Gene_locus Report for: artsp-hod

Protein fold adaptation to novel enzymatic reactions is a fundamental evolutionary process. Cofactor-independent oxygenases degrading N-heteroaromatic substrates belong to the alpha/beta-hydrolase (ABH) fold superfamily that typically does not catalyze oxygenation reactions. Here, we have integrated crystallographic analyses under normoxic and hyperoxic conditions with molecular dynamics and quantum mechanical calculations to investigate its prototypic 1-H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) member. O2 localization to the 'oxyanion hole', where catalysis occurs, is an unfavorable event and the direct competition between dioxygen and water for this site is modulated by the 'nucleophilic elbow' residue. A hydrophobic pocket that overlaps with the organic substrate binding site can act as a proximal dioxygen reservoir. Freeze-trap pressurization allowed the structure of the ternary complex with a substrate analogue and O2 bound at the oxyanion hole to be determined. Theoretical calculations reveal that O2 orientation is coupled to the charge of the bound organic ligand. When 1-H-3-hydroxy-4-oxoquinaldine is uncharged, O2 binds with its molecular axis along the ligand's C2C4 direction in full agreement with the crystal structure. Substrate activation triggered by deprotonation of its 3-OH group by the His-Asp dyad, rotates O2 by approximately 60 deg. This geometry maximizes the charge transfer between the substrate and O2, thus weakening the double bond of the latter. Electron density transfer to the O2(pi*) orbital promotes the formation of the peroxide intermediate via intersystem crossing that is rate-determining. Our work provides a detailed picture of how evolution has repurposed the ABH-fold architecture and its simple catalytic machinery to accomplish metal-independent oxygenation***LongTextEnd*** Paper "Ahmadi-Soleimani_2023_Life.Sci__122100" Author "Ahmadi-Soleimani SM" Author "Amiry GY" Author "Khordad E" Author "Masoudi M" Author "Beheshti F" Year "2023" Title "Omega-3 fatty acids prevent nicotine withdrawal-induced impairment of learning and memory via affecting oxidative status, inflammatory response, cholinergic activity, BDNF and amyloid-B in rat hippocampal tissues" Journal "Life Sciences" Volume "" Page "122100" "" Medline "37722588" Abstract "Ahmadi-Soleimani_2023_Life.Sci__122100" LongText "Ahmadi-Soleimani_2023_Life.Sci__122100" In the present study, the main objective was to reveal whether treatment by Omega-3 fatty acids could prevent the adverse effects of adolescent nicotine withdrawal on spatial and avoidance memory in male rats. For this purpose, Morris water maze and passive avoidance tests were performed on male Wistar rats and the hippocampal levels of oxidative stress markers, inflammatory indices, brain-derived neurotrophic factor, nitrite, amyloid-B and acetylcholinesterase (AChE) were measured. Moreover, density of dark neurons were assessed in CA(1) and CA(3) regions. Results showed that adolescent nicotine exposure followed by a period of drug cessation exacerbates the behavioral indices of learning and memory through affecting a variety of biochemical markers within the hippocampal tissues. These changes lead to elevation of oxidative and inflammatory markers, reduction of neurotrophic capacity and increased AChE activity in hippocampal tissues. In addition, it was observed that co-administration of nicotine with Omega-3 fatty acids significantly prevents nicotine withdrawal-induced adverse effects through restoration of the mentioned biochemical disturbances. Therefore, we suggest administration of Omega-3 fatty acids as a safe, inexpensive and effective therapeutic strategy for prevention of memory dysfunctions associated with nicotine abstinence during adolescence.

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

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.

Protein fold adaptation to novel enzymatic reactions is a fundamental evolutionary process. Cofactor-independent oxygenases degrading N-heteroaromatic substrates belong to the alpha/beta-hydrolase (ABH) fold superfamily that typically does not catalyze oxygenation reactions. Here, we have integrated crystallographic analyses under normoxic and hyperoxic conditions with molecular dynamics and quantum mechanical calculations to investigate its prototypic 1-H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) member. O2 localization to the 'oxyanion hole', where catalysis occurs, is an unfavorable event and the direct competition between dioxygen and water for this site is modulated by the 'nucleophilic elbow' residue. A hydrophobic pocket that overlaps with the organic substrate binding site can act as a proximal dioxygen reservoir. Freeze-trap pressurization allowed the structure of the ternary complex with a substrate analogue and O2 bound at the oxyanion hole to be determined. Theoretical calculations reveal that O2 orientation is coupled to the charge of the bound organic ligand. When 1-H-3-hydroxy-4-oxoquinaldine is uncharged, O2 binds with its molecular axis along the ligand's C2C4 direction in full agreement with the crystal structure. Substrate activation triggered by deprotonation of its 3-OH group by the His-Asp dyad, rotates O2 by approximately 60 deg. This geometry maximizes the charge transfer between the substrate and O2, thus weakening the double bond of the latter. Electron density transfer to the O2(pi*) orbital promotes the formation of the peroxide intermediate via intersystem crossing that is rate-determining. Our work provides a detailed picture of how evolution has repurposed the ABH-fold architecture and its simple catalytic machinery to accomplish metal-independent oxygenation***LongTextEnd*** Paper "Ahmadi-Soleimani_2023_Life.Sci__122100" Author "Ahmadi-Soleimani SM" Author "Amiry GY" Author "Khordad E" Author "Masoudi M" Author "Beheshti F" Year "2023" Title "Omega-3 fatty acids prevent nicotine withdrawal-induced impairment of learning and memory via affecting oxidative status, inflammatory response, cholinergic activity, BDNF and amyloid-B in rat hippocampal tissues" Journal "Life Sciences" Volume "" Page "122100" "" Medline "37722588" Abstract "Ahmadi-Soleimani_2023_Life.Sci__122100" LongText "Ahmadi-Soleimani_2023_Life.Sci__122100" In the present study, the main objective was to reveal whether treatment by Omega-3 fatty acids could prevent the adverse effects of adolescent nicotine withdrawal on spatial and avoidance memory in male rats. For this purpose, Morris water maze and passive avoidance tests were performed on male Wistar rats and the hippocampal levels of oxidative stress markers, inflammatory indices, brain-derived neurotrophic factor, nitrite, amyloid-B and acetylcholinesterase (AChE) were measured. Moreover, density of dark neurons were assessed in CA(1) and CA(3) regions. Results showed that adolescent nicotine exposure followed by a period of drug cessation exacerbates the behavioral indices of learning and memory through affecting a variety of biochemical markers within the hippocampal tissues. These changes lead to elevation of oxidative and inflammatory markers, reduction of neurotrophic capacity and increased AChE activity in hippocampal tissues. In addition, it was observed that co-administration of nicotine with Omega-3 fatty acids significantly prevents nicotine withdrawal-induced adverse effects through restoration of the mentioned biochemical disturbances. Therefore, we suggest administration of Omega-3 fatty acids as a safe, inexpensive and effective therapeutic strategy for prevention of memory dysfunctions associated with nicotine abstinence during adolescence.

Protein fold adaptation to novel enzymatic reactions is a fundamental evolutionary process. Cofactor-independent oxygenases degrading N-heteroaromatic substrates belong to the alpha/beta-hydrolase (ABH) fold superfamily that typically does not catalyze oxygenation reactions. Here, we have integrated crystallographic analyses at normoxic and hyperoxic conditions with molecular dynamics and quantum mechanical calculations to investigate its prototypic 1-H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) member. O2 localization to the oxyanion hole, where catalysis occurs, is an unfavorable event and the direct competition between dioxygen and water for this site is modulated by the nucleophilic elbow residue. A hydrophobic pocket that overlaps with the organic substrate binding site can act as a proximal dioxygen reservoir. Freeze-trap pressurization allowed to determine the structure of the ternary complex with a substrate analogue and O2 bound at the oxyanion hole. Theoretical calculations reveal that O2 orientation is coupled to the charge of the bound organic ligand. When 1-H-3-hydroxy-4-oxoquinaldine is uncharged, O2 binds with its molecular axis along the ligands C2-C4 direction in full agreement with the crystal structure. Substrate activation triggered by deprotonation of its 3-OH group by the His-Asp dyad, rotates O2 by approximately 60 degrees. This geometry maximizes the charge-transfer between the substrate and O2 thus weakening the double bond of the latter. Electron density transfer to the O2(Pi*) orbital promotes the formation of the peroxide intermediate via intersystem crossing that is rate-determining. Our work provides a detailed picture of how evolution has repurposed the ABH-fold architecture and its simple catalytic machinery to accomplish metal-independent oxygenation. Significance: Many of the current O2-dependent enzymes have evolved from classes that existed prior to the switch from a reducing to an oxidative atmosphere and whose original functions are unrelated to dioxygen chemistry. A group of bacterial dioxygenases belong to the alpha/beta-hydrolase (ABH) fold superfamily that typically does not catalyze oxygenation reactions. These enzymes degrade their N-heteroaromatic substrates in a cofactor-independent manner relying only on the simple nucleophile-histidine-acid ABH-fold catalytic toolbox. In this work we show how O2 localizes at the catalytic site by taking advantage of multiple strategies that minimize the strong competition by water, the co-substrate in the ancestral hydrolytic enzyme. We also show that substrate activation by the His-Asp catalytic dyad leads a ligand-O2 complex that maximizes the electron transfer from the organic substrate to O2, thus promoting intersystem crossing and circumventing the spin-forbiddeness of the reaction. Overall, our work explains how evolution has repurposed the ABH-fold architecture and its simple catalytic machinery to accomplish spin-restricted metal-independent oxygenation.

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

Dioxygenases catalyze a diverse range of chemical reactions that involve the incorporation of oxygen into a substrate and typically use a transition metal or organic cofactor for reaction. Bacterial (1H)-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD) belongs to a class of oxygenases able to catalyze this energetically unfavorable reaction without any cofactor. In the quinaldine metabolic pathway, HOD breaks down its natural N-heteroaromatic substrate using a mechanism that is still incompletely understood. Experimental and computational approaches were combined to study the initial step of the catalytic cycle. We have investigated the role of the active site His-251/Asp-126 dyad, proposed to be involved in substrate hydroxyl group deprotonation, a critical requirement for subsequent oxygen reaction. The pH profiles obtained under steady-state conditions for the H251A and D126A variants show a strong pH effect on their kcat and kcat/Km constants, with a decrease in kcat/Km of 5500- and 9-fold at pH 10.5, respectively. Substrate deprotonation studies under transient-state conditions show that this step is not rate-limiting and yield a pKa value of approximately 7.2 for WT HOD. A large solvent isotope effect was found, and the pKa value was shifted to approximately 8.3 in D2O. Crystallographic and computational studies reveal that the mutations have a minor effect on substrate positioning. Computational work shows that both His-251 and Asp-126 are essential for the proton transfer driving force of the initial reaction. This multidisciplinary study offers unambiguous support to the view that substrate deprotonation, driven by the His/Asp dyad, is an essential requirement for its activation.

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.

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.

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.

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.

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.

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.

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.

The ring cleaving enzyme 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD)) of Arthrobacter spec. R61a is part of the quinaldine degradation pathway. Carbon monoxide and N-acetyl-anthranilate are the products formed by dioxygenolytic cleavage of two C-C bonds in the substrate's pyridine ring. The gene coding for HOD was cloned and sequenced. An isoelectric point of pH 5.40 and a molecular mass of 31,838 Da was deduced from the sequence. HOD is shown to be remarkably similar to 1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (QDO) of Pseudomonas putida 33/1, but not to other dioxygenases described so far. Consensus regions indicative for any chromophoric cofactor or any catalytically relevant metal were not detected. Sequence comparisons and secondary structure predictions revealed HOD as a new member of the alpha/beta hydrolase fold family. Expression in E. coli yielded recombinant catalytically active His-tagged HOD. S101A and D233A, two mutants of HOD, were obtained by site-directed mutagenesis. Since their residual activity is 43.1% and 62.6%, respectively, they probably are of no catalytic relevance although they might play a role in the interaction between enzyme and substrate.

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.