Functional expression of the quinoline 2-oxidoreductase genes (qorMSL) in Pseudomonas putida KT2440 pUF1 and in P-putida 86-1 Δqor 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.mg(-1), the specific activity of Qor purified from P. putida KT2440 pUF1 was only 0.8-2.5 U.mg(-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 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.