Functional expression of the quinoline 2-oxidoreductase genes (qorMSL) in Pseudomonas putida KT2440 pUF1 and in P. putida 86-1 deltaqor pUF1 and analysis of the Qor proteins.
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 deltaqor were used as recipients for pUF1. Whereas Qor from the wild-type strain showed a specific activity of 19-23 U x mg(-1), the specific activity of Qor purified from P. putida KT2440 pUF1 was only 0.8-2.5 U x mg(-1), and its apparent k(cat) (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 deltaqor 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.
Frerichs-Deeken
Ursula
U
AG Mikrobiologie, Institut für Chemie und Biologie des Meeres, Carl von Ossietzky Universität Oldenburg, Germany.
Goldenstedt
Birgit
B
Gahl-Janssen
Renate
R
Kappl
Reinhard
R
Hüttermann
Jürgen
J
Fetzner
Susanne
S
D001426:Bacterial Proteins / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites / Q000502:physiology; D002384:Catalysis; D003001:Cloning, Molecular; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation / Q000502:physiology; D015870:Gene Expression; D007501:Iron / Q000032:analysis; D008982:Molybdenum / Q000032:analysis; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000737:chemistry* / Q000235:genetics; D044925:Oxidoreductases Acting on CH-CH Group Donors; D016958:Pseudomonas putida / Q000201:enzymology*; D013056:Spectrophotometry, Ultraviolet; D013455:Sulfur / Q000032:analysis
D001426:Bacterial Proteins; D013455:Sulfur; D008982:Molybdenum; D007501:Iron; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C068667:quinoline 2-oxidoreductase