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 Dqor were used as recipients for pUF1. Whereas Qor from the wild-type strain showed a specific activity of 19-23 UAEmg )1 , the specific activity of Qor purified from P. putida KT2440 pUF1 was only 0.8-2.5 UAEmg )1 , and its apparent k cat (quinoline) was about ninefold lower than that of wild-type Qor. The apparent K m 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 Dqor 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.Keywords: quinoline 2-oxidoreductase; molybdenum hydroxylase; expression cloning; molybdopterin cytosine dinucleotide; Pseudomonas sp.Quinoline 2-oxidoreductase (Qor) from Pseudomonas putida 86 catalyses the formation of 1H-2-oxoquinoline (2-hydroxyquinoline) from quinoline [1,2]. Besides quinoline, some quinoline derivatives and the benzodiazines quinazoline and quinoxaline are accepted as substrates [1,3]. Like other enzymes catalysing the hydroxylation of N-heteroaromatic rings at positions that are susceptible to nucleophilic attack, Qor belongs to the family of molybdenum hydroxylases that introduce an oxygen atom (originating from water) into their substrate according to the following stoichiometry: R-H + H 2 O fi R-OH + 2[e -] + 2H + . Due to a common structure of their molybdenum center and due to significant amino acid sequence similarity to xanthine oxidases/xanthine dehydrogenases, the molybdenum hydroxylases have also been classified as enzymes belonging to the Ôxanthine oxidase familyÕ [4][5][6][7]. Molybdenum hydroxylases basically contain the same type of redox centers constituting an intramolecular electron transport chain, namely a molybdenum ion, that is the site of substrate hydroxylation, two distinct [2Fe)2S] clusters, and -in most cases -FAD [5,8,9]. The molybdenum is bound to the sulfur atoms of the ene-dithiolate function of a unique pyranopterin cofactor. Other coordination positions to the molybdenum are occupied by a sulfido and an oxo ligand, and a catalytically l...