Fetzner S

References (41)

Title : Stabilizing AqdC, a Pseudomonas Quinolone Signal-Cleaving Dioxygenase from Mycobacteria, by FRESCO-Based Protein Engineering - Wullich_2021_Chembiochem_22_733
Author(s) : Wullich SC , Wijma HJ , Janssen DB , Fetzner S
Ref : Chembiochem , 22 :733 , 2021
Abstract : The mycobacterial PQS dioxygenase AqdC, a cofactor-less protein with an alpha/beta-hydrolase fold, inactivates the virulence-associated quorum-sensing signal molecule 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS) produced by the opportunistic pathogen Pseudomonas aeruginosa and is therefore a potential anti-virulence tool. We have used computational library design to predict stabilizing amino acid replacements in AqdC. While 57 out of 91 tested single substitutions throughout the protein led to stabilization, as judged by increases in T/ of >2 degreesC, they all impaired catalytic activity. Combining substitutions, the proteins AqdC-G40K-A134L-G220D-Y238W and AqdC-G40K-G220D-Y238W showed extended half-lives and the best trade-off between stability and activity, with increases in T/ of 11.8 and 6.1 degreesC and relative activities of 22 and 72%, respectively, compared to AqdC. Molecular dynamics simulations and principal component analysis suggested that stabilized proteins are less flexible than AqdC, and the loss of catalytic activity likely correlates with an inability to effectively open the entrance to the active site.
ESTHER : Wullich_2021_Chembiochem_22_733
PubMedSearch : Wullich_2021_Chembiochem_22_733
PubMedID: 33058333
Gene_locus related to this paper: mycab-x8en65

Title : Enzyme-Mediated Quenching of the Pseudomonas Quinolone Signal (PQS): A Comparison between Naturally Occurring and Engineered PQS-Cleaving Dioxygenases - Arranz San Martin_2022_Biomolecules_12_170
Author(s) : Arranz San Martin A , Vogel J , Wullich SC , Quax WJ , Fetzner S
Ref : Biomolecules , 12 :170 , 2021
Abstract : The opportunistic pathogen Pseudomonas aeruginosa employs quorum sensing to govern the production of many virulence factors. Interference with quorum sensing signaling has therefore been put forward as an attractive approach to disarm this pathogen. Here, we analyzed the quorum quenching properties of natural and engineered (2-alkyl-)3-hydroxy-4(1H)-quinolone 2,4-dioxygenases (HQDs) that inactivate the P. aeruginosa signal molecule PQS (Pseudomonas quinolone signal; 2-heptyl-3-hydroxy-4(1H)-quinolone). When added exogenously to P. aeruginosa cultures, all HQDs tested significantly reduced the levels of PQS and other alkylquinolone-type secondary metabolites deriving from the biosynthetic pathway, such as the respiratory inhibitor 2-heptyl-4-hydroxyquinoline N-oxide. HQDs from Nocardia farcinica and Streptomyces bingchenggensis, which combine low KM values for PQS with thermal stability and resilience in the presence of P. aeruginosa exoproducts, respectively, attenuated production of the virulence factors pyocyanin and pyoverdine. A delay in mortality was observed when Galleria mellonella larvae were infected with P. aeruginosa suspensions treated with the S. bingchenggensis HQD or with inhibitors of alkylquinolone biosynthesis. Our data indicate that quenching of PQS signaling has potential as an anti-virulence strategy; however, an efficient anti-virulence therapy against P. aeruginosa likely requires a combination of agents addressing multiple targets
ESTHER : Arranz San Martin_2022_Biomolecules_12_170
PubMedSearch : Arranz San Martin_2022_Biomolecules_12_170
PubMedID:
Gene_locus related to this paper: artsp-hod , strbb-d7bw96 , mycab-x8en65 , nocfa-q5yp20

Title : Definition of an alpha\/beta-hydrolase fold subfamily comprising Pseudomonas quinolone signal cleaving dioxygenases - Wullich_2020_Appl.Environ.Microbiol__
Author(s) : Wullich SC , Arranz San Martin A , Fetzner S
Ref : Applied Environmental Microbiology , : , 2020
Abstract : The quinolone ring is a common core structure of natural products exhibiting antimicrobial, cytotoxic, and signaling activities. A prominent example is the Pseudomonas quinolone signal (PQS), a quorum sensing signal molecule involved in the regulation of virulence of P. aeruginosa The key reaction to quinolone inactivation and biodegradation is the cleavage of the 3-hydroxy-4(1H)-quinolone ring, catalyzed by dioxygenases (HQDs) which are members of the alpha/beta-hydrolase fold superfamily. The alpha/beta-hydrolase fold core domain consists of a beta-sheet surrounded by alpha-helices, with an active site usually containing a catalytic triad comprising a nucleophilic residue, an acidic residue, and a histidine. The nucleophile is located at the tip of a sharp turn called the "nucleophilic elbow". In this work, we developed a search workflow for the identification of HQD proteins from databases. Search and validation criteria include a [H-x(2)-W] motif at the nucleophilic elbow, a [HFP-x(4)-P] motif comprising the catalytic histidine, the presence of a helical cap domain, the positioning of the triad's acidic residue at the end of beta-strand 6, and a set of conserved hydrophobic residues contributing to the substrate cavity. The 161 candidate proteins identified from the UniProtKB database originate from environmental and plant-associated microorganisms from all domains of life. Verification and characterization of HQD activity of 9 new candidate proteins confirmed the reliability of the search strategy, and suggested residues correlating with distinct substrate preferences. Among the new HQDs, PQS dioxygenases from Nocardia farcinica, N. cyriacigeorgica, and Streptomyces bingchenggensis likely are part of a catabolic pathway for alkylquinolone utilization.ImportanceFunctional annotation of protein sequences is a major requirement for the investigation of metabolic pathways and the identification of sought-after biocatalysts. To identify heterocyclic ring-cleaving dioxygenases within the huge superfamily of alpha/beta-hydrolase fold proteins, we defined search and validation criteria for the primarily motif-based identification of 3-hydroxy-4(1H)-quinolone 2,4-dioxygenases (HQD). HQDs are key enzymes for the inactivation of metabolites which can have signaling, antimicrobial, or cytotoxic functions. The HQD candidates detected in this study occur particularly in environmental and plant-associated microorganisms. Because HQDs active towards the Pseudomonas quinolone signal (PQS) likely contribute to interactions within microbial communities and modulate the virulence of Pseudomonas aeruginosa, we analyzed the catalytic properties of a PQS-cleaving subset of HQDs, and specified characteristics to identify PQS-cleaving dioxygenases within the HQD family.
ESTHER : Wullich_2020_Appl.Environ.Microbiol__
PubMedSearch : Wullich_2020_Appl.Environ.Microbiol__
PubMedID: 32086305

Title : Structural basis for recognition and ring-cleavage of the Pseudomonas quinolone signal (PQS) by AqdC, a mycobacterial dioxygenase of the alpha\/beta-hydrolase fold family - Wullich_2019_J.Struct.Biol_207_287
Author(s) : Wullich SC , Kobus S , Wienhold M , Hennecke U , Smits SHJ , Fetzner S
Ref : J Struct Biol , 207 :287 , 2019
Abstract : The cofactor-less dioxygenase AqdC of Mycobacteroides abscessus catalyzes the cleavage and thus inactivation of the Pseudomonas quinolone signal (PQS, 2-heptyl-3-hydroxy-4(1H)-quinolone), which plays a central role in the regulation of virulence factor production by Pseudomonas aeruginosa. We present here the crystal structures of AqdC in its native state and in complex with the PQS cleavage product N-octanoylanthranilic acid, and of mutant AqdC proteins in complex with PQS. AqdC possesses an alpha/beta-hydrolase fold core domain with additional helices forming a cap domain. The protein is traversed by a bipartite tunnel, with a funnel-like entry section leading to an elliptical substrate cavity where PQS positioning is mediated by a combination of hydrophobic interactions and hydrogen bonds, with the substrate's C4 carbonyl and C3 hydroxyl groups tethered by His97 and the catalytic His246, respectively. The side chain of the AqdC-bound product extends deeper into the "alkyl tail section" of the tunnel than PQS, tentatively suggesting product exit via this part of the tunnel. AqdC prefers PQS over congeners with shorter alkyl substituents at C2. Kinetic data confirmed the strict requirement of the active-site base His246 for catalysis, and suggested that evolution of the canonical nucleophile/His/Asp catalytic triad of the hydrolases to an Ala/His/Asp triad is favorable for catalyzing dioxygenolytic PQS ring cleavage.
ESTHER : Wullich_2019_J.Struct.Biol_207_287
PubMedSearch : Wullich_2019_J.Struct.Biol_207_287
PubMedID: 31228546
Gene_locus related to this paper: mycab-x8en65

Title : Synthesis and biological activity of methylated derivatives of the Pseudomonas metabolites HHQ, HQNO and PQS - Thierbach_2019_Beilstein.J.Org.Chem_15_187
Author(s) : Thierbach S , Wienhold M , Fetzner S , Hennecke U
Ref : Beilstein J Org Chem , 15 :187 , 2019
Abstract : Selectively methylated analogues of naturally occurring 2-heptyl-4(1H)-quinolones, which are alkaloids common within the Rutaceae family and moreover are associated with quorum sensing and virulence of the human pathogen Pseudomonas aeruginosa, have been prepared. While the synthesis by direct methylation was successful for 3-unsubstituted 2-heptyl-4(1H)-quinolones, methylated derivatives of the Pseudomonas quinolone signal (PQS) were synthesized from 3-iodinated quinolones by methylation and iodine-metal exchange/oxidation. The two N- and O-methylated derivatives of the PQS showed strong quorum sensing activity comparable to that of PQS itself. Staphylococcus aureus, another pathogenic bacterium often co-occurring with P. aeruginosa especially in the lung of cystic fibrosis patients, was inhibited in planktonic growth and cellular respiration by the 4-O-methylated derivatives of HQNO and HHQ, respectively.
ESTHER : Thierbach_2019_Beilstein.J.Org.Chem_15_187
PubMedSearch : Thierbach_2019_Beilstein.J.Org.Chem_15_187
PubMedID: 30745993

Title : Chemical Modification and Detoxification of the Pseudomonas aeruginosa Toxin 2-Heptyl-4-hydroxyquinoline N-Oxide by Environmental and Pathogenic Bacteria - Thierbach_2017_ACS.Chem.Biol_12_2305
Author(s) : Thierbach S , Birmes FS , Letzel MC , Hennecke U , Fetzner S
Ref : ACS Chemical Biology , 12 :2305 , 2017
Abstract : 2-Heptyl-4-hydroxyquinoline N-oxide (HQNO), a major secondary metabolite and virulence factor produced by the opportunistic pathogen Pseudomonas aeruginosa, acts as a potent inhibitor of respiratory electron transfer and thereby affects host cells as well as microorganisms. In this study, we demonstrate the previously unknown capability of environmental and pathogenic bacteria to transform and detoxify this compound. Strains of Arthrobacter and Rhodococcus spp. as well as Staphylococcus aureus introduced a hydroxyl group at C-3 of HQNO, whereas Mycobacterium abscessus, M. fortuitum, and M. smegmatis performed an O-methylation, forming 2-heptyl-1-methoxy-4-oxoquinoline as the initial metabolite. Bacillus spp. produced the glycosylated derivative 2-heptyl-1-(beta-d-glucopyranosydyl)-4-oxoquinoline. Assaying the effects of these metabolites on cellular respiration and on quinol oxidase activity of membrane fractions revealed that their EC50 values were up to 2 orders of magnitude higher than that of HQNO. Furthermore, cellular levels of reactive oxygen species were significantly lower in the presence of the metabolites than under the influence of HQNO. Therefore, the capacity to transform HQNO should lead to a competitive advantage against P. aeruginosa. Our findings contribute new insight into the metabolic diversity of bacteria and add another layer of complexity to the metabolic interactions which likely contribute to shaping polymicrobial communities comprising P. aeruginosa.
ESTHER : Thierbach_2017_ACS.Chem.Biol_12_2305
PubMedSearch : Thierbach_2017_ACS.Chem.Biol_12_2305
PubMedID: 28708374

Title : Mycobacterium abscessus subsp. abscessus Is Capable of Degrading Pseudomonas aeruginosa Quinolone Signals - Birmes_2017_Front.Microbiol_8_339
Author(s) : Birmes FS , Wolf T , Kohl TA , Ruger K , Bange F , Kalinowski J , Fetzner S
Ref : Front Microbiol , 8 :339 , 2017
Abstract : Pseudomonas aeruginosa employs 2-heptyl-3-hydroxy-4(1H)-quinolone (the Pseudomonas quinolone signal, PQS) and 2-heptyl-4(1H)-quinolone (HHQ) as quorum sensing signal molecules, which contribute to a sophisticated regulatory network controlling the production of virulence factors and antimicrobials. We demonstrate that Mycobacterium abscessus(T) and clinical M. abscessus isolates are capable of degrading these alkylquinolone signals. Genome sequences of 50 clinical M. abscessus isolates indicated the presence of aqdRABC genes, contributing to fast degradation of HHQ and PQS, in M. abscessus subsp. abscessus strains, but not in M. abscessus subsp. bolletii and M. abscessus subsp. massiliense isolates. A subset of 18 M. a. subsp. abscessus isolates contained the same five single nucleotide polymorphisms (SNPs) compared to the aqd region of the type strain. Interestingly, representatives of these isolates showed faster PQS degradation kinetics than the M. abscessus type strain. One of the SNPs is located in the predicted promoter region of the aqdR gene encoding a putative transcriptional regulator, and two others lead to a variant of the AqdC protein termed AqdC(II), which differs in two amino acids from AqdC(I) of the type strain. AqdC, the key enzyme of the degradation pathway, is a PQS dioxygenase catalyzing quinolone ring cleavage. While transcription of aqdR and aqdC is induced by PQS, transcript levels in a representative of the subset of 18 isolates were not significantly altered despite the detected SNP in the promoter region. However, purified recombinant AqdC(II) and AqdC(I) exhibit different kinetic properties, with approximate apparent Km values for PQS of 14 muM and 37 muM, and kcat values of 61 s(-1) and 98 s(-1), respectively, which may (at least in part) account for the observed differences in PQS degradation rates of the strains. In co-culture experiments of P. aeruginosa PAO1 and M. abscessus, strains harboring the aqd genes reduced the PQS levels, whereas mycobacteria lacking the aqd gene cluster even boosted PQS production. The results suggest that the presence and expression of the aqd genes in M. abscessus lead to a competitive advantage against P. aeruginosa.
ESTHER : Birmes_2017_Front.Microbiol_8_339
PubMedSearch : Birmes_2017_Front.Microbiol_8_339
PubMedID: 28303132
Gene_locus related to this paper: rhoer-aqdC1 , rhoer-aqdC2

Title : Enzyme-Mediated Quenching of the Pseudomonas Quinolone Signal (PQS) Promotes Biofilm Formation of Pseudomonas aeruginosa by Increasing Iron Availability - Tettmann_2016_Front.Microbiol_7_1978
Author(s) : Tettmann B , Niewerth C , Kirschhofer F , Neidig A , Dotsch A , Brenner-Weiss G , Fetzner S , Overhage J
Ref : Front Microbiol , 7 :1978 , 2016
Abstract : The 2-alkyl-3-hydroxy-4(1H)-quinolone 2,4-dioxygenase HodC was previously described to cleave the Pseudomonas quinolone signal, PQS, which is exclusively used in the complex quorum sensing (QS) system of Pseudomonas aeruginosa, an opportunistic pathogen employing QS to regulate virulence and biofilm development. Degradation of PQS by exogenous addition of HodC to planktonic cells of P. aeruginosa attenuated production of virulence factors, and reduced virulence in planta. However, proteolytic cleavage reduced the efficacy of HodC. Here, we identified the secreted protease LasB of P. aeruginosa to be responsible for HodC degradation. In static biofilms of the P. aeruginosa PA14 lasB::Tn mutant, the catalytic activity of HodC led to an increase in viable biomass in newly formed but also in established biofilms, and reduced the expression of genes involved in iron metabolism and siderophore production, such as pvdS, pvdL, pvdA, and pvdQ. This is likely due to an increase in the levels of bioavailable iron by degradation of PQS, which is able to sequester iron from the surrounding environment. Thus, HodC, despite its ability to quench the production of virulence factors, is contraindicated for combating P. aeruginosa biofilms.
ESTHER : Tettmann_2016_Front.Microbiol_7_1978
PubMedSearch : Tettmann_2016_Front.Microbiol_7_1978
PubMedID: 28018312

Title : PqsE of Pseudomonas aeruginosa Acts as Pathway-Specific Thioesterase in the Biosynthesis of Alkylquinolone Signaling Molecules - Drees_2015_Chem.Biol_22_611
Author(s) : Drees SL , Fetzner S
Ref : Chemical Biology , 22 :611 , 2015
Abstract : Pseudomonas aeruginosa uses the alkylquinolones PQS (2-heptyl-3-hydroxy-4(1H)-quinolone) and HHQ (2-heptyl-4(1H)-quinolone) as quorum-sensing signal molecules, controlling the expression of many virulence genes as a function of cell population density. The biosynthesis of HHQ is generally accepted to require the pqsABCD gene products. We now reconstitute the biosynthetic pathway in vitro, and demonstrate that in addition to PqsABCD, PqsE has a role in HHQ synthesis. PqsE acts as thioesterase, hydrolyzing the biosynthetic intermediate 2-aminobenzoylacetyl-coenzyme A to form 2-aminobenzoylacetate, the precursor of HHQ and 2-aminoacetophenone. The role of PqsE can be taken over to some extent by the broad-specificity thioesterase TesB, explaining why the pqsE deletion mutant of P. aeruginosa still synthesizes HHQ. Interestingly, the pqsE mutant produces increased levels of 2,4-dihydroxyquinoline, resulting from intramolecular cyclization of 2-aminobenzoylacetyl-coenzyme A. Overall, our data suggest that PqsE promotes the efficiency of alkylquinolone signal molecule biosynthesis in P. aeruginosa and balances the levels of secondary metabolites deriving from the alkylquinolone biosynthetic pathway.
ESTHER : Drees_2015_Chem.Biol_22_611
PubMedSearch : Drees_2015_Chem.Biol_22_611
PubMedID: 25960261

Title : Biotic inactivation of the Pseudomonas aeruginosa quinolone signal molecule - Soh_2015_Environ.Microbiol_17_4352
Author(s) : Soh EY , Chhabra SR , Halliday N , Heeb S , Muller C , Birmes FS , Fetzner S , Camara M , Chan KG , Williams P
Ref : Environ Microbiol , 17 :4352 , 2015
Abstract : In Pseudomonas aeruginosa, quorum sensing (QS) regulates the production of secondary metabolites, many of which are antimicrobials that impact on polymicrobial community composition. Consequently, quenching QS modulates the environmental impact of P. aeruginosa. To identify bacteria capable of inactivating the QS signal molecule 2-heptyl-3-hydroxy-4(1H)-quinolone (PQS), a minimal medium containing PQS as the sole carbon source was used to enrich a Malaysian rainforest soil sample. This yielded an Achromobacter xylosoxidans strain (Q19) that inactivated PQS, yielding a new fluorescent compound (I-PQS) confirmed as PQS-derived using deuterated PQS. The I-PQS structure was elucidated using mass spectrometry and nuclear magnetic resonance spectroscopy as 2-heptyl-2-hydroxy-1,2-dihydroquinoline-3,4-dione (HHQD). Achromobacter xylosoxidans Q19 oxidized PQS congeners with alkyl chains ranging from C1 to C5 and also N-methyl PQS, yielding the corresponding 2-hydroxy-1,2-dihydroquinoline-3,4-diones, but was unable to inactivate the PQS precursor HHQ. This indicates that the hydroxyl group at position 3 in PQS is essential and that A. xylosoxidans inactivates PQS via a pathway involving the incorporation of oxygen at C2 of the heterocyclic ring. The conversion of PQS to HHQD also occurred on incubation with 12/17 A. xylosoxidans strains recovered from cystic fibrosis patients, with P. aeruginosa and with Arthrobacter, suggesting that formation of hydroxylated PQS may be a common mechanism of inactivation.
ESTHER : Soh_2015_Environ.Microbiol_17_4352
PubMedSearch : Soh_2015_Environ.Microbiol_17_4352
PubMedID: 25809238

Title : Rhodococcus erythropolis BG43 Genes Mediating Pseudomonas aeruginosa Quinolone Signal Degradation and Virulence Factor Attenuation - Muller_2015_Appl.Environ.Microbiol_81_7720
Author(s) : Muller C , Birmes FS , Ruckert C , Kalinowski J , Fetzner S
Ref : Applied Environmental Microbiology , 81 :7720 , 2015
Abstract : Rhodococcus erythropolis BG43 is able to degrade the Pseudomonas aeruginosa quorum sensing signal molecules PQS (Pseudomonas quinolone signal) [2-heptyl-3-hydroxy-4(1H)-quinolone] and HHQ [2-heptyl-4(1H)-quinolone] to anthranilic acid. Based on the hypothesis that degradation of HHQ might involve hydroxylation to PQS followed by dioxygenolytic cleavage of the heterocyclic ring and hydrolysis of the resulting N-octanoylanthranilate, the genome was searched for corresponding candidate genes. Two gene clusters, aqdA1B1C1 and aqdA2B2C2, each predicted to code for a hydrolase, a flavin monooxygenase, and a dioxygenase related to 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, were identified on circular plasmid pRLCBG43 of strain BG43. Transcription of all genes was upregulated by PQS, suggesting that both gene clusters code for alkylquinolone-specific catabolic enzymes. An aqdR gene encoding a putative transcriptional regulator, which was also inducible by PQS, is located adjacent to the aqdA2B2C2 cluster. Expression of aqdA2B2C2 in Escherichia coli conferred the ability to degrade HHQ and PQS to anthranilic acid; however, for E. coli transformed with aqdA1B1C1, only PQS degradation was observed. Purification of the recombinant AqdC1 protein verified that it catalyzes the cleavage of PQS to form N-octanoylanthranilic acid and carbon monoxide and revealed apparent Km and kcat values for PQS of approximately 27 muM and 21 s(-1), respectively. Heterologous expression of the PQS dioxygenase gene aqdC1 or aqdC2 in P. aeruginosa PAO1 quenched the production of the virulence factors pyocyanin and rhamnolipid and reduced the synthesis of the siderophore pyoverdine. Thus, the toolbox of quorum-quenching enzymes is expanded by new PQS dioxygenases.
ESTHER : Muller_2015_Appl.Environ.Microbiol_81_7720
PubMedSearch : Muller_2015_Appl.Environ.Microbiol_81_7720
PubMedID: 26319870
Gene_locus related to this paper: rhoer-aqdC1 , rhoer-aqdC2 , rhoer-aqda1

Title : Complete genome sequence of Rhodococcus erythropolis BG43 (DSM 46869), a degrader of Pseudomonas aeruginosa quorum sensing signal molecules - Ruckert_2015_J.Biotechnol_211_99
Author(s) : Ruckert C , Birmes FS , Muller C , Niewerth H , Winkler A , Fetzner S , Kalinowski J
Ref : J Biotechnol , 211 :99 , 2015
Abstract : Rhodococcus erythropolis BG43 was isolated from soil and characterized as a degrader of the quorum sensing signal molecules 2-heptyl-3-hydroxy-4(1H)-quinolone (the Pseudomonas quinolone signal, PQS) and 2-heptyl-4(1H)-quinolone, produced by Pseudomonas aeruginosa. The complete genome of R. erythropolis BG43 consists of a circular chromosome and three plasmids, one of them circular and two linear ones. In total, 6158 protein-coding regions were identified. With this genome sequence, the genetic basis of its quorum-quenching ability and possible biotechnological applications can be explored further.
ESTHER : Ruckert_2015_J.Biotechnol_211_99
PubMedSearch : Ruckert_2015_J.Biotechnol_211_99
PubMedID: 26210289
Gene_locus related to this paper: rhoer-aqdC2 , rhoer-aqda1

Title : Crystal structure analysis of EstA from Arthrobacter sp. Rue61a--an insight into catalytic promiscuity - Wagner_2014_FEBS.Lett_588_1154
Author(s) : Wagner UG , DiMaio F , Kolkenbrock S , Fetzner S
Ref : FEBS Letters , 588 :1154 , 2014
Abstract : In this article we analyze the reasons for catalytic promiscuity of a type VIII esterase with beta-lactamase fold and the ability to cleave beta-lactams. We compared the structure of this enzyme to those of an esterase of the same type without any lactamase ability, an esterase with moderate lactamase ability, and a class C beta-lactamase with similar fold. Our results show that for these enzymes, the difference in the substrate specificity is sterically driven.
ESTHER : Wagner_2014_FEBS.Lett_588_1154
PubMedSearch : Wagner_2014_FEBS.Lett_588_1154
PubMedID: 24613918

Title : Conversion of the Pseudomonas aeruginosa Quinolone Signal and Related Alkylhydroxyquinolines by Rhodococcus sp. Strain BG43 - Muller_2014_Appl.Environ.Microbiol_80_7266
Author(s) : Muller C , Birmes FS , Niewerth H , Fetzner S
Ref : Applied Environmental Microbiology , 80 :7266 , 2014
Abstract : A bacterial strain, which based on the sequences of its 16S rRNA, gyrB, catA, and qsdA genes, was identified as a Rhodococcus sp. closely related to Rhodococcus erythropolis, was isolated from soil by enrichment on the Pseudomonas quinolone signal [PQS; 2-heptyl-3-hydroxy-4(1H)-quinolone], a quorum sensing signal employed by the opportunistic pathogen Pseudomonas aeruginosa. The isolate, termed Rhodococcus sp. strain BG43, cometabolically degraded PQS and its biosynthetic precursor 2-heptyl-4(1H)-quinolone (HHQ) to anthranilic acid. HHQ degradation was accompanied by transient formation of PQS, and HHQ hydroxylation by cell extracts required NADH, indicating that strain BG43 has a HHQ monooxygenase isofunctional to the biosynthetic enzyme PqsH of P. aeruginosa. The enzymes catalyzing HHQ hydroxylation and PQS degradation were inducible by PQS, suggesting a specific pathway. Remarkably, Rhodococcus sp. BG43 is also capable of transforming 2-heptyl-4-hydroxyquinoline-N-oxide to PQS. It thus converts an antibacterial secondary metabolite of P. aeruginosa to a quorum sensing signal molecule.
ESTHER : Muller_2014_Appl.Environ.Microbiol_80_7266
PubMedSearch : Muller_2014_Appl.Environ.Microbiol_80_7266
PubMedID: 25239889
Gene_locus related to this paper: rhoer-aqdC1 , rhoer-aqdC2

Title : Substrate-assisted O2 activation in a cofactor-independent dioxygenase - Thierbach_2014_Chem.Biol_21_217
Author(s) : Thierbach S , Bui N , Zapp J , Chhabra SR , Kappl R , Fetzner S
Ref : Chemical Biology , 21 :217 , 2014
Abstract : In contrast to the majority of O2-activating enzymes, which depend on an organic cofactor or a metal ion for catalysis, a particular group of structurally unrelated oxygenases is functional without any cofactor. In this study, we characterized the mechanism of O2 activation in the reaction pathway of a cofactor-independent dioxygenase with an alpha/beta-hydrolase fold, which catalyzes the oxygenolytic cleavage of 2-alkyl-3-hydroxy-4(1H)-quinolones. Chemical analysis and electron paramagnetic resonance spectroscopic data revealed that O2 activation in the enzyme's active site is substrate-assisted, relying on single electron transfer from the bound substrate anion to O2 to form a radical pair, which recombines to a C2-peroxide intermediate. Thus, an oxygenase can function without a cofactor, if the organic substrate itself, after activation to a (carb)anion by an active-site base, is intrinsically reactive toward molecular oxygen.
ESTHER : Thierbach_2014_Chem.Biol_21_217
PubMedSearch : Thierbach_2014_Chem.Biol_21_217
PubMedID: 24388758

Title : Complete genome sequence and metabolic potential of the quinaldine-degrading bacterium Arthrobacter sp. Rue61a - Niewerth_2012_BMC.Genomics_13_534
Author(s) : Niewerth H , Schuldes J , Parschat K , Kiefer P , Vorholt JA , Daniel R , Fetzner S
Ref : BMC Genomics , 13 :534 , 2012
Abstract : BACKGROUND: Bacteria of the genus Arthrobacter are ubiquitous in soil environments and can be considered as true survivalists. Arthrobacter sp. strain Rue61a is an isolate from sewage sludge able to utilize quinaldine (2-methylquinoline) as sole carbon and energy source. The genome provides insight into the molecular basis of the versatility and robustness of this environmental Arthrobacter strain.
RESULTS: The genome of Arthrobacter sp. Rue61a consists of a single circular chromosome of 4,736,495 bp with an average G + C content of 62.32%, the circular 231,551-bp plasmid pARUE232, and the linear 112,992-bp plasmid pARUE113 that was already published. Plasmid pARUE232 is proposed to contribute to the resistance of Arthrobacter sp. Rue61a to arsenate and Pb2+, whereas the linear plasmid confers the ability to convert quinaldine to anthranilate. Remarkably, degradation of anthranilate exclusively proceeds via a CoA-thioester pathway. Apart from quinaldine utilization, strain Rue61a has a limited set of aromatic degradation pathways, enabling the utilization of 4-hydroxy-substituted aromatic carboxylic acids, which are characteristic products of lignin depolymerization, via ortho cleavage of protocatechuate. However, 4-hydroxyphenylacetate degradation likely proceeds via meta cleavage of homoprotocatechuate. The genome of strain Rue61a contains numerous genes associated with osmoprotection, and a high number of genes coding for transporters. It encodes a broad spectrum of enzymes for the uptake and utilization of various sugars and organic nitrogen compounds. A. aurescens TC-1 is the closest sequenced relative of strain Rue61a.
CONCLUSIONS: The genome of Arthrobacter sp. Rue61a reflects the saprophytic lifestyle and nutritional versatility of the organism and a strong adaptive potential to environmental stress. The circular plasmid pARUE232 and the linear plasmid pARUE113 contribute to heavy metal resistance and to the ability to degrade quinaldine, respectively.
ESTHER : Niewerth_2012_BMC.Genomics_13_534
PubMedSearch : Niewerth_2012_BMC.Genomics_13_534
PubMedID: 23039946
Gene_locus related to this paper: artat-a1r5d9 , artat-a1r6c9 , 9micc-j7lr62 , 9micc-j7lsz4

Title : Hydrolase-like properties of a cofactor-independent dioxygenase - Thierbach_2012_Chembiochem_13_1125
Author(s) : Thierbach S , Buldt-Karentzopoulos K , Dreiling A , Hennecke U , Konig S , Fetzner S
Ref : Chembiochem , 13 :1125 , 2012
Abstract : Mechanistic promiscuity: The (2-alkyl)-3-hydroxy-4(1H)-quinolone-cleaving dioxygenase Hod has an alpha/beta-hydrolase fold and a Ser/His/Asp triad in its active site. Isatoic anhydride, a suicide substrate of serine hydrolases, inactivates Hod by covalent modification of the active-site serine, thus indicating that the alpha/beta-hydrolase fold can accommodate dioxygenase chemistry without completely abandoning hydrolase-like properties.
ESTHER : Thierbach_2012_Chembiochem_13_1125
PubMedSearch : Thierbach_2012_Chembiochem_13_1125
PubMedID: 22549932

Title : Structural basis for cofactor-independent dioxygenation of N-heteroaromatic compounds at the alpha\/beta-hydrolase fold - Steiner_2010_Proc.Natl.Acad.Sci.U.S.A_107_657
Author(s) : Steiner RA , Janssen HJ , Roversi P , Oakley AJ , Fetzner S
Ref : Proc Natl Acad Sci U S A , 107 :657 , 2010
Abstract : Enzymatic catalysis of oxygenation reactions in the absence of metal or organic cofactors is a considerable biochemical challenge. The CO-forming 1-H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) from Arthrobacter nitroguajacolicus Ru61a and 1-H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (QDO) from Pseudomonas putida 33/1 are homologous cofactor-independent dioxygenases involved in the breakdown of N-heteroaromatic compounds. To date, they are the only dioxygenases suggested to belong to the alpha/beta-hydrolase fold superfamily. Members of this family typically catalyze hydrolytic processes rather than oxygenation reactions. We present here the crystal structures of both HOD and QDO in their native state as well as the structure of HOD in complex with its natural 1-H-3-hydroxy-4-oxoquinaldine substrate, its N-acetylanthranilate reaction product, and chloride as dioxygen mimic. HOD and QDO are structurally very similar. They possess a classical alpha/beta-hydrolase fold core domain additionally equipped with a cap domain. Organic substrates bind in a preorganized active site with an orientation ideally suited for selective deprotonation of their hydroxyl group by a His/Asp charge-relay system affording the generation of electron-donating species. The "oxyanion hole" of the alpha/beta-hydrolase fold, typically employed to stabilize the tetrahedral intermediate in ester hydrolysis reactions, is utilized here to host and control oxygen chemistry, which is proposed to involve a peroxide anion intermediate. Product release by proton back transfer from the catalytic histidine is driven by minimization of intramolecular charge repulsion. Structural and kinetic data suggest a nonnucleophilic general-base mechanism. Our analysis provides a framework to explain cofactor-independent dioxygenation within a protein architecture generally employed to catalyze hydrolytic reactions.
ESTHER : Steiner_2010_Proc.Natl.Acad.Sci.U.S.A_107_657
PubMedSearch : Steiner_2010_Proc.Natl.Acad.Sci.U.S.A_107_657
PubMedID: 20080731
Gene_locus related to this paper: artsp-hod , psepu-QDO

Title : Cofactor-independent oxidases and oxygenases - Fetzner_2010_Appl.Microbiol.Biotechnol_86_791
Author(s) : Fetzner S , Steiner RA
Ref : Applied Microbiology & Biotechnology , 86 :791 , 2010
Abstract : Whereas the majority of O(2)-metabolizing enzymes depend on transition metal ions or organic cofactors for catalysis, a significant number of oxygenases and oxidases neither contain nor require any cofactor. Among the cofactor-independent oxidases, urate oxidase, coproporphyrinogen oxidase, and formylglycine-generating enzyme are of mechanistic as well as medical interest. Formylglycine-generating enzyme is also a promising tool for protein engineering as it can be used to equip proteins with a reactive aldehyde function. PqqC, an oxidase in the biosynthesis of the bacterial cofactor pyrroloquinoline quinone, catalyzes an eight-electron ring-closure oxidation reaction. Among bacterial oxygenases, quinone-forming monooxygenases involved in the tailoring of polyketides, the dioxygenase DpgC found in the biosynthesis of a building block of vancomycin and teicoplanin antibiotics, luciferase monooxygenase from Renilla sp., and bacterial ring-cleaving 2,4-dioxygenases active towards 3-hydroxy-4(1H)-quinolones have been identified as cofactor-independent enzymes. Interestingly, the 3-hydroxy-4(1H)-quinolone 2,4-dioxygenases as well as Renilla luciferase use an alpha/beta-hydrolase architecture for oxygenation reactions. Cofactor-independent oxygenases and oxidases catalyze very different reactions and belong to several different protein families, reflecting their diverse origin. Nevertheless, they all may share the common mechanistic concept of initial base-catalyzed activation of their organic substrate and "substrate-assisted catalysis".
ESTHER : Fetzner_2010_Appl.Microbiol.Biotechnol_86_791
PubMedSearch : Fetzner_2010_Appl.Microbiol.Biotechnol_86_791
PubMedID: 20157809

Title : Dioxygenase-mediated quenching of quinolone-dependent quorum sensing in Pseudomonas aeruginosa - Pustelny_2009_Chem.Biol_16_1259
Author(s) : Pustelny C , Albers A , Buldt-Karentzopoulos K , Parschat K , Chhabra SR , Camara M , Williams P , Fetzner S
Ref : Chemical Biology , 16 :1259 , 2009
Abstract : 2-Heptyl-3-hydroxy-4(1H)-quinolone (PQS) is a quorum-sensing signal molecule used by Pseudomonas aeruginosa. The structural similarity between 3-hydroxy-2-methyl-4(1H)-quinolone, the natural substrate for the 2,4-dioxygenase, Hod, and PQS prompted us to investigate whether Hod quenched PQS signaling. Hod is capable of catalyzing the conversion of PQS to N-octanoylanthranilic acid and carbon monoxide. In P. aeruginosa PAO1 cultures, exogenously supplied Hod protein reduced expression of the PQS biosynthetic gene pqsA, expression of the PQS-regulated virulence determinants lectin A, pyocyanin, and rhamnolipids, and virulence in planta. However, the proteolytic cleavage of Hod by extracellular proteases, competitive inhibition by the PQS precursor 2-heptyl-4(1H)-quinolone, and PQS binding to rhamnolipids reduced the efficiency of Hod as a quorum-quenching agent. Nevertheless, these data indicate that enzyme-mediated PQS inactivation has potential as an antivirulence strategy against P. aeruginosa.
ESTHER : Pustelny_2009_Chem.Biol_16_1259
PubMedSearch : Pustelny_2009_Chem.Biol_16_1259
PubMedID: 20064436

Title : EstA from Arthrobacter nitroguajacolicus Ru61a, a thermo- and solvent-tolerant carboxylesterase related to class C beta-lactamases - Schutte_2007_Curr.Microbiol_54_230
Author(s) : Schutte M , Fetzner S
Ref : Curr Microbiol , 54 :230 , 2007
Abstract : The estA gene encoding a novel cytoplasmic carboxylesterase from Arthrobacter nitroguajacolicus Ru61a was expressed in Escherichia coli. Sequence analysis and secondary structure predictions suggested that EstA belongs to the family VIII esterases, which are related to class C beta-lactamases. The S-x-x-K motif that in beta-lactamases contains the catalytic nucleophile, and a putative active-site tyrosine residue are conserved in EstA. The native molecular mass of hexahistidine-tagged (His6) EstA, purified by metal chelate affinity chromatography, was estimated to be 95 kDa by gel filtration, whereas the His6EstA peptide has a calculated molecular mass of 42.1 kDa. The enzyme catalyzes the hydrolysis of short-chain phenylacyl esters and triglycerides, and shows weak activity toward 2-hydroxy- and 2-nitroacetanilide. Its catalytic activity was inhibited by the serine-specific effector phenylmethylsulfonyl fluoride, and by Cd2+ and Hg2+ ions. Maximum activity of His6EstA was observed at a pH of 9.5 and a temperature of 50 degrees C to 60 degrees C. The enzyme was fairly thermostable. After 19 days at 50 degrees C and after 24 hours at 60 degrees C, its residual relative esterase activity toward phenylacetate was still 53% and 30%, respectively. Exposure of His6EstA to buffer-solvent mixtures showed that the enzyme was inactivated by several high log P (hydrophobic) solvents, whereas it showed remarkable stability and activity in up to 30% (by volume) of polar (low log P) organic solvents such as dimethylsulfoxide, methanol, acetonitrile, acetone, and propanol.
ESTHER : Schutte_2007_Curr.Microbiol_54_230
PubMedSearch : Schutte_2007_Curr.Microbiol_54_230
PubMedID: 17294326

Title : Cofactor-independent oxygenases go it alone -
Author(s) : Fetzner S
Ref : Nat Chemical Biology , 3 :374 , 2007
PubMedID: 17576423

Title : A new monocupin quercetinase of Streptomyces sp. FLA: identification and heterologous expression of the queD gene and activity of the recombinant enzyme towards different flavonols - Merkens_2007_Arch.Microbiol_187_475
Author(s) : Merkens H , Sielker S , Rose K , Fetzner S
Ref : Arch Microbiol , 187 :475 , 2007
Abstract : The gene queD encoding quercetinase of Streptomyces sp. FLA, a soil isolate related to S. eurythermus (T), was identified. Quercetinases catalyze the 2,4-dioxygenolytic cleavage of 3,5,7,3',4'-pentahydroxyflavone to 2-protocatechuoylphloroglucinol carboxylic acid and carbon monoxide. The queD gene was expressed in S. lividans and E. coli, and the recombinant hexahistidine-tagged protein (QueDHis(6)) was purified. Several flavonols were converted by QueDHis(6), whereas CO formation from the 2,3-dihydroflavonol taxifolin and the flavone luteolin were not observed. In contrast to bicupin quercetinases from Aspergillus japonicus and Bacillus subtilis, and bicupin pirins showing quercetinase activity, QueD of strain FLA is a monocupin exhibiting 35.9% sequence identity to the C-terminal domain of B. subtilis quercetinase. Its native molecular mass of 63 kDa suggests a multimeric protein. A queD-specific probe hybridized with fragments of genomic DNA of four other quercetin degrading Streptomyces strains, but not with DNA of B. subtilis. Potential ORFs upstream of queD probably code for a serine protease and an endoribonuclease; two ORFs downstream of queD may encode an amidohydrolase and a carboxylesterase. This arrangement suggests that queD is not part of a catabolic gene cluster. Quercetinases might play a major role as detoxifying rather than catabolic enzymes.
ESTHER : Merkens_2007_Arch.Microbiol_187_475
PubMedSearch : Merkens_2007_Arch.Microbiol_187_475
PubMedID: 17516049

Title : Crystallization and preliminary X-ray analysis of 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase from Arthrobacter nitroguajacolicus Ru61a: a cofactor-devoid dioxygenase of the alpha\/beta-hydrolase-fold superfamily - Steiner_2007_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_63_382
Author(s) : Steiner RA , Frerichs-Deeken U , Fetzner S
Ref : Acta Crystallographica Sect F Struct Biol Cryst Commun , 63 :382 , 2007
Abstract : 1H-3-Hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) is a cofactor-devoid dioxygenase that is involved in the anthranilate pathway of quinaldine degradation. HOD has been proposed to belong to the alpha/beta-hydrolase-fold superfamily of enzymes. N-terminally His6-tagged HOD has been crystallized by the hanging-drop vapour-diffusion method using sodium/potassium tartrate as a precipitant and CuCl2 as an additive. The structure was solved by the single anomalous dispersion (SAD) technique using data collected to 3.5 A resolution at the Cu absorption peak wavelength. The crystals belong to the primitive tetragonal space group P4(3)2(1)2, with unit-cell parameters a = b = 153.788, c = 120.872 A.
ESTHER : Steiner_2007_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_63_382
PubMedSearch : Steiner_2007_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_63_382
PubMedID: 17565176
Gene_locus related to this paper: artsp-hod

Title : Complete nucleotide sequence of the 113-kilobase linear catabolic plasmid pAL1 of Arthrobacter nitroguajacolicus Ru61a and transcriptional analysis of genes involved in quinaldine degradation - Parschat_2007_J.Bacteriol_189_3855
Author(s) : Parschat K , Overhage J , Strittmatter AW , Henne A , Gottschalk G , Fetzner S
Ref : Journal of Bacteriology , 189 :3855 , 2007
Abstract : The nucleotide sequence of the linear catabolic plasmid pAL1 from the 2-methylquinoline (quinaldine)-degrading strain Arthrobacter nitroguajacolicus Ru61a comprises 112,992 bp. A total of 103 open reading frames (ORFs) were identified on pAL1, 49 of which had no annotatable function. The ORFs were assigned to the following functional groups: (i) catabolism of quinaldine and anthranilate, (ii) conjugation, and (iii) plasmid maintenance and DNA replication and repair. The genes for conversion of quinaldine to anthranilate are organized in two operons that include ORFs presumed to code for proteins involved in assembly of the quinaldine-4-oxidase holoenzyme, namely, a MobA-like putative molybdopterin cytosine dinucleotide synthase and an XdhC-like protein that could be required for insertion of the molybdenum cofactor. Genes possibly coding for enzymes involved in anthranilate degradation via 2-aminobenzoyl coenzyme A form another operon. These operons were expressed when cells were grown on quinaldine or on aromatic compounds downstream in the catabolic pathway. Single-stranded 3' overhangs of putative replication intermediates of pAL1 were predicted to form elaborate secondary structures due to palindromic and superpalindromic terminal sequences; however, the two telomeres appear to form different structures. Sequence analysis of ORFs 101 to 103 suggested that pAL1 codes for one or two putative terminal proteins, presumed to be covalently bound to the 5' termini, and a multidomain telomere-associated protein (Tap) comprising 1,707 amino acids. Even if the putative proteins encoded by ORFs 101 to 103 share motifs with the Tap and terminal proteins involved in telomere patching of Streptomyces linear replicons, their overall sequences and domain structures differ significantly.
ESTHER : Parschat_2007_J.Bacteriol_189_3855
PubMedSearch : Parschat_2007_J.Bacteriol_189_3855
PubMedID: 17337569
Gene_locus related to this paper: artsp-hod

Title : Stability, unfolding, and structural changes of cofactor-free 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase - Beermann_2007_Biochemistry_46_4241
Author(s) : Beermann B , Guddorf J , Boehm K , Albers A , Kolkenbrock S , Fetzner S , Hinz HJ
Ref : Biochemistry , 46 :4241 , 2007
Abstract : Stability, unfolding mechanism, spectroscopic, densimetric, and structural characteristics of the oxidatively stable C69S variant (HodC) of 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) have been determined by classical and pressure modulation scanning calorimetry (DSC and PMDSC, respectively), circular dichroism (CD) spectroscopy, differential scanning densimetry (DSD), and dynamic light scattering measurements. At 25 degrees C, hexahistidine-tagged HodC has a hydrodynamic radius of 2.3 nm and is characterized by an unusually high degree of alpha-helical structure of approximately 60%, based on deconvolution of CD spectra. The percentage of beta-sheets and -turns is expected to be relatively low in view of its sequence similarity to proteins of the alpha/beta-hydrolase fold superfamily. His6HodC exhibits three-state unfolding (N <--> I <--> D) with an intermediate state I that exhibits at the transition temperature a volume larger than that of the native or denatured state. The intermediate state I is also associated with the highest isothermal expansion coefficient, alphaP, of the three states and exhibits a significantly lower percentage of alpha-helical structure than the native state. The stability difference between the native and intermediate state is rather small which makes I a potential candidate for reactions with various ligands, particularly those having a preference for the apparently preserved beta-type motifs.
ESTHER : Beermann_2007_Biochemistry_46_4241
PubMedSearch : Beermann_2007_Biochemistry_46_4241
PubMedID: 17371045

Title : Crystallization and diffraction data of 1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase: a cofactor-free oxygenase of the alpha\/beta-hydrolase family - Qi_2007_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_63_378
Author(s) : Qi R , Fetzner S , Oakley AJ
Ref : Acta Crystallographica Sect F Struct Biol Cryst Commun , 63 :378 , 2007
Abstract : 1H-3-Hydroxy-4-oxoquinoline 2,4-dioxygenase (QDO) from Pseudomonas putida 33/1 catalyses the oxygenolysis of 1H-3-hydroxy-4-oxoquinoline to form N-formylanthranilic acid and carbon monoxide without the aid of cofactors. Both N-terminally His6-tagged and native QDO were overexpressed in Escherichia coli and purified by conventional chromatographic procedures. Untagged QDO, but not His6-tagged QDO, was crystallized by the vapour-diffusion method, giving hexagonal bipyramid crystals belonging to space group P6(1)22. Selenomethionine-containing native QDO was prepared and crystallized under identical conditions. The unit-cell parameters were a = b = 90.1, c = 168.6 A, alpha = beta = 90, gamma = 120 degrees. Using synchrotron radiation, these crystals diffract to 2.5 A. The expression, purification and crystallization of QDO are reported here.
ESTHER : Qi_2007_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_63_378
PubMedSearch : Qi_2007_Acta.Crystallogr.Sect.F.Struct.Biol.Cryst.Commun_63_378
PubMedID: 17565175

Title : N-acetylanthranilate amidase from Arthrobacter nitroguajacolicus Ru61a, an alpha\/beta-hydrolase-fold protein active towards aryl-acylamides and -esters, and properties of its cysteine-deficient variant - Kolkenbrock_2006_J.Bacteriol_188_8430
Author(s) : Kolkenbrock S , Parschat K , Beermann B , Hinz HJ , Fetzner S
Ref : Journal of Bacteriology , 188 :8430 , 2006
Abstract : N-acetylanthranilate amidase (Amq), a 32.8-kDa monomeric amide hydrolase, is involved in quinaldine degradation by Arthrobacter nitroguajacolicus Ru61a. Sequence analysis and secondary structure predictions indicated that Amq is related to carboxylesterases and belongs to the alpha/beta-hydrolase-fold superfamily of enzymes; inactivation of (His(6)-tagged) Amq by phenylmethanesulfonyl fluoride and diethyl pyrocarbonate and replacement of conserved residues suggested a catalytic triad consisting of S155, E235, and H266. Amq is most active towards aryl-acetylamides and aryl-acetylesters. Remarkably, its preference for ring-substituted analogues was different for amides and esters. Among the esters tested, phenylacetate was hydrolyzed with highest catalytic efficiency (k(cat)/K(m) = 208 mM(-1) s(-1)), while among the aryl-acetylamides, o-carboxy- or o-nitro-substituted analogues were preferred over p-substituted or unsubstituted compounds. Hydrolysis by His(6)Amq of primary amides, lactams, N-acetylated amino acids, azocoll, tributyrin, and the acylanilide and urethane pesticides propachlor, propham, carbaryl, and isocarb was not observed; propanil was hydrolyzed with 1% N-acetylanthranilate amidase activity. The catalytic properties of the cysteine-deficient variant His(6)AmqC22A/C63A markedly differed from those of His(6)Amq. The replacements effected some changes in K(m)s of the enzyme and increased k(cat)s for most aryl-acetylesters and some aryl-acetylamides by factors of about three to eight while decreasing k(cat) for the formyl analogue N-formylanthranilate by several orders of magnitude. Circular dichroism studies indicated that the cysteine-to-alanine replacements resulted in significant change of the overall fold, especially an increase in alpha-helicity of the cysteine-deficient protein. The conformational changes may also affect the active site and may account for the observed changes in kinetic properties.
ESTHER : Kolkenbrock_2006_J.Bacteriol_188_8430
PubMedSearch : Kolkenbrock_2006_J.Bacteriol_188_8430
PubMedID: 17041061
Gene_locus related to this paper: 9micc-q7wsq8

Title : Dioxygenases without requirement for cofactors: identification of amino acid residues involved in substrate binding and catalysis, and testing for rate-limiting steps in the reaction of 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase - Frerichs-Deeken_2005_Curr.Microbiol_51_344
Author(s) : Frerichs-Deeken U , Fetzner S
Ref : Curr Microbiol , 51 :344 , 2005
Abstract : 1H-3-Hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod), catalyzing cleavage of its heteroaromatic substrate to form carbon monoxide and N-acetylanthranilate, belongs to the alpha/beta hydrolase fold family of enzymes. Analysis of protein variants suggested that Hod has adapted active-site residues of the alpha/beta hydrolase fold for the dioxygenolytic reaction. H251 was recently shown to act as a general base to abstract a proton from the organic substrate. Residue S101, which corresponds to the nucleophile of the catalytic triad of alpha/beta-hydrolases, presumably participates in binding the heteroaromatic substrate. H102 and residues located in the topological region of the triad's acidic residue appear to influence O2 binding and reactivity. A tyrosine residue might be involved in the turnover of the ternary complex [HodH+-3,4-dioxyquinaldine dianion-O2]. Absence of viscosity effects and kinetic solvent isotope effects suggests that turnover of the ternary complex, rather than substrate binding, product release, or proton movements, involves the rate-determining step in the reaction catalyzed by Hod.
ESTHER : Frerichs-Deeken_2005_Curr.Microbiol_51_344
PubMedSearch : Frerichs-Deeken_2005_Curr.Microbiol_51_344
PubMedID: 16187153
Gene_locus related to this paper: artsp-hod

Title : Sequence and transcriptional analysis of a gene cluster of Pseudomonas putida 86 involved in quinoline degradation - Carl_2004_Gene_331_177
Author(s) : Carl B , Arnold A , Hauer B , Fetzner S
Ref : Gene , 331 :177 , 2004
Abstract : Although quinoline 2-oxidoreductase (Qor) and 1H-2-oxoquinoline 8-monooxygenase (OxoOR), which catalyse the first two steps of quinoline degradation by Pseudomonas putida 86, and their genes have been investigated in some detail, the genetic organization and regulation of the catabolic pathway are not known yet. A gene cluster involved in quinoline degradation was characterized. Upstream of oxoO encoding the oxygenase component of OxoOR, the gene oxoS coding for a XylS-type protein is located. The DNA region downstream of oxoO comprises potential open reading frames (ORFs) that may code for further catabolic enzymes (an alpha/beta-hydrolase fold protein, and an amidase), and for accessory proteins presumably required for the assembly of metal cofactor containing holoenzymes (XdhC-like protein, MoeC- and MobA-like protein(s), IscS and IscU). The potential iscU gene is followed by the genes qorMSL that encode the structural subunits of Qor. Three potential ORFs (ORFs7-9) are located between qorMSL and oxoR, which codes for the reductase component of OxoOR. ORFs7-9 have counterparts in the cox (CO oxidizing system) and nic (nicotine degradation) gene clusters. Transcription of all these genes and ORFs located downstream of oxoS is induced by quinoline or 1H-2-oxoquinoline. Insertional inactivation of oxoS abolished quinoline-induced transcription. However, weak transcription of ORFs7-9 also occurred independent of quinoline and OxoS. The typical tandem recognition site for a XylS-type transcriptional activator was identified in the putative promoter region of qorM, and archetypal XylS indeed was found to activate synthesis of Qor. Motifs corresponding to single half-sites of a XylS-type binding site are located upstream of oxoO, the xdhC-like gene, and oxoR. Putative quinoline-specific transcriptional start sites were identified for these genes, and for qorM. The gene cluster probably is transcribed from several promoters, resulting in multiple overlapping polycistronic mRNAs.
ESTHER : Carl_2004_Gene_331_177
PubMedSearch : Carl_2004_Gene_331_177
PubMedID: 15094204
Gene_locus related to this paper: psepu-oxoH

Title : Dioxygenases without requirement for cofactors and their chemical model reaction: compulsory order ternary complex mechanism of 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase involving general base catalysis by histidine 251 and single-electron oxidation of the substrate dianion - Frerichs-Deeken_2004_Biochemistry_43_14485
Author(s) : Frerichs-Deeken U , Ranguelova K , Kappl R , Huttermann J , Fetzner S
Ref : Biochemistry , 43 :14485 , 2004
Abstract : 1H-3-Hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) is a cofactor-less dioxygenase belonging to the alpha/beta hydrolase fold family, catalyzing the cleavage of 1H-3-hydroxy-4-oxoquinaldine (I) and 1H-3-hydroxy-4-oxoquinoline (II) to N-acetyl- and N-formylanthranilate, respectively, and carbon monoxide. Bisubstrate steady-state kinetics and product inhibition patterns of HodC, the C69A protein variant of Hod, suggested a compulsory-order ternary-complex mechanism, in which binding of the organic substrate precedes dioxygen binding, and carbon monoxide is released first. The specificity constants, k(cat)/K(m,A) and k(cat)/K(m,O)()2, were 1.4 x 10(8) and 3.0 x 10(5) M(-1) s(-1) with I and 1.2 x 10(5) and 0.41 x 10(5) M(-1) s(-1) with II, respectively. Whereas HodC catalyzes formation of the dianion of its organic substrate prior to dioxygen binding, HodC-H251A does not, suggesting that H251, which aligns with the histidine of the catalytic triad of the alpha/beta hydrolases, acts as general base in catalysis. Investigation of base-catalyzed dioxygenolysis of I by electron paramagnetic resonance (EPR) spectroscopy revealed formation of a resonance-stabilized radical upon exposure to dioxygen. Since in D(2)O spectral properties are not affected, exchangeable protons are not involved, confirming that the dianion is the reactive intermediate that undergoes single-electron oxidation. We suggest that in the ternary complex of the enzyme, direct single-electron transfer from the substrate dianion to dioxygen may occur, resulting in a radical pair. Based on the estimated spin distribution within the radical anion (observed in the model reaction of I), radical recombination may produce a C4- or C2-hydroperoxy(di)anion. Subsequent intramolecular attack would result in the 2,4-endoperoxy (di)anion that may collapse to the reaction products.
ESTHER : Frerichs-Deeken_2004_Biochemistry_43_14485
PubMedSearch : Frerichs-Deeken_2004_Biochemistry_43_14485
PubMedID: 15533053
Gene_locus related to this paper: artsp-hod

Title : Gene cluster of Arthrobacter ilicis Ru61a involved in the degradation of quinaldine to anthranilate: characterization and functional expression of the quinaldine 4-oxidase qoxLMS genes - Parschat_2003_J.Biol.Chem_278_27483
Author(s) : Parschat K , Hauer B , Kappl R , Kraft R , Huttermann J , Fetzner S
Ref : Journal of Biological Chemistry , 278 :27483 , 2003
Abstract : A genetic analysis of the anthranilate pathway of quinaldine degradation was performed. A 23-kb region of DNA from Arthrobacter ilicis Ru61a was cloned into the cosmid pVK100. Although Escherichia coli clones containing the recombinant cosmid did not transform quinaldine, cosmids harboring the 23-kb region, or a 10.8-kb stretch of this region, conferred to Pseudomonas putida KT2440 the ability to cometabolically convert quinaldine to anthranilate. The 10.8-kb fragment thus contains the genes coding for quinaldine 4-oxidase (Qox), 1H-4-oxoquinaldine 3-monooxygenase, 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, and N-acetylanthranilate amidase. The qoxLMS genes coding for the molybdopterin cytosine dinucleotide-(MCD-), FeSI-, FeSII-, and FAD-containing Qox were inserted into the expression vector pJB653, generating pKP1. Qox is the first MCD-containing enzyme to be synthesized in a catalytically fully competent form by a heterologous host, P. putida KT2440 pKP1; the catalytic properties and the UV-visible and EPR spectra of Qox purified from P. putida KT2440 pKP1 were essentially like those of wild-type Qox. This provides a starting point for the construction of protein variants of Qox by site-directed mutagenesis. Downstream of the qoxLMS genes, a putative gene whose deduced amino acid sequence showed 37% similarity to the cofactor-inserting chaperone XdhC was located. Additional open reading frames identified on the 23-kb segment may encode further enzymes (a glutamyl tRNA synthetase, an esterase, two short-chain dehydrogenases/reductases, an ATPase belonging to the AAA family, a 2-hydroxyhepta-2,4-diene-1,7-dioate isomerase/5-oxopent-3-ene-1,2,5-tricarboxylate decarboxylase-like protein, and an enzyme of the mandelate racemase group) and hypothetical proteins involved in transcriptional regulation, and metabolite transport.
ESTHER : Parschat_2003_J.Biol.Chem_278_27483
PubMedSearch : Parschat_2003_J.Biol.Chem_278_27483
PubMedID: 12730200
Gene_locus related to this paper: artsp-hod

Title : Oxygenases without requirement for cofactors or metal ions - Fetzner_2002_Appl.Microbiol.Biotechnol_60_243
Author(s) : Fetzner S
Ref : Applied Microbiology & Biotechnology , 60 :243 , 2002
Abstract : Mono- and dioxygenases usually depend on a transition metal or an organic cofactor to activate dioxygen, or their organic substrate, or both. This review points out that there are at least two separate families of oxygenases without any apparent requirement for cofactors or metal ions: the quinone-forming monooxygenases which are important 'tailoring enzymes' in the biosynthesis of several types of aromatic polyketide antibiotics, and the bacterial dioxygenases involved in the degradation of distinct quinoline derivatives, catalyzing the 2,4-dioxygenolytic cleavage of 3-hydroxy-4-quinolones with concomitant release of carbon monoxide. The quinone-forming monooxygenases might be useful for the modification of polyketide structures, either by using them as biocatalysts, or by employing combinatorial biosynthesis approaches. Cofactor-less oxygenases present the mechanistically intriguing problem of how dioxygen is activated for catalysis. However, the reactions catalyzed by these enzymes are poorly understood in mechanistic terms. Formation of a protein radical and a substrate-derived radical, or direct electron transfer from a deprotonated substrate to molecular oxygen to form a caged radical pair may be discussed as hypothetical mechanisms. The latter reaction route is expected for substrates that can easily donate an electron to dioxygen, and requires the ability of the enzyme to stabilize anionic intermediates. Histidine residues found to be catalytically relevant in both types of cofactor-less oxygenases might be involved in substrate deprotonation and/or electrostatic stabilization.
ESTHER : Fetzner_2002_Appl.Microbiol.Biotechnol_60_243
PubMedSearch : Fetzner_2002_Appl.Microbiol.Biotechnol_60_243
PubMedID: 12436305
Gene_locus related to this paper: artsp-hod , psepu-QDO

Title : Site-directed mutagenesis of potential catalytic residues in 1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase, and hypothesis on the catalytic mechanism of 2,4-dioxygenolytic ring cleavage - Fischer_2000_FEMS.Microbiol.Lett_190_21
Author(s) : Fischer F , Fetzner S
Ref : FEMS Microbiology Letters , 190 :21 , 2000
Abstract : 1H-3-Hydroxy-4-oxoquinoline 2,4-dioxygenase (Qdo) is a cofactor-free dioxygenase proposed to belong to the alpha/beta hydrolase fold superfamily of enzymes. Alpha/beta Hydrolases contain a highly conserved catalytic triad (nucleophile-acidic residue-histidine). We previously identified a corresponding catalytically essential histidine residue in Qdo. However, as shown by amino acid replacements through site-directed mutagenesis, nucleophilic and acidic residues of Qdo considered as possible triad residues were not absolutely required for activity. This suggests that Qdo does not contain the canonical catalytic triad of the alpha/beta hydrolase fold enzymes. Some radical trapping agents affected the Qdo-catalyzed reaction. A hypothetical mechanism of Qdo-catalyzed dioxygenation of 1H-3-hydroxy-4-oxoquinoline is compared with the dioxygenation of FMNH2 catalyzed by bacterial luciferase, which also uses a histidine residue as catalytic base.
ESTHER : Fischer_2000_FEMS.Microbiol.Lett_190_21
PubMedSearch : Fischer_2000_FEMS.Microbiol.Lett_190_21
PubMedID: 10981684
Gene_locus related to this paper: psepu-QDO

Title : Enzymes Involved in the Aerobic Bacterial Degradation of N-Heteroaromatic Compounds: Molybdenum Hydroxylases and Ring-Opening 2,4-Dioxygenases - Fetzner_2000_Naturwiss_87_59
Author(s) : Fetzner S
Ref : Naturwissenschaften , 87 :59 , 2000
Abstract : Many N-heteroaromatic compounds are utilized by micro-organisms as a source of carbon (and nitrogen) and energy. The aerobic bacterial degradation of these growth substrates frequently involves several hydroxylation steps and subsequent dioxygenolytic cleavage of (di)hydroxy-substituted heteroaromatic intermediates to aliphatic metabolites which finally are channeled into central metabolic pathways. As a rule, the initial bacterial hydroxylation of a N-heteroaromatic compound is catalyzed by a molybdenum hydroxylase, which uses a water molecule as source of the incorporated oxygen. The enzyme's redox-active centers - the active site molybdenum ion coordinated to a distinct pyranopterin cofactor, two different [2Fe2S] centers, and in most cases, flavin adenine dinucleotide - transfer electrons from the N-heterocyclic substrate to an electron acceptor, which for many molybdenum hydroxylases is still unknown. Ring-opening 2,4-dioxygenases involved in the bacterial degradation of quinaldine and 1H-4-oxoquinoline catalyze the cleavage of two carbon-carbon bonds with concomitant formation of carbon monoxide. Since they contain neither a metal center nor an organic cofactor, and since they do not show any sequence similarity to known oxygenases, these unique dioxygenases form a separate enzyme family. Quite surprisingly, however, they appear to be structurally and mechanistically related to enzymes of the alpha/beta hydrolase fold superfamily. Microbial enzymes are a great resource for biotechnological applications. Microbial strains or their enzymes may be used for degradative (bioremediation) or synthetic (biotransformation) purposes. Modern bioremediation or biotransformation strategies may even involve microbial catalysts or strains designed by protein engineering or pathway engineering. Prerequisite for developing such modern tools of biotechnology is a comprehensive understanding of microbial metabolic pathways, of the structure and function of enzymes, and of the molecular mechanisms of biocatalysis.
ESTHER : Fetzner_2000_Naturwiss_87_59
PubMedSearch : Fetzner_2000_Naturwiss_87_59
PubMedID: 10663136

Title : Cloning, sequence analysis, and expression of the Pseudomonas putida 33\/1 1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase gene, encoding a carbon monoxide forming dioxygenase - Max_1999_Biochim.Biophys.Acta_1431_547
Author(s) : Max N , Betz A , Facey S , Lingens F , Hauer B , Fetzner S
Ref : Biochimica & Biophysica Acta , 1431 :547 , 1999
Abstract : 1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (Qdo) from the 1H-4-oxoquinoline utilizing Pseudomonas putida strain 33/1, which catalyzes the cleavage of 1H-3-hydroxy-4-oxoquinoline to carbon monoxide and N-formylanthranilate, is devoid of any transition metal ion or other cofactor and thus represents a novel type of ring-cleavage dioxygenase. Gene qdo was cloned and sequenced. Its overexpression in Escherichia coli yielded recombinant His-tagged Qdo which was catalytically active. Qdo exhibited 36% and 16% amino acid identity to 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) and atropinesterase (a serine hydrolase), respectively. Qdo as well as Hod possesses a SXSHG motif, resembling the motif GXSXG of the serine hydrolases which comprises the active-site nucleophile (X=arbitrary residue).
ESTHER : Max_1999_Biochim.Biophys.Acta_1431_547
PubMedSearch : Max_1999_Biochim.Biophys.Acta_1431_547
PubMedID: 10350631
Gene_locus related to this paper: psepu-QDO

Title : Bacterial 2,4-dioxygenases: new members of the alpha\/beta hydrolase-fold superfamily of enzymes functionally related to serine hydrolases - Fischer_1999_J.Bacteriol_181_5725
Author(s) : Fischer F , Kunne S , Fetzner S
Ref : Journal of Bacteriology , 181 :5725 , 1999
Abstract : 1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (Qdo) from Pseudomonas putida 33/1 and 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (Hod) from Arthrobacter ilicis Ru61a catalyze an N-heterocyclic-ring cleavage reaction, generating N-formylanthranilate and N-acetylanthranilate, respectively, and carbon monoxide. Amino acid sequence comparisons between Qdo, Hod, and a number of proteins belonging to the alpha/beta hydrolase-fold superfamily of enzymes and analysis of the similarity between the predicted secondary structures of the 2,4-dioxygenases and the known secondary structure of haloalkane dehalogenase from Xanthobacter autotrophicus GJ10 strongly suggested that Qdo and Hod are structurally related to the alpha/beta hydrolase-fold enzymes. The residues S95 and H244 of Qdo were found to be arranged like the catalytic nucleophilic residue and the catalytic histidine, respectively, of the alpha/beta hydrolase-fold enzymes. Investigation of the potential functional significance of these and other residues of Qdo through site-directed mutagenesis supported the hypothesis that Qdo is structurally as well as functionally related to serine hydrolases, with S95 being a possible catalytic nucleophile and H244 being a possible catalytic base. A hypothetical reaction mechanism for Qdo-catalyzed 2,4-dioxygenolysis, involving formation of an ester bond between the catalytic serine residue and the carbonyl carbon of the substrate and subsequent dioxygenolysis of the covalently bound anionic intermediate, is discussed.
ESTHER : Fischer_1999_J.Bacteriol_181_5725
PubMedSearch : Fischer_1999_J.Bacteriol_181_5725
PubMedID: 10482514
Gene_locus related to this paper: psepu-QDO

Title : Bacterial dehalogenation - Fetzner_1998_Appl.Microbiol.Biotechnol_50_633
Author(s) : Fetzner S
Ref : Applied Microbiology & Biotechnology , 50 :633 , 1998
Abstract : Halogenated organic compounds are produced industrially in large quantities and represent an important class of environmental pollutants. However, an abundance of haloorganic compounds is also produced naturally. Bacteria have evolved several strategies for the enzyme-catalyzed dehalogenation and degradation of both haloaliphatic and haloaromatic compounds: (i) Oxidative dehalogenation is the result of mono- or dioxygenase-catalyzed, co-metabolic or metabolic reactions. (ii) In dehydrohalogenase-catalyzed dehalogenation, halide elimination leads to the formation of a double bond. (iii) Substitutive dehalogenation in most cases is a hydrolytic process, catalyzed by halidohydrolases, but there also is a "thiolytic" mechanism with glutathione as cosubstrate. Dehalogenation by halohydrin hydrogen-halide lyases is the result of an intramolecular substitution reaction. (iv) A distinct dechlorination mechanism involves methyl transfer from chloromethane onto tetrahydrofolate. (v) Reductive dehalogenations are co-metabolic processes, or they are specific reactions involved in substrate utilization (carbon metabolism), or reductive dehalogenation is coupled to energy conservation: some anaerobic bacteria use a specific haloorganic compound as electron acceptor of a respiratory process. This review discusses the mechanisms of enzyme-catalyzed dehalogenation reactions, describes some pathways of the bacterial degradation of haloorganic compounds, and indicates some trends in the biological treatment of organohalogen-polluted air, groundwater, soil, and sediments.
ESTHER : Fetzner_1998_Appl.Microbiol.Biotechnol_50_633
PubMedSearch : Fetzner_1998_Appl.Microbiol.Biotechnol_50_633
PubMedID: 9891928

Title : 2-oxo-1,2-dihydroquinoline 8-monooxygenase: phylogenetic relationship to other multicomponent nonheme iron oxygenases - Rosche_1997_J.Bacteriol_179_3549
Author(s) : Rosche B , Tshisuaka B , Hauer B , Lingens F , Fetzner S
Ref : Journal of Bacteriology , 179 :3549 , 1997
Abstract : 2-Oxo-1,2-dihydroquinoline 8-monooxygenase, an enzyme involved in quinoline degradation by Pseudomonas putida 86, had been identified as a class IB two-component nonheme iron oxygenase based on its biochemical and biophysical properties (B. Rosche, B. Tshisuaka, S. Fetzner, and F. Lingens, J. Biol. Chem. 270:17836-17842, 1995). The genes oxoR and oxoO, encoding the reductase and the oxygenase components of the enzyme, were sequenced and analyzed. oxoR was localized approximately 15 kb downstream of oxoO. Expression of both genes was detected in a recombinant Pseudomonas strain. In the deduced amino acid sequence of the NADH:(acceptor) reductase component (OxoR, 342 amino acids), putative binding sites for a chloroplast-type [2Fe-2S] center, for flavin adenine dinucleotide, and for NAD were identified. The arrangement of these cofactor binding sites is conserved in all known class IB reductases. A dendrogram of reductases confirmed the similarity of OxoR to other class IB reductases. The oxygenase component (OxoO, 446 amino acids) harbors the conserved amino acid motifs proposed to bind the Rieske-type [2Fe-2S] cluster and the mononuclear iron. In contrast to known class IB oxygenase components, which are composed of differing subunits, OxoO is a homomultimer, which is typical for class IA oxygenases. Sequence comparison of oxygenases indeed revealed that OxoO is more related to class IA than to class IB oxygenases. Thus, 2-oxo-1,2-dihydroquinoline 8-monooxygenase consists of a class IB-like reductase and a class IA-like oxygenase. These results support the hypothesis that multicomponent enzymes may be composed of modular elements having different phylogenetic origins.
ESTHER : Rosche_1997_J.Bacteriol_179_3549
PubMedSearch : Rosche_1997_J.Bacteriol_179_3549
PubMedID: 9171399
Gene_locus related to this paper: psepu-oxoH

Title : 2,4-dioxygenases catalyzing N-heterocyclic-ring cleavage and formation of carbon monoxide. Purification and some properties of 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase from Arthrobacter sp. Ru61a and comparison with 1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase from Pseudomonas putida 33\/1 - Bauer_1996_Eur.J.Biochem_240_576
Author(s) : Bauer I , Max N , Fetzner S , Lingens F
Ref : European Journal of Biochemistry , 240 :576 , 1996
Abstract : 1H-3-Hydroxy-4-oxoquinaldine 2,4-dioxygenase (MeQDO) was purified from quinaldine-grown Arthrobacter sp. Ru61a. It was enriched 59-fold in a yield of 22%, and its properties were compared with 1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (QDO) purified from Pseudomonas putida 33/1. The enzyme-catalyzed conversions were performed in an (18O)O2/(16O)O2 atmosphere. Two oxygen atoms of either (18O)O2 or (16O)O2 were incorporated at C2 and C4 of the respective substrates, indicating that these unusual enzymes, which catalyze the cleavage of two carbon-carbon bonds concomitant with CO formation, indeed are 2,4-dioxygenases. Both enzymes are small monomeric proteins of 32 kDa (MeQDO) and 30 kDa (QDO). The apparent K(m) values of MeQDO for 1H-3-hydroxy-4-oxoquinaldine and QDO for 1H-3-hydroxy-4-oxoquinoline were 30 microM and 24 microM, respectively. In both 2,4-dioxygenases, there was no spectral evidence for the presence of a chromophoric cofactor. EPR analyses of MeQDO did not reveal any signal that could be assigned to an organic radical species or to a metal, and X-ray fluorescence spectrometry of both enzymes did not show any metal present in stoichiometric amounts. Ethylxanthate, metal-chelating agents (tiron, alpha, alpha'-bipyridyl, 8-hydroxyquinoline, o-phenanthroline, EDTA, diphenylthiocarbazone, diethyldithiocarbamate), reagents that modify sulfhydryl groups (iodoacetamide, N-ethylmaleimide, p-hydroxymercuribenzoate), and reducing agents (sodium dithionite, dithiothreitol, mercaptoethanol) either did not affect 2,4-dioxygenolytic activities at all or inhibited at high concentrations only. With respect to the supposed lack of any cofactor and with respect to the inhibitors of dioxygenolytic activities, MeQDO and QDO resemble aci-reductone oxidase (CO-forming) from Klebsiella pneumoniae, which catalyzes 1,3-dioxygenolytic cleavage of 1,2-dihydroxy-3-keto-S-methylthiopentene anion (Wray, J. W. & Abeles, R. H. (1993) J. Biol. Chem. 268, 21466-21469; Wray, J. W. & Abeles, R. H. (1995) J. Biol. Chem. 270, 3147-3153). 1H-3-Hydroxy-4-oxoquinaldine and 1H-3-hydroxy-4-oxoquinoline were reactive towards molecular oxygen in the presence of the base catalyst potassium-tert.-butoxide in the aprotic solvent N,N-dimethylformamide. Base-catalyzed oxidation, yielding the same products as the enzyme-catalyzed conversions, provides a non-enzymic model reaction for 2,4-dioxygenolytic release of CO from 1H-3-hydroxy-4-oxoquinaldine and 1H-3-hydroxy-4-oxoquinoline.
ESTHER : Bauer_1996_Eur.J.Biochem_240_576
PubMedSearch : Bauer_1996_Eur.J.Biochem_240_576
PubMedID: 8856057
Gene_locus related to this paper: artsp-hod , psepu-QDO

Title : Bacterial dehalogenases: biochemistry, genetics, and biotechnological applications - Fetzner_1994_Microbiol.Rev_58_641
Author(s) : Fetzner S , Lingens F
Ref : Microbiol Rev , 58 :641 , 1994
Abstract : This review is a survey of bacterial dehalogenases that catalyze the cleavage of halogen substituents from haloaromatics, haloalkanes, haloalcohols, and haloalkanoic acids. Concerning the enzymatic cleavage of the carbon-halogen bond, seven mechanisms of dehalogenation are known, namely, reductive, oxygenolytic, hydrolytic, and thiolytic dehalogenation; intramolecular nucleophilic displacement; dehydrohalogenation; and hydration. Spontaneous dehalogenation reactions may occur as a result of chemical decomposition of unstable primary products of an unassociated enzyme reaction, and fortuitous dehalogenation can result from the action of broad-specificity enzymes converting halogenated analogs of their natural substrate. Reductive dehalogenation either is catalyzed by a specific dehalogenase or may be mediated by free or enzyme-bound transition metal cofactors (porphyrins, corrins). Desulfomonile tiedjei DCB-1 couples energy conservation to a reductive dechlorination reaction. The biochemistry and genetics of oxygenolytic and hydrolytic haloaromatic dehalogenases are discussed. Concerning the haloalkanes, oxygenases, glutathione S-transferases, halidohydrolases, and dehydrohalogenases are involved in the dehalogenation of different haloalkane compounds. The epoxide-forming halohydrin hydrogen halide lyases form a distinct class of dehalogenases. The dehalogenation of alpha-halosubstituted alkanoic acids is catalyzed by halidohydrolases, which, according to their substrate and inhibitor specificity and mode of product formation, are placed into distinct mechanistic groups. beta-Halosubstituted alkanoic acids are dehalogenated by halidohydrolases acting on the coenzyme A ester of the beta-haloalkanoic acid. Microbial systems offer a versatile potential for biotechnological applications. Because of their enantiomer selectivity, some dehalogenases are used as industrial biocatalysts for the synthesis of chiral compounds. The application of dehalogenases or bacterial strains in environmental protection technologies is discussed in detail.
ESTHER : Fetzner_1994_Microbiol.Rev_58_641
PubMedSearch : Fetzner_1994_Microbiol.Rev_58_641
PubMedID: 7854251