Role of Active Site Residues in Catalytic Activities of Ascorbate Peroxidase from Leishmania Major
IR@IICB: CSIR-Indian Institute of Chemical Biology, Kolkata
View Archive Info| Field | Value | |
| Title |
Role of Active Site Residues in Catalytic
Activities of Ascorbate Peroxidase from
Leishmania Major
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| Creator |
Yadav, Rajesh K
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| Subject |
Structural Biology & Bioinformatics
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| Description |
Peroxidases hold a venerable position in the history of enzymology and
predominantly in enzyme kinetics. This is one of the most extensively studied group of
enzymes and literature has been enriched with the reviews and a large number of basic
research papers dating back to the early part of the nineteenth century. The plant peroxidase,
particularly horseradish peroxidase C (HRPC) from horseradish root, helped to usher in the
modern era of enzymology dating back to 1920s and 1930s. The formation of
spectroscopically distinct intermediates during the peroxide catalyzed cycle is helped to
shape the development of the methodologies on the steady state and rapid reaction kinetics in
the 1940s. Peroxidases therefore bear considerable historical significance. A major
breakthrough in peroxidase research has come through the X-ray crystal structure of yeast
cytochrome c peroxidase (CCP) in the year 1980. From their discovery both HRP and CCP
are considered as model peroxidases to carry out structure function study of other
peroxidases. During 1940s to 1960s a large number of peroxidases are reported from animal
sources and the nature and physiological functions of these enzymes were the subject of
research. Animal peroxidases are shown to play many important physiological functions like
hormone synthesis, phagocytic killing of bacteria, antibacterial functions in saliva and milk,
catabolism of estradiol in the uterus and fertilization of ovum to cite as examples. A large
body of literature shows that peroxidases in conjunction to catalase and glutathione
peroxidase metabolize the physiologically produced cellular hydrogen peroxide (H2O2), and
thus protect cells from the detrimental effect of H2O2. Ascorbate dependent peroxidase
(APX) was discovered in 1979. APX from pea and soybean plant are well-characterized
biochemically and their crystal structures have been solved in great detail in the year 1995. In
plants different isoforms of APX are shown to localize in chloroplast, cytosol and microbody
where they eliminate photosynthetically generated H2O2 by glutathione/ascorbate cycle and
control H2O2 mediated redox signaling. With the advent of science and technology the
genome sequence project of several pathogenic microorganisms are taken to better
understand their biology. The genome sequence of Leishmania major has revealed the
presence of a single copy putative plant like ascorbate dependent heme peroxidase gene
(LmAPX). This gene has been cloned, expressed and purified in our laboratory for the first
time. Earlier study from this laboratory showed that the recombinant protein could catabolize
H2O2 in vitro. Due to its digenetic life cycle Leishmania has to encounter rigorous oxidizing
environment as host immune response along with reactive oxygen species (ROS) generated
by its own physiological process. But they lack catalase and selenium containing glutathione
peroxidase, the enzymes of first line defense against H2O2 in the most of organisms. Hence,
the mechanism by which it withstands the toxic effects of H2O2 is still unclear. This has
actually stimulated us to take up this problem and to identify the role of LmAPX enzyme in
parasite H2O2 metabolism and survival by detail biochemical and cell biology approach.
LmAPX decompose H2O2 to H2O by peroxidative cycle. Interestingly LmAPX oxidizes both
small molecule ascorbate as well as macromolecule cytochrome c. It is well known that CCP
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oxidizes cytochrome c using protein radical but APX oxidizes ascorbate using porphyrin
radical intermediate. Naturally our first aim will be to identify actual reaction intermediate in
LmAPX. Therefore “Chapter-I” of this dissertation deals with, detailed mechanism and the
nature of it. Not only this, how protein architecture controls the orientation of both heme group
and active site residues. Hence in the “Chapter-II” of this dissertation we will discuss the role
of surface acidic residue in proper maintenance of active site conformation of LmAPX.
Moreover, ascorbate oxidation rate of LmAPX is slower than plant APX. Finally “Chapter-
III” of this dissertation unravels the reason of slow ascorbate turnover by creating Phe / Arg
switch in LmAPX.
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| Date |
2010
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| Type |
Thesis
NonPeerReviewed |
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| Format |
application/pdf
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| Identifier |
http://www.eprints.iicb.res.in/445/1/Thesis_Rajesh.pdf
Yadav, Rajesh K (2010) Role of Active Site Residues in Catalytic Activities of Ascorbate Peroxidase from Leishmania Major. PhD thesis, Jadavpur University. |
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| Relation |
http://www.eprints.iicb.res.in/445/
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