Molecular characterization of the mde operon involved in L-methionine catabolism of Pseudomonas putida

A 15-kb region of Pseudomonas putida chromosomal DNA containing the rode operon and an upstream regulatory gene (mdeR) has been cloned and sequenced. The mde operon contains two structural genes involved in L-methionine degradative metabolism: the already-identified mdeA, which encodes L- methionine γ-lyase (H. Inoue, K. Inagaki, M. Sugimoto, N. Esaki, K. Soda, and H. Tanaka. J. Biochem. (Tokyo) 117:1120-1125, 1995), and mdeB, which encodes a homologous protein to the homodimeric-type E1 component of pyruvate dehydrogenase complex. A rho-independent terminator was present just downstream of mdeB, and open reading frames corresponding to other components of α-keto acid dehydrogenase complex were not found. When MdeB was overproduced in Escherichia coli, the cell extract showed the E1 activity with high specificity for α-ketobutyrate rather than pyruvate. These results suggest that MdeB plays an important rule in the metabolism of α- ketobutyrate produced by MdeA from L-methionine. Accordingly, mdeB encodes a novel E1 component, α-ketobutyrate dehydrogenase E1 component, of an unknown α-keto acid dehydrogenase complex in P. putida. In addition, we found that the ruder gene was located on the opposite strand and began at 127 bp from the translational start site of mdeA. The mdeR gene product has been identified as a member of the leucine-responsive regulatory protein (Lrp) family and revealed to act as an essential positive regulator allowing the expression of the mdeAB operon.