Cloning, expression and characterization of the catalase–peroxidase (KatG) gene from a fast-growing Mycobacterium sp. strain JC1 DSM 3803

摘要

The gene encoding a catalase–peroxidase (KatG) was cloned from chromosomal DNA of a fast-growing Mycobacterium sp. strain JC1 DSM 3803. The nucleotide sequence of a 5.7 kb EcoRI fragment containing the katG and its flanking regions was determined. The fragment (5,706 bps) contained two complete open reading frames (ORFs) encoding putative ferric uptake regulator A (FurA) and KatG proteins. The cloned gene, katG, had an ORF of 2241 nt, encoding a protein with calculated molecular mass of 81,748 Da. The furA was located in the upstream of the katG with the same transcriptional direction and there was a 38 bp gap space between them. The deduced KatG and FurA protein sequences showed significant homologies to KatG2 and Fur2 of Mycobacterium smegmatis and clustered with other mycobacterial KatG and Fur-like proteins in phylogenetic trees, respectively. The recombinant KatG overproduced in Escherichia coli was nearly indistinguishable from the native JC1 catalase–peroxidase in enzymatic properties and also possessed the resistance to organic solvents, indicating that the cloned katG truly encodes the Mycobacterium sp. JC1 catalase–peroxidase. Difference spectroscopy revealed Mn(II) binding near the haem of the KatG. Transcript analysis of the furA–katG using RT–PCR suggests that the katG is independently transcribed from the furA.