2,4‐Dioxygenases Catalyzing N‐Heterocyclic‐Ring Cleavage and Formation of Carbon Monoxide

Abstract

1H-3-Hydroxy-4-oxoquinaldine 2,4-dioxygenase (MeQDO) was purified from quinaldine-grown Arthrobacter sp. Ru6la. It was enriched %fold in a yield of 22%, and its properties were compared with 1 H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (QDO) purified from Pseudomonas putida 3311. The enzyme-catalyzed conversions were performed in an ('80)0,/('60)0, atmosphere. Two oxygen atoms of either ('*O)O, or (l60)O, 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,, values of MeQDO for 1 H-3-hydroxy-4-oxoquinaldine and QDO for lH-3-hydroxy-4-oxoquinoline were 30 pM and 24 pM, 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 Xray fluorescence spectrometry of both enzymes did not show any metal present in stoichiometric amounts. Ethylxanthate, metal-chelating agents (tiron, n,d-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 j 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 Klebsiellu pneumoniae, which catalyzes 1,3-dioxygenolytic cleavage of 1,2-dihydroxy-3-keto-S-methylthiopentene anion ( Chem. 270,[3147][3148][3149][3150][3151][3152][3153]. 1H-3-Hydroxy-4-oxoquinaldine and 1H-3-hydroxy-4-oxoquinoline were reactive towards molecular oxygen in the presence of the base catalyst potassiumtert.-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 1 H-3-hydroxy-4-oxoquinaldine and 1 H-3-hydroxy-4-oxoquinoline.Keywords: carbon monoxide ; heterocyclic ring cleavage; 3-hydroxy-4( 1H)-quinolone(derivatives) ; 1 H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (decyclizing, CO-forming) ; 1 H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase (decyclizing, CO-forming).Arfhrobacfer sp. strain Rii61 a and Pseudomonas putida strain 33/1 utilize quinaldine (2-methylquinoline) and 1H-4-0~0-quinoline, respectively, as sole source of carbon, nitrogen, and energy. In the gram-positive strain Rii61 a, initial hydroxylation

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“…In parallel, Fetzner and coworkers reported PQS-degrading activity by conversion of PQS to N-octanoylanthranilic acid by the dioxygenase Hod (1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase) of Arthrobacter nitroguajacolicus (162). Hod and the Pseudomonas putida homologue QDO (1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase) (8) belong to an unusual family of cofactor-independent dioxygenases involved in the breakdown of N-heteroaromatic compounds (187). A major drawback in the use of Hod against P. aeruginosa infections is its sensitivity to degradation by P. aeruginosa exoproteases (162), a limiting factor that may be overcome in the following years by protease-resistant variants selected via rational or random mutagenesis.…”

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“…These compounds are widespread in the environment as naturally occurring plant materials, industrially important precursors of dyes, pharmaceuticals and antibiotics, and components of coal tar. The pathways for 1H-4-oxoquinoline and 2-methylquinoline degradation from P. putida 33/1 and Arthrobacter nitroguajacolicus Rü61, respectively, were found to have novel and very interesting ring cleavage dioxygenase enzymes, 1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (Qdo) and 1H-3-hydroxyoxoquinaldine 2,4-dioxygenase (Hod) (7,29). The enzymes are members of the ␣/␤ hydrolase fold superfamily and are related to serine hydrolases (30,82,103).…”

Crystallization and preliminary X-ray analysis of 1H-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase fromArthrobacter nitroguajacolicusRü61a: a cofactor-devoid dioxygenase of the α/β-hydrolase-fold superfamily

“…For instance, oxygenation is often used to make lipids and particularly aromatic molecules amenable to further biochemical transformations. 1H-3-Hydroxy-4-oxoquinaldine 2,4-dioxygenase (HOD; EC 1.13.11.48) from Arthrobacter nitroguajacolicus Rü 61a is a 32 kDa enzyme that is involved in the anthranilate pathway of quinaldine degradation (Bauer et al, 1996). It catalyses the O 2 -dependent N-heteroaromatic ring cleavage of 1H-3-hydroxy-4-oxoquinaldine to N-acetylanthranilate and carbon monoxide (Fig.…”

“…HOD displays no sequence similarities to other known oxygenases, except for the 1H-3-hydroxy-4-oxoquinoline 2,4-dioxygenase (QDO; EC 1.13.11.47) involved in 1H-4-oxoquinoline degradation by Pseudomonas putida 33/1 (Qi et al, 2007). QDO catalyses a reaction very similar to that catalysed by HOD; namely, the dioxygenolytic N-heteroaromatic ring cleavage of 1H-3-hydroxy-4-oxoquinoline to N-formylanthranilate and carbon monoxide (Bauer et al, 1996). HOD and QDO share 37% identity at the sequence level and these two enzymes are the only dioxygenases that have been proposed to be members of the / -hydrolase-fold enzymes.…”

“…The mechanism used by nonhaem iron-dependent (Que, 1999), copper-dependent (Steiner et al, 2002), flavin-dependent and pterin-dependent (Massey, 1994;Palfey et al, 1995) oxygenases have been studied and various reviews are available that discuss the general mechanisms of oxygen and substrate activation in enzymatic oxygenation reactions. Biochemical and spectroscopic studies have shown that neither HOD nor QDO contain organic cofactors or stoichiometric amounts of any metal (Bauer et al, 1996;Fetzner, 2002). The mechanism(s) employed by these dioxygenases are therefore very interesting from the viewpoint of fundamental enzymology.…”