Selectivity Studies of the Benzene cis -Dihydrodiol Dehydrogenase Enzyme from Pseudomonas putida ML2 with Vicinal Diol Substrates

The enantiopure (1 S, 2 S )- cis -dihydrodiol metabolites 2B - 5B have been obtained in low yield from the corresponding monosubstituted halobenzene substrates 2A - 5A, using a wild-type strain of Pseudomonas putida (ML2) containing benzene dioxygenase (BDO). Benzene cis -dihydrodiol dehydrogenase (BCD) from P. putida ML2 and naphthalene cis -dihydrodiol dehydrogenase (NCD) from P. putida 8859 were purified and used in a comparative study of the stereoselective biotransformation of cis -dihydrodiol enantiomers 2B - 5B. The BCD and NCD enzymes were found to accept cis -dihydrodiol enantiomers of monosubstituted benzene cis -dihydrodiol substrates 2B - 5B of opposite absolute configuration. The acyclic alkene 1,2-diols 10 - 17 were also found to be acceptable substrates for BCD.     

Selectivity Studies of the Benzene cis -Dihydrodiol Dehydrogenase Enzyme from Pseudomonas putida ML2 with Vicinal Diol Substrates

The enantiopure (1 S , 2 S )- cis -dihydrodiol metabolites 2B - 5B have been obtained in low yield from the corresponding monosubstituted halobenzene substrates 2A - 5A , using a wild-type strain of Pseudomonas putida (ML2) containing benzene dioxygenase (BDO). Benzene cis -dihydrodiol dehydrogenase (BCD) from P. putida ML2 and naphthalene cis -dihydrodiol dehydrogenase (NCD) from P. putida 8859 were purified and used in a comparative study of the stereoselective biotransformation of cis -dihydrodiol enantiomers 2B - 5B . The BCD and NCD enzymes were found to accept cis -dihydrodiol enantiomers of monosubstituted benzene cis -dihydrodiol substrates 2B - 5B of opposite absolute configuration. The acyclic alkene 1,2-diols 10 - 17 were also found to be acceptable substrates for BCD.     

Metabolism of benzo[a]pyrene VI. Stereoselective metabolism of benzo[a]pyrene and benzo[a]pyrene 7,8-dihydrodiol to diol epoxides

(±)-7β,8α-Dihydroxy-9β,10β-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-1) and (±)-7β,8α-dihydroxy-9α,10α-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (diol epoxide-2) are highly mutagenic diol epoxide diastereomers that are formed during metabolism of the carcinogen (±)-trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. Remarkable stereoselectivity has been observed on metabolism of the optically pure (+)- and (?)-enantiomers of the dihydrodiol which are obtained by separation of the diastereomeric diesters with (?)-α-methoxy-α-trifluoromethylphenylacetic acid. The high stereoselectivity in the formation of diol epoxide-1 relative to diol epoxide-2 was observed with liver microsomes from 3-methylcholanthrene-treated rats and with a purified cytochrome P-448-containing monoxygenase system where the (?)-enantiomer produced a diol epoxide-2 to diol epoxide-1 ratio of 6 : 1 and the (+)-enantiomer produced a ratio of 1 : 22. Microsomes from control and phenobarbital-treated rats were less stereospecific in the metabolism of enantiomers of BP 7,8-dihydrodiol. The ratio of diol epoxide-2 to diol epoxide-1 formed from the (?)- and (+)-enantiomers with microsomes from control rats was 2 : 1 and 1 : 6, respectively. Both enantiomers of BP 7,8-dihydrodiol were also metabolized to a phenolic derivative, tentatively identified as 6,7,8-trihydroxy-7,8-dihydrobenzo[a]pyrene, which accounted for ～30% of the total metabolites formed by microsomes from control and phenobarbital-pretreated rats whereas this metabolite represents ～5% of the total metabolites with microsomes from 3-methylcholanthrene-treated rats. With benzo[a]pyrene as substrate, liver microsomes produced the 4,5-, 7,8- and 9,10-dihydrodiol with high optical purity (>85%), and diol epoxides were also formed. Most of the optical activity in the BP 7,8-dihydrodiol was due to metabolism by the monoxygenase system rather than by epoxide hydrase, since hydration of (±)-benzo[a]pyrene 7,8-oxide by liver microsomes produced dihydrodiol which was only 8% optically pure. Thus, the stereospecificity of both the monoxygenase system and, to a lesser extent, epoxide hydrase plays important roles in the metabolic activation of benzo[a]pyrene to carcinogens and mutagens.     

Absolute configuration of trans-7,8-dihydroxy-7,8-dihydro-7-methylbenzo[a]pyrene enantiomer and the unusual quasidiequatorial conformation of the diacetate and dimenthoxyacetate derivatives

The enantiomers of trans-7,8-dihydroxy-7,8-dihydro-7-methylbenzo[a]pyrene (7-MBaP 7,8-dihydrodiol) and of trans-7,8-dihydroxy-7,8,9,10-tetrahydro-7-methylbenzo[a]pyrene (7-MBaP 7,8-tetrahydrodiol) were directly resolved by high-performance liquid chromatography (HPLC) using a commercially available column packed with an (R)-N-(3,5-dinitrobenzoyl)-phenylglycine derivative of gamma-aminopropylsilanized silica. The absolute configurations of the resolved enantiomers were determined by the exciton chirality method. Circular dichroism (CD) spectral analysis of the quasidiequatorial benzo[a]pyrene 7R,8R-dihydrodiol enantiomer and its diacetate and dimenthoxyacetate derivatives indicated conformational changes were induced upon derivatization. However, the characteristic CD Cotton effects of the quasidiequatorial 7-MBaP 7,8-dihydrodiol and its diacetate and dimenthoxyacetate derivatives were similar indicating that the conformation of 7-MBaP trans-7,8-dihydrodiol was not altered upon derivatization. Proton nuclear magnetic resonance (NMR) spectral analyses confirmed that 7-MBaP 7,8-dihydrodiol, its diacetate and dimenthoxyacetate derivatives all have quasidiequatorial conformations. The results indicate that the methyl substituent of 7-MBaP 7,8-dihydrodiol maintains a quasiaxial position regardless of the size of the acyl derivatives linked to the hydroxyl groups.     

8-Oxo-7,8-dihydro-2'-deoxyguanosine is not salvaged for DNA synthesis in human leukemic U937 cells

8-Oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), the most common oxidatively modified nucleoside, is released from oxidized DNA and oxidized nucleotide pool. However, little information is available regarding the metabolic pathway of free 8-oxo-dG. In this study, we generated radiolabeled 8-oxo-dG to track its metabolic fate. We report that 8-oxo-dG is neither phosphorylated to 8-oxo-dGMP nor degraded to the free base, 8-oxo-7,8-dihydroguanine (8-oxo-Gua), indicating that 8-oxo-dG is not a substrate for nucleotide synthesis. This result was confirmed by the finding that no radioactivity was detected in the DNA of U937 cells after incubating the cells with radiolabeled 8-oxo-dG. These observations indicate that 8-oxo-dG produced by oxidative stress is not reutilized for DNA synthesis.     

Effects of a 6-fluoro substituent on the metabolism of benzo(a)pyrene 7,8-dihydrodiol to bay-region diol epoxides by rat liver enzymes

Metabolism of trans-7,8-dihydroxy-7,8-dihydro-6-fluorobenzo(a)pyrene by liver microsomes from 3-methylcholanthrene-treated rats and by a highly purified monooxygenase system, reconstituted with cytochrome P-450c, has been examined. Although both the fluorinated and unfluorinated 7,8-dihydrodiol formed from benzo(a)pyrene by liver microsomes share (R,R)-absolute configuration, the fluorinated dihydrodiol prefers the conformation in which the hydroxyl groups are pseudodiaxial due to the proximate fluorine. The fluorinated 4,5- and 9,10-dihydrodiols are also greater than 97% the (R,R)-enantiomers. For benzo(a)pyrene, metabolism of the (7R,8R)-dihydrodiol to a bay-region 7,8-diol-9,10-epoxide in which the benzylic hydroxyl group and epoxide oxygen are trans constitutes the only known pathway to an ultimate carcinogen. With the microsomal and the purified monooxygenase system, this pathway accounts for 76-82% of the total metabolites from the 7,8-dihydrodiol. In contrast, only 32-49% of the corresponding diol epoxide is obtained from the fluorinated dihydrodiol and this fluorinated diol epoxide has altered conformation in that its hydroxyl groups prefer to be pseudodiaxial. Much smaller amounts of the diastereomeric 7,8-diol-9,10-epoxides in which the benzylic hydroxyl groups and the epoxide oxygen are cis are formed from both dihydrodiols. As the fluorinated diol epoxides are weaker mutagens toward bacteria and mammalian cells relative to the unfluorinated diol epoxides, conformation appears to be an important determinant in modulating the biological activity of diol epoxides. One of the more interesting metabolites of 6-fluorinated 7,8-dihydrodiol was a relatively stable arene oxide, probably the 4,5-oxide, which is resistant to the action of epoxide hydrolase.     

Benzo{a}pyrene-induced DNA damage in Mytilus galloprovincialis: measurement of bulky DNA adducts and DNA oxidative damage in terms of 8-oxo-7,8-dihydro-2´-deoxyguanosine formation

Bulky DNA adducts and 8-oxo-7,8-dihydro-2´-deoxyguanosine (8-oxodGuo) were measured in gill DNA of benzo[a]pyrene (B[a]P)-exposed mussels (50 mg kg^-1 dw day^-1), respectively by the ³²P-post-labelling technique and high performance liquid chromatography coupled to electrochemical detection assay. A time-course study was performed for both biomarkers and their potential use for marine biomonitoring discussed for the sentinel species studied. In gills, B[a]P-related DNA adducts were positively correlated with B[a     

The role of the Ah receptor and p38 in benzo[a]pyrene-7,8-dihydrodiol and benzo[a]pyrene-7,8-dihydrodiol-9,10-epoxide-induced apoptosis

Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous contaminants in the environment. Benzo[a]pyrene (B[a]P), a prototypical member of this class of chemicals, affects cellular signal transduction pathways and induces apoptosis. In this study, the proximate carcinogen of B[a]P metabolism, trans-7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene (B[a]P-7,8-dihydrodiol) and the ultimate carcinogen, B[a]P-r-7,t-8-dihydrodiol-t-9,10-epoxide(+/-) (BPDE-2) were found to induce apoptosis in human HepG2 cells. Apoptosis initiated by B[a]P-7,8-dihydrodiol was linked to activation of the Ah receptor and induction of CYP1A1, an event that can lead to the formation of BPDE-2. With both B[a]P-7,8-dihydrodiol and BPDE-2 treatment, changes in anti- and pro-apoptotic events in the Bcl-2 family of proteins correlated with the release of mitochondrial cytochrome c and caspase activation. The onset of apoptosis as monitored by caspase activation was linked to mitogen-activated protein (MAP) kinases. Utilizing mouse hepa1c1c7 cells and the Arnt-deficient BPRc1 cells, activation of MAP kinase p38 by B[a]P-7,8-dihydrodiol was shown to be Ah receptor-dependent, indicating that metabolic activation by CYP1A1 was required. This was in contrast to p38 activation by BPDE-2, an event that was independent of Ah receptor function. Confirmation that MAP kinases play a critical role in BPDE-2-induced apoptosis was shown by inhibiting caspase activation of poly(ADP-ribose)polymerase 1 (PARP-1) by chemical inhibitors of p38 and ERK1/2. Furthermore, mouse embryo p38-/- fibroblasts were shown to be resistant to the actions of BPDE-2-induced apoptosis as determined by annexin V analysis, cytochrome c release, and cleavage of PARP-1. These results confirm that the Ah receptor plays a critical role in B[a]P-7,8-dihydrodiol-induced apoptosis while p38 MAP kinase links the actions of an electrophilic metabolite like BPDE-2 to the regulation of programmed cell death.     

Combination of azathioprine and UVA irradiation is a major source of cellular 8-oxo-7,8-dihydro-2'-deoxyguanosine

Thiopurine antimetabolites, such as azathioprine (Aza) and 6-thioguanine (6-TG), are widely used in the treatment of cancer, inflammatory conditions and organ transplantation patients. Recent work has shown that cells treated with 6-TG and UVA generate ROS, with implied oxidatively generated modification of DNA. In a study of urinary 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) in renal transplant patients, we provided the first in vivo evidence linking Aza and oxidatively damaged DNA. Using the hOGG1 comet assay, we herein demonstrate high levels of 8-oxodG and alkali-labile sites (ALS) in cells treated with biologically relevant doses of 6-TG, or Aza, plus UVA. This damage was induced dose-dependently. Surprisingly, given the involvement of 6-TG incorporation into DNA in its therapeutic effect, significant amounts of 8-oxodG and ALS were induced in quiescent cells, although less than in proliferating cells. We speculate that some activity of hOGG1 towards unirradiated, 6-TG treated cells, implies possible recognition of 6-TG or derivatives thereof. This is the first report to conclusively demonstrate oxidatively damaged DNA in cells treated with thiopurines and UVA. These data indicate that Aza-derived oxidative stress will occur in the skin of patients on Aza, following even low level UVA exposure. This is a probable contributor to the increased risk of non-melanoma skin cancer in these patients. However, as oxidative stress is unlikely to be involved in the therapeutic effects of Aza, intercepting ROS production in the skin could be a viable route by which this side effect may be minimised.     

Mutagenicity of secondary oxidation products of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-hydroxy-2'- deoxyguanosine 5'-triphosphate)

8-Oxo-7,8-dihydroguanine (8-hydroxyguanine) is oxidized more easily than normal nucleobases, which can produce spiroiminodihydantoin (Sp) and guanidinohydantoin (Gh). These secondary oxidation products of 8-oxo-7,8-dihydroguanine are highly mutagenic when formed within DNA. To evaluate the mutagenicity of the corresponding oxidation products of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate (8-hydroxy-2'- deoxyguanosine 5'-triphosphate) in the nucleotide pool, Escherichia coli cells deficient in the mutT gene were treated with H(2)O(2), and the induced mutations were analyzed. Moreover, the 2'-deoxyriboside 5'-triphosphate derivatives of Sp and Gh were also introduced into competent E. coli cells. The H(2)O(2) treatment of mutT E. coli cells resulted in increase of G:C → T:A and A:T → T:A mutations. However, the incorporation of exogenous Sp and Gh 2'-deoxyribonucleotides did not significantly increase the mutation frequency. These results suggested that the oxidation product(s) of 8-oxo-7,8-dihydro-2'-deoxyguanosine 5'-triphosphate induces G:C → T:A and A:T → T:A mutations, and that the 2'-deoxyriboside 5'-triphosphate derivatives of Sp and Gh exhibit quite weak mutagenicity, in contrast to the bases in DNA.     

Isotope dilution high-performance liquid chromatography–electrospray tandem mass spectrometry assay for the measurement of 8-oxo-7,8-dihydro-2′-deoxyguanosine in biological samples

A sensitive and specific assay aimed at measuring 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) has been developed by associating a reversed-phase liquid chromatographic separation with an electrospray tandem mass spectrometric detection. The HPLC–MS approach in the single ion monitoring (SIM) mode and the HPLC–MS/MS assay in the multiple reaction monitoring (MRM) mode have been compared, using isotopically labeled [M+4] 8-oxodGuo as the internal standard. The limit of detection of 8-oxodGuo was found to be around 5 pmol and 20 fmol for the HPLC–MS and HPLC–MS/MS methods, respectively. The HPLC–MS/MS assay is sensitive enough to allow the determination of the level of 8-oxodGuo in cellular liver DNA and in urine samples.     

Induction and repair of DNA strand breaks in cultured human fibroblasts exposed to various phenols and dihydrodiols of benzo[a]pyrene

Cultured human fibroblasts from healthy donors were incubated for 30 min with nine different benzo[a]pyrene (BP) derivatives in the presence or absence of liver microsomes from 3-methylcholanthrene treated rats. The induction and repair of DNA strand breaks were analysed by alkaline unwinding and separation of double and single stranded DNA (SS-DNA) by hydroxylapatite chromatography immediately after the incubation or at various times after the treatment. In the absence of microsomes DNA stand breaks were detected in fibroblasts exposed to 30 microM of each of the six BP phenols (1-, 2-, 3-, 7-, 9- or 11-OH-BP) and the three BP dihydrodiols (BP-4,5-, BP-7,8- or BP-9,10-dihydrodiol). After removal of the BP derivatives from the medium the DNA strand breaks disappeared within 24 h. alpha-Naphthoflavone (alpha-NF) caused a decrease in the induction of strand breaks by 1-, 3- and 9-OH-BP but did not affect the induction of strand breaks in cells exposed to BP-7,8-dihydrodiol. In the presence of microsomes DNA strand breaks were found after exposure to 30 microM of each of the six BP phenols (1-, 2-, 3-, 7-, 9- or 11-OH-BP), as well as BP-7,8- and 9,10-dihydrodiol. In contrast BP-4,5-dihydrodiol did not induce strand breaks under these conditions. The induction of strand breaks by BP-7,8-dihydrodiol was enhanced in the presence of cytosine-1-beta-D-arabinofuranoside (AraC). In all cases the DNA strand breaks had disappeared 24 h after removal of the BP derivatives and microsomes except after treatment with BP-7,8-dihydrodiol.     

Oxidation of carbazole to 3-hydroxycarbazole by naphthalene 1,2-dioxygenase and biphenyl 2,3-dioxygenase

Abstract Naphthalene 1,2-dioxygenase from Pseudomonas sp. NCIB 9816-4 and biphenyl dioxygenase from Beijerinckia sp. B8/36 oxidized the aromatic N-heterocycle carbazole to 3-hydroxycarbazole. Toluene dioxygenase from Pseudomonas putida F39/D did not oxidize carbazole. Transformations were carried out by mutant strains which oxidize naphthalene and biphenyl to cis -dihydrodiols, and with a recombinant E. coli strain expressing the structural genes of naphthalene 1,2-dioxygenase from Pseudomonas sp. NCIB 9816-4. 3-Hydroxycarbazole is presumed to result from the dehydration of an unstable cis -dihydrodiol.     

Microbial metabolism of quinoline and related compounds. III. Degradation of 3-chloroquinoline-8-carboxylic acid by Pseudomonas spec. EK III

Bacteria have been isolated with the ability to use 3-chloroquinoline-8-carboxylic acid as sole source of carbon and energy. According to their physiological properties, these bacteria have been classified as Pseudomonas spec. Two metabolites of the degradation pathway have been isolated and identified. The first metabolite was 3-(3-carboxy-3-oxopropenyl)-2-hydroxy-5-chloropyridine, the meta-cleavage product of 3-chloro-7,8-dihydroxyquinoline. The second metabolite, 5-chloro-2-hydroxynicotinic acid, was not further metabolized by this organisms.     

Evaluation of enzyme-linked immunosorbent assay and liquid chromatography-tandem mass spectrometry methodology for the analysis of 8-oxo-7,8-dihydro-2'-deoxyguanosine in saliva and urine

While ELISA is a frequently used means of assessing 8-oxo-7,8-dihydro-2-deoxyguanosine (8-oxodG) in biological fluids, differences in baseline urinary 8-oxodG levels, compared to chromatographic techniques, have raised questions regarding the specificity of immunoassays. Recently, ELISA of salivary 8-oxodG has been used to report on periodontal disease. We compared salivary 8-oxodG levels, determined by two commercial ELISA kits, to liquid chromatography-tandem mass spectrometry (LC-MS/MS) with prior purification using solid-phase extraction. While values were obtained with both ELISA kits, salivary 8-oxodG values were below or around the limit of detection of our LC-MS/MS assay. As the limit of detection for the LC-MS/MS procedure is much lower than ELISA, we concluded that the assessment of salivary 8-oxodG by ELISA is not accurate. In contrast to previous studies, ELISA levels of urinary 8-oxodG (1.67 ± 0.53 pmol/μmol creatinine) were within the range reported previously only for chromatographic assays, although still significantly different than LC-MS/MS (0.41 ± 0.39 pmol/μmol creatinine; p = 0.002). Furthermore, no correlation with LC-MS/MS was seen. These results question the ability of ELISA approaches, at present, to specifically determine absolute levels of 8-oxodG in saliva and urine. Ongoing investigation in our laboratories aims to identify the basis of the discrepancy between ELISA and LC-MS/MS.     

8-Oxo-7,8-dihydroguanine and 8-oxo-7,8-dihydro-2′-deoxyguanosine levels in human urine do not depend on diet

In the present study, we used the method involving HPLC pre-purification followed by gas chromatography with isotope dilution mass spectrometric detection for the determination of 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodGuo) and 8-oxo-7,8-dihydroguanine (8-oxoGua) in human urine. The mean levels of 8-oxoGua and 8-oxodGuo in the urine samples of the subjects on unrestricted diet were respectively 1.87 nmol/kg 24 h (±0.90) and 0.83 nmol/kg 24h (±0.49), and in the case of the groups studied, they did not depend on the applied diet. The sum of the amounts of both compounds in urine can give information about the formation rate of 8-oxoGua in cellular DNA. It is also likely that the levels of modified nucleo-base/side in urine sample are reflective of the involvement of different repair pathways responsible for the removal of 8-oxodGuo from DNA, namely base excision repair (BER) and nucleotide excision repair (NER).     

Microbial metabolism of quinoline and related compounds. VII. Quinoline oxidoreductase from Pseudomonas putida: a molybdenum-containing enzyme

The quinoline oxidoreductase from Pseudomonas putida was purified 50-fold to homogeneity with 21% recovery, using ammonium sulfate precipitation, hydrophobic interaction-, anion exchange-, and gel chromatography. The Mr of the native enzyme was calculated to be 300,000 by gel filtration. SDS-polyacrylamide gel electrophoresis of the enzyme revealed three protein bands corresponding to Mr 85,000, 30,000 and 20,000. The enzyme contained 8 atoms of iron, 8 atoms of acid-labile sulfide, 2 molecules of FAD, and the molybdenum cofactor, molybdopterin. Besides quinoline, the quinoline oxidoreductase also catalysed the conversion of 5-, 6-, 7- and 8-hydroxyquinoline and 8-chloroquinoline to the corresponding 2-oxo compounds. The incorporated oxygen atom was derived from water. Cyanide and methanol were effective inhibitors.     

Functional expression of the quinoline 2-oxidoreductase genes (qorMSL) in Pseudomonas putida KT2440 pUF1 and in P. putida 86-1 deltaqor pUF1 and analysis of the Qor proteins.

The availability of a system for the functional expression of genes coding for molybdenum hydroxylases is a prerequisite for the construction of enzyme variants by mutagenesis. For the expression cloning of quinoline 2-oxidoreductase (Qor) from Pseudomonas putida 86--that contains the molybdopterin cytosine dinucleotide molybdenum cofactor (Mo-MCD), two distinct [2Fe-2S] clusters and FAD--the qorMSL genes were inserted into the broad host range vector, pJB653, generating pUF1. P. putida KT2440 and P. putida 86-1 deltaqor were used as recipients for pUF1. Whereas Qor from the wild-type strain showed a specific activity of 19-23 U x mg(-1), the specific activity of Qor purified from P. putida KT2440 pUF1 was only 0.8-2.5 U x mg(-1), and its apparent k(cat) (quinoline) was about ninefold lower than that of wild-type Qor. The apparent Km values for quinoline were similar for both proteins. UV/visible and EPR spectroscopy indicated the presence of the full set of [2Fe-2S] clusters and FAD in Qor from P. putida KT2440 pUF1, however, the very low intensity of the Mo(V)-rapid signal, that occurs in the presence of quinoline, as well as metal analysis indicated a deficiency of the molybdenum center. In contrast, the metal content, and the spectroscopic and catalytic properties of Qor produced by P. putida 86-1 deltaqor pUF1 were essentially like those of wild-type Qor. Release of CMP upon acidic hydrolysis of the Qor proteins suggested the presence of the MCD form of the pyranopterin cofactor; the CMP contents of the three enzymes were similar.     

Expression of the iorAB genes from Brevundimonas diminuta 7 encoding the molybdenum hydroxylase isoquinoline 1-oxidoreductase in Pseudomonas putida

Isoquinoline 1-oxidoreductase (Ior) from Brevundimonas diminuta 7, encoded by iorAB, is a molybdenum hydroxylase containing a molybdopterin cytosine dinucleotide molybdenum cofactor (Mo-MCD) and two distinct [2Fe2S] clusters. The iorAB genes were inserted into pJB653, generating pIL1. Pseudomonas putida KT2440, and P. putida 86 which produces a Mo-MCD-containing quinoline 2-oxidoreductase when grown on quinoline, were used as recipients for pIL1. Upon induction of gene expression, both clones produced Ior protein, but Ior activity was not detectable in P. putida KT2440 pIL1. In P. putida 86 pIL1, formation of catalytically active Ior required the presence of quinoline, suggesting that accessory gene(s) encoding product(s) essential for the assembly of catalytically competent Ior is (are) part of the quinoline regulon in P. putida 86.