Comparative EPR and Redox Studies of Three Prokaryotic Enzymes of the Xanthine Oxidase Family:  Quinoline 2-Oxidoreductase, Quinaldine 4-Oxidase, and Isoquinoline 1-Oxidoreductase

Canne, Christoph; Stephan, Ingrid; Finsterbusch, J.; Lingens, Franz; Kappl, Reinhard; Fetzner, Susanne; Hüttermann, Jürgen

doi:10.1021/bi970581d

Cited by 30 publications

(38 citation statements)

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“…Until recently, studies on Qox [12,16,50] and other Mo-containing dehydrogenases [12,31,52] focused mainly on structural and biochemical investigations. This study constitutes the first biotechnological in vivo application of Qox-containing recombinant P. putida strains for productive biocatalytic C-H oxyfunctionalizations and represents the potential of Qox to achieve carbon oxyfunctionalizations without active aeration, reducing the process complexicity, costs, and limitations in technical applications.…”

Section: Introductionmentioning

confidence: 99%

Regioselective aromatic hydroxylation of quinaldine by water using quinaldine 4-oxidase in recombinant Pseudomonas putida

Ütkür

Gaykawad

Bühler

et al. 2010

J Ind Microbiol Biotechnol

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Biocatalytic hydrocarbon oxyfunctionalizations are typically accomplished using oxygenases in living bacteria as biocatalysts. These processes are often limited by either oxygen mass transfer, cofactor regeneration, and/or enzyme instabilities due to the formation of reactive oxygen species. Here, we discuss an alternative approach based on molybdenum (Mo)-containing dehydrogenases, which produce, rather than consume, reducing equivalents in the course of substrate hydroxylation and use water as the oxygen donor. Mo-containing dehydrogenases have a high potential for overcoming limitations encountered with oxygenases. In order to evaluate the suitability and efficiency of a Mo-containing dehydrogenase-based biocatalyst, we investigated quinaldine 4-oxidase (Qox)-containing Pseudomonas strains and the conversion of quinaldine to 4-hydroxyquinaldine. Host strain and carbon source selection proved to be crucial factors influencing biocatalyst efficiency. Resting P. putida KT2440 (pKP1) cells, grown on and induced with benzoate, showed the highest Qox activity and were used for process development. To circumvent substrate and product toxicity/inhibition, a two-liquid phase approach was chosen. Without active aeration and with 1-dodecanol as organic carrier solvent a productivity of 0.4 g l (tot) (-1) h(-1) was achieved, leading to the accumulation of 2.1 g l (tot) (-1) 4-hydroxyquinaldine in 6 h. The process efficiency compares well with values reported for academic and industrially applied biocatalytic oxyfunctionalization processes emphasizing the potential and feasibility of the Qox-containing recombinant cells for heteroaromatic carbon oxyfunctionalizations without the necessity for active aeration.

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Section: Introductionmentioning

confidence: 99%

Regioselective aromatic hydroxylation of quinaldine by water using quinaldine 4-oxidase in recombinant Pseudomonas putida

Ütkür

Gaykawad

Bühler

et al. 2010

J Ind Microbiol Biotechnol

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“…The S subunit (157 residues) comprises 2 domains, each coordinating 1 [2Fe-2S] cluster. The N-terminal domain (residues 1-79) is similar to ''plant-type ferredoxins'' and binds the [2Fe-2S] cluster close to the FAD, which is designated as Fe/S II or type II on the basis of its EPR characteristics in homologous molybdenum hydroxylases (31)(32)(33). The C-terminal domain (residues 80-157) displays a 4-helix bundle with 2-fold symmetry unique to molybdenum hydroxylases (19) and coordinates the [2Fe-2S] cluster (type I) closest to the molybdopterin.…”

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confidence: 99%

The Mo-Se active site of nicotinate dehydrogenase

Wagener

Pierik

Ibdah

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

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Nicotinate dehydrogenase (NDH) from Eubacterium barkeri is a molybdoenzyme catalyzing the hydroxylation of nicotinate to 6-hydroxynicotinate. Reactivity of NDH critically depends on the presence of labile (nonselenocysteine) selenium with an as-yetunidentified form and function. We have determined the crystal structure of NDH and analyzed its active site by multiple wavelengths anomalous dispersion methods. We show that selenium is bound as a terminal MoASe ligand to molybdenum and that it occupies the position of the terminal sulfido ligand in other molybdenum hydroxylases. The role of selenium in catalysis has been assessed by model calculations, which indicate an acceleration of the critical hydride transfer from the substrate to the selenido ligand in the course of substrate hydroxylation when compared with an active site containing a sulfido ligand. The MoO(OH)Se active site of NDH shows a novel type of utilization and reactivity of selenium in nature.S elenium is an essential component of several enzymes and has a key role in various biological redox processes. Usually selenium occurs in proteins as selenocysteine, which is cotranslationally inserted as the 21st amino acid (1) and is found in a variety of proteins in all 3 kingdoms of life (2). Selenium also finds a natural use as 5-methylaminomethyl-2-selenouridine in the ''wobble'' position of some tRNAs (3). The ionic radii and electronegativities of selenium and sulfur are similar, but selenide is a stronger reducing agent than sulfide. Because of the lower pK a value of selenols compared with thiols selenocysteine is deprotonated under physiological conditions, whereas cysteine is mostly protonated (4). The ionization state together with the better polarizability of selenium makes selenocysteine a good nucleophile. Several molybdenum-and tungsten-containing enzymes have been shown to contain selenium (5), and a recent comprehensive genomic analysis has revealed a clear relationship between selenium and molybdenum utilization across all 3 domains of life (6). Selenium is found as selenocysteine in some prokaryotic molybdenum-containing oxotransferases like formate dehydrogenase H from Escherichia coli, where it coordinates molybdenum in the oxidized state of the enzyme (7-9). In other enzymes, notably members of the molybdenum hydroxylase family (10, 11), like nicotinate dehydrogenase (NDH) from Eubacterium barkeri (12-14) and the xanthine oxidoreductases (XORs) of Clostridium purinolyticum (15), Clostridium acidiurici (16) and Eubacterium barkeri (17) and the purine hydroxylase from C. purinolyticum (15) contain a labile (nonselenocysteine) selenium in an unidentified form essential for their reactivity (18).Molybdenum hydroxylases catalyze the hydroxylation of various organic molecules following the general scheme:This hydroxylation reaction is unique in biology as it uses water as the source for the hydroxyl oxygen and not dioxygen (10). The active site of molybdenum hydroxylases contains molybdenum coordinated by the enedithiolate group of a pyrano...

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“…For other related enzymes (Qor and Ior), small shifts of the g factors of FeS clusters have been reported depending on the mode of reduction (9). An indication of a magnetic interaction between the FeS centers as found for Ior was not observed.…”

Section: Degradative Capacities Of P Putida Kt2440 Cosmid Clones Andmentioning

confidence: 82%

“…Only minor traces of a rapid (r) species are detectable. In comparison, the g 2 and g 3 components of the very rapid signals found in Qor from P. putida 86 and Ior from B. diminuta are clearly different (9). The FAD signals in all Qox samples display a line width of 1.6 mT typical for the anionic (red) FAD radical.…”

Section: Degradative Capacities Of P Putida Kt2440 Cosmid Clones Andmentioning

confidence: 84%

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Gene Cluster of Arthrobacter ilicis Rü61a Involved in the Degradation of Quinaldine to Anthranilate

Parschat

Hauer

Kappl

et al. 2003

Journal of Biological Chemistry

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A genetic analysis of the anthranilate pathway of quinaldine degradation was performed. A 23-kb region of DNA from Arthrobacter ilicis Rü 61a 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.The genetic diversity and flexibility of prokaryotes has led to the evolution of an impressive variety of metabolic pathways to transform or degrade natural as well as numerous xenobiotic compounds. The genes coding for enzymes involved in degradative pathways are often organized as operons and supraoperonic clusters comprising "pathway modules" (1-4).N-Heteroaromatic compounds are metabolized and even mineralized by various bacteria (for a review, see Ref. 5 and references therein). Quinaldine (2-methylquinoline) is utilized by Arthrobacter ilicis Rü 61a as a source of carbon, nitrogen, and energy; its degradation proceeds via the "anthranilate pathway" (5, 6). The initial step, the hydroxylation of quinaldine in para position to the N-heteroatom, is catalyzed by the inducible enzyme quinaldine 4-oxidase (Qox). 1 Qox is a molybdo-iron/sulfur-flavoprotein with an (LMS) 2 subunit structure and has been classified to belong to the xanthine oxidase family of molybdenum enzymes (7-10; for reviews on molybdenum enzymes, see Refs. 11-13). Like many other bacterial molybdenum hydroxylases, e.g. quinoline 2-oxidoreductase (Qor) from Pseudomonas putida 86 (14, 15), isoquinoline 1-oxidoreductase (Ior...

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Comparative EPR and Redox Studies of Three Prokaryotic Enzymes of the Xanthine Oxidase Family: Quinoline 2-Oxidoreductase, Quinaldine 4-Oxidase, and Isoquinoline 1-Oxidoreductase

Cited by 30 publications

References 29 publications

Regioselective aromatic hydroxylation of quinaldine by water using quinaldine 4-oxidase in recombinant Pseudomonas putida

Regioselective aromatic hydroxylation of quinaldine by water using quinaldine 4-oxidase in recombinant Pseudomonas putida

The Mo-Se active site of nicotinate dehydrogenase

Gene Cluster of Arthrobacter ilicis Rü61a Involved in the Degradation of Quinaldine to Anthranilate

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