TAILIEUCHUNG - Báo cáo khoa học: Functional expression of the quinoline 2-oxidoreductase genes (qorMSL) in Pseudomonas putida KT2440 pUF1 and in P. putida 86-1 Dqor pUF1 and analysis of the Qor proteins

The availability of a system for the functional expression of genes coding formolybdenumhydroxylases is a prerequisite for the construction of enzyme variants bymutagenesis. For the expression cloning of quinoline 2-oxidoreductase (Qor) fromPseudomonas putida86 – that contains the molybdo-pterin cytosine dinucleotide molybdenum cofactor (Mo-MCD), two distinct [2Fe)2S] clusters and FAD – the qorMSLgenes were inserted into the broad host range vector, pJB653, generating . putidaKT2440 and P. putida86-1Dqor were used as recipients for pUF1 | Eur. J. Biochem. 270 1567-1577 2003 FEBS 2003 doi Functional expression of the quinoline 2-oxidoreductase genes qorMSL in Pseudomonas putida KT2440 pUF1 and in P. putida 86-1 Dqor pUF1 and analysis of the Qor proteins Ursula Frerichs-Deeken1 Birgit Goldenstedt1 2 Renate Gahl-JanBen1 Reinhard KaDDl3. Jurgen Huttermann3 and Susanne Fetzner1 2 1AG Mikrobiologie Institut fur Chemie und Biologie des Meeres Carl von Ossietzky Universitat Oldenburg Germany 2Institut fur Mikrobiologie Westfalische Wilhelms-Universitat Munster Germany 3Fachrichtung Biophysik und Physikalische Grundlagen der Medizin Universitdt des Saarlandes Homburg Saar Germany 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 molybdo-pterin 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 U-mg-1 the specific activity of Qor purified from P. putida KT2440 pUF1 was only U-mg-1 and its apparent kcat 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 Dqor pUF1 were essentially .

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