Studies On The Functions Of Gene Cluster In Klebsiella Pneumoniae M5al Encoding Enzymes For 3-hydroxybenzoate Catabolism

Gentisate (2,5-dihydroxybenzoate) pathway is one of the three typical routes for bacterial aromatic catabolism. This pathway plays an important role in bacterial aromatic catabolism, but little is known at the biochemical and molecular levels compared with the other two pathways through catechol metabolism. Klebsiella pneumoniae M5a1 metabolizes 3-hydroxybenzoate via gentisate to central metabolites. We have determined the sequence of a 8kb region spanning the mhb genes. The individual genes from the cluster mhbRTMDIH of 3-hydroxybenzoate-degrading Klebsiella pneumoniae M5a1 were cloned and overexpressed. By cloning into expression vectors and by biochemical analyses, four of these genes (mhbMDIH) have been shown to encode the enzymes involved in the catabolism of 3-hydroxybenzoate to fumarate and pyruvate through the gentisate pathway. MhbM is a 3-hydroxybenzoate-6-hydroxylase that catalyzed the para-hydroxylation of 3-hydroxybenzoate(3-HBA) to 2,5-dihydroxybenzoate (gentisate). MhbD is a gentisate 1,2-dioxygenase which converts gentisate to maleylpyruvate. MhbI is a reduced glutathione-dependent maleylpyruvate isomerase catalyzing the isomerization of maleylpyruvate to fumarylpyruvate. MhbH is a fumarylpyruvate hydrolase which hydrolyzes fumarylpyruvate to fumarate and pyruvate. MhbM is also purified and its dynamics characterization was performed. Frame-shift mutants of mhbR and mhbT were also made to investigate their possible roles in 3-hydroxybenzoate catabolism. This project is funded by the National Natural Science Foundation of China (No. 30170036). The current result reported here is the second example of the research onthe genes encoding enzymes involved in the entire gentisate pathway and the 3-hydroxybenzoate-6-hydroxylase was studied at molecular level for the first time.