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

Abstract

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.

Manganese(II) complexes with thiosemicarbazones as potential anti-Mycobacterium tuberculosis agents

“…For 5, a third band is observed at 369.00 nm, corresponding to a major contribution from phenyl ring π-π* internal transitions. As expected, spin-forbidden d-d bands were not observed in the spectra of the high-spin d 5 Mn(II) complexes presented in this work.…”

“…Catalase-peroxidase (CP or KatG) is a heme protein that exhibits both catalase and peroxidase activities and plays a crucial role in defending against oxidative stress generated by aerobic oxygen metabolism or phagocytic attack from macrophages or neutrophils [5,6]. In MTB the KatG is responsible for the conversion of Mn(II) to Mn(III) in metalloenzymes containing manganese in active sites and has attracted special attention due to its participation in mycobacterial virulence [7].…”

“…In this context, it was demonstrated that the redox reversible metal complex [Fe II (CN) 5 (INH)] 3 − [10] containing the pro-drug was able to overcome the drug resistance. This complex presents a minimal inhibitory concentration (MIC, defined as the lowest concentration resulting in 90% of growth inhibition of the bacteria) value similar to that of free isoniazid as well as a good selectivity index.…”

Molecular cloning, purification, and characterization of a superoxide dismutase from a fast-growing Mycobacterium sp. Strain JC1 DSM 3803

“…This KatG exhibited typical enzymatic characters of mycobacterial KatGs and the antibodies raised against it strongly cross-reacted with other mycobacterial KatGs (Ro et al, 2003). Recently, we cloned a DNA fragment containing two complete open reading frames (ORFs) encoding a putative ferric uptake regulator A (FurA) and a putative KatG protein (Lee et al, 2010). Genetic analysis of the cloned furA and katG genes revealed that their gene organization and sequence homology were highly homologous to those of other mycobacteria.…”

“…Two nested primers, Sod-NF (5 -CAGATCAACGAGCTTCACCA-3 ) and Sod-NR (5 -TAGTCCGCCTTCACGTTCTT-3 ), were synthesized to screen the previously constructed JC1 genomic cosmid library (Lee et al, 2010), based on the real JC1 partial SOD gene sequence identified above. The PCR screening was carried out in a 20 l reaction mixture containing 1 l of JC1 cosmid DNA, 50 pmol of each primer, and 1 unit of HotStar Taq plus DNA polymerase.…”