STRESS RESPONSES - Cyanobacteria - page 688

A . variabilis ATCC 29413, No . spumigena CCY9414, N . punctiforme PCC 73102 and Anabaena sp. strain
PCC 7120 KatG gene sequences are absent (Bernroitner et al ., 2009). The cyanobacterial KatG enzyme
is dimeric (Obinger et al ., 1997; Jakopitsch et al ., 1999) as also KatGs from Bacillus stearothermophilus
(Loprasert et al ., 1989) and Streptomyces sp. (Youn et al ., 1995). Monomeric ( Halobacterium halobium ,
Fukumori et al. , 1985) and tetrameric KatGs ( E . coli ; Claiborne and Fridovich, 1979; Mycobacterium
smegmatis ; Marcinkeviciene et al ., 1995 and Rhodobacter capsulatus ; Hochman and Shemesh, 1987;
Forkl et al ., 1993) are also known in the respective bacterial species mentioned in parentheses.
Cloning, nucleotide sequencing, expression of recombinant protein in E . coli of katG gene and
KatG enzyme and its kinetic properties from S . elongatus PCC 7942 (Mutsuda et al ., 1996) and
Synechocystis sp. strain PCC 6803 (Jakopitsch et al ., 1999; Regelsberger et al ., 1999) have been reported.
The S . elongatus PCC 7942 KatG exhibits high homology with Synechocystis sp. strain PCC 6803 KatG
enzyme. The ORF of katG of S . elongatus PCC 7942 is 2160 bp long that encodes a protein of 720
amino acids long with a molecular mass of ~79.9 kDa. The native enzyme purifi ed to homogeneity
also revealed a molecular mass of 150 kDa composed of two identical subunits with a mass of 79
kDa. The absorption spectrum revealed the presence of a typical of protohaem molecule per dimer.
The native enzyme purifi ed from the cells and the enzyme overexpressed in E . coli [BL21(DE3)pLyS]
exhibited similar kinetic properties for catalase activity and the same ratio of catalase to peroxidase
activity (Mutsuda et al ., 1996). The purifi cation and kinetic properties of KatG enzyme from cytosolic
extracts of A . nidulans ( S . elongatus PCC 7942) revealed that the enzyme possesses two identical
subunits of equal size (80.5 kDa). It exhibits both catalase and O-dianisidine peroxidase activity
(as it accepts electrons from O-dianisidine but not from ascorbate, glutathione and NADH). The
catalase activity has been found to be very effi cient with a pH optimum between pH 6.5 and 7.5
(in contrast to broad pH range shown by monofunctional catalases) and a K m for H 2 O 2 of 4.3 mM with a
calculated turnover number of 7200 s -1 . The KatG of A . nidulans is insensitive to the eukaryotic catalase
inhibitor 3-amino-1,2,4-triazole (in contrast to the sensitive nature of monofunctional catalases) but
sensitive to cyanide (K i =27.2 µM; Obinger et al ., 1997). Synechocystis sp. strain PCC 6803 katG gene that
encodes a bifunctional catalase-peroxidase has been cloned and the coding sequence was extended by
attaching a hexahistidine tag at the C-terminus and the gene sequence was used to transform E . coli
[BL 21(DE3)pLyS] strain using the pET-3a vector. The enzyme overexpressed in E . coli was purifi ed
to homogeneity and shown to exist in a homodimeric state with a molecular mass of 170 kDa. As
typical of bifunctional catalase-peroxidase, the KatG enzyme showed a high catalase activity with
a Km of 4.9 ± 0.25 mM for H 2 O 2 and turnover number of 3500 s -1 . It also showed peroxidase activity
with O-dianisidine, guaiacol and pyrogallol but not with NAD(P)H, ferricytochrome c , ascorbate or
glutathione as electron donors (Jakopitsch et al ., 1999). The KatG enzyme from cytosolic extracts of
Synechocystis sp. strain PCC 6803 purifi ed to homogeneity by a six-step purifi cation procedure also
exhibited almost similar kinetic parameters described by Jakopitsch et al . (1999) for the recombinant
enzyme produced in E . coli . Spectroscopy of the pyridine ferrochrome revealed iron protoporphyrin
IX as the prosthetic group. Peptide mass mapping (by matrix-assisted laser desorption ionization
time-of-fl ight mass spectrometry) established identity between the amino acid sequence of purifi ed
KatG from Synechocystis sp. strain PCC 6803 and the deduced amino acid sequence from katG gene
sequence from the same organism (Regelsberger et al ., 1999).The in vivo role of KatG from Synechocystis
sp. strain PCC 6803 was investigated by the isolation of a katG deletion mutant. Surprisingly the
mutant showed resistance to H 2 O 2 and MV-induced stress because of its residual H 2 O 2 scavenging
activity (30 times lower than that of the wild-type), though the mutant was indistinguishable from
the wild-type in doubling time and other growth characteristics. The residual peroxidase activity
seemed to be suffi cient to protect the cells from oxidative stress and the protective role of KatG