Cloning, expression and characterization of the catalase-peroxidase (KatG) gene from a fast-growing Mycobacterium sp. strain JC1 DSM 3803.
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.
Lee
Hyun-Il
HI
Department of Biochemistry, Graduate School of Medicine, Konkuk University, Seoul 134-701, Korea.
Yoon
Ji-Hyun
JH
Nam
Ji-Sun
JS
Kim
Young-Min
YM
Ro
Young-Tae
YT
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D001665:Binding Sites; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D004795:Enzyme Stability; D015870:Gene Expression; D005798:Genes, Bacterial; D008345:Manganese / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009161:Mycobacterium / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D020102:Mycobacterium smegmatis / Q000235:genetics; D010544:Peroxidases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010802:Phylogeny; D011993:Recombinant Fusion Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012997:Solvents
D001426:Bacterial Proteins; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; D012997:Solvents; C068477:ferric uptake regulating proteins, bacterial; D008345:Manganese; D010544:Peroxidases; C081610:catalase-peroxidase, bacteria