2,4-DIOXYENASES CATALYZING N-HETEROCYCLIC-RING CLEAVAGE AND FORMATIONOF 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/

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-ox oquinoline 2,4-dioxygenase (QDO) purified from Pseudomonas putida 33/1 . The enzyme-catalyzed conversions were performed in an (O-18(2))O-2/( O-16)O-2 atmosphere. Two oxygen atoms of either (O-18)O-2 or (O-16)O-2 were incorporated at C2 and C4 of the respective substrates, indicati ng that these unusual enzymes, which catalyze the cleavage of two carb on-carbon bonds concomitant with CO formation, indeed are 2,4-dioxygen ases. Both enzymes are small monomeric proteins of 32 kDa (MeQDO) and 30 kDa (QDO). The apparent K-m values of MeQDO fur 1H-3-hydroxy-4-oxoq uinaldine and QDO for 1H-3-hydroxy-4-oxoyuinoline were 30 mu M and 24 mu M, respectively. In both 2,4-dioxygenases, there was no spectral ev idence for the presence of a chromophoric cofactor. EPR analyses of Me QDO did not reveal any signal that could be assigned to an organic rad ical species or to a metal, and X-ray fluorescence spectrometry; of bo th enzymes did not show any metal present in stoichiometric amounts. E thylxanthate, metal-chelating agents (tiron, alpha,alpha'-bipyridyl, 8 -hydroxyquinoline, o-phenanthroline, EDTA, diphenylthiocarbazone, diet hyldithiocarbamate), reagents that modify sulfhydryl groups (iodoaceta mide, N-ethylmaleimide, p-hydroxymercuribenzoate), and reducing agents (sodium dithionite, dithiothreitol, mercaptoethanol) either did not a ffect 2,4-dioxygenolytic activities at all or inhibited at high concen trations only. With respect to the supposed lack of any cofactor and w ith respect to the inhibitors of dioxygenolytic activities, MeQDO and QDO resemble aci-reductone oxidase (CO-forming) from Klebsiella pneumo niae, which catalyzes 1,3-dioxygenolytic cleavage of 1,2-dihydroxy-3-k eto-S-methylthiopentene anion (Wray, J. W. & Abeles, R. H. (1993) J. B iol. Chem. 268, 21466-21469; Wray, J. W. & Abeles, R. H. (1995) J. Bio l. Chem. 270, 3147-3153). 1H-3-Hydroxy-4-oxoquinaldine and 1H-3-hydrox y-4-oxoquinoline were reactive towards molecular oxygen in the presenc e of the base catalyst potassium-tert.-butoxide in the aprotic solvent N,N-dimethylformamide. Base-catalyzed oxidation, yielding the same pr oducts as the enzyme-catalyzed conversions, provides a non-enzymic mod el reaction for 2,4-dioxygenolytic release of CO from 1H-3-hydroxy-4-o xoquinaldine and 1H-3-hydroxy-4-oxoquinoline.