADH to the substrate, resulting in the cleavage of the nitrogen double bond. In this study, we report the identification of amino acid residues critical for FMN binding in AzoA. FMN is stabilized by 22 amino acid residues, eight of which, Trp-105, Asn-106, Leu-107, Gly-150, Gly-151, Tyr-153, Asn-121 and Tyr-129, are involved in binding the FMN isoalloxazine ring. In silico analysis of the amino acid residues revealed that the Trp residue at position 105 of AzoA is the most likely significant contributor to the binding of FMN to the enzyme and is involved in FMN stabilization and destabilization. Site-directed mutagenesis analysis of Trp-105 was performed to determine the role of this amino acid residue in FMN binding and azo dye reductive activity. The mutant proteins were overexpressed in Escherichia coli and purified by anion-exchange and size-exclusion chromatography. The replacement of Trp-105 by the small side-chain amino acids Ala and Gly caused complete loss of both affinity for FMN and enzyme activity. Substitution of Tyr for Trp-105 did not significantly decrease the V(max) of the enzyme (22 % reduction). Substitutions with three bulky side-chain amino acids, Gln, Phe and His, produced enzymes with lower V(max) values (decreases of 68.2, 30.6 and 8.2-fold, respectively). However, these mutated enzymes maintained K(m) values similar to the wild-type enzyme. This study provides an insight into the catalytic properties of AzoA in FMN stabilization and enzyme activity.