Investigation of calcium-dependent activity and conformational dynamics of zebra fish 12-lipoxygenase
(2017) In Biochimica et Biophysica Acta - General Subjects 1861(8). p.2099-2111- Abstract
Background A 12-lipoxygenase in zebra fish (zf12-LOX) was found to be required for normal embryonic development and LOXs are of great interest for targeted drug designing. In this study, we investigate the structural-functional aspects of zf12-LOX in response to calcium. Methods A soluble version of zf12-LOX was created by mutagenesis. Based on multiple sequence alignment, we mutated the putative calcium-responsive amino acids in N-PLAT domain of soluble zf12-LOX. Using a series of biophysical methods, we ascertained the oligomeric state, stability, structural integrity and conformational changes of zf12-LOX in response to calcium. We also compared the biophysical properties of soluble zf12-LOX with the mutant in the absence and... (More)
Background A 12-lipoxygenase in zebra fish (zf12-LOX) was found to be required for normal embryonic development and LOXs are of great interest for targeted drug designing. In this study, we investigate the structural-functional aspects of zf12-LOX in response to calcium. Methods A soluble version of zf12-LOX was created by mutagenesis. Based on multiple sequence alignment, we mutated the putative calcium-responsive amino acids in N-PLAT domain of soluble zf12-LOX. Using a series of biophysical methods, we ascertained the oligomeric state, stability, structural integrity and conformational changes of zf12-LOX in response to calcium. We also compared the biophysical properties of soluble zf12-LOX with the mutant in the absence and presence of calcium. Results Here we provide a detailed characterization of soluble zf12-LOX and the mutant. Both proteins exist as compact monomers in solution, however the enzyme activity of soluble zf12-LOX is significantly increased in presence of calcium. We find that the stimulatory effect of calcium on zf12-LOX is related to a change in protein structure as observed by SAXS, adopting an open-state. In contrast, enzyme with a mutated calcium regulatory site has reduced activity-response to calcium and restricted large re-modeling, suggesting that it retains a closed-state in response to calcium. Taken together, our study suggests that Ca2 +-dependent regulation is associated with different domain conformation(s) that might change the accessibility to substrate-binding site in response to calcium. General significance The study can be broadly implicated in better understanding the mode(s) of action of LOXs, and the enzymes regulated by calcium in general.
(Less)
- author
- Mittal, Monica ; Hasan, Mahmudul LU ; Balagunaseelan, Navisraj ; Fauland, Alexander ; Wheelock, Craig E. ; Rådmark, Olof ; Haeggström, Jesper Z and Rinaldo-Matthis, Agnes
- organization
- publishing date
- 2017-08-01
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- Activity, Calcium, Dynamic light scattering, Eicosanoids, Lipoxygenases, SAXS
- in
- Biochimica et Biophysica Acta - General Subjects
- volume
- 1861
- issue
- 8
- pages
- 13 pages
- publisher
- Elsevier
- external identifiers
-
- pmid:28528958
- wos:000404702600020
- scopus:85020125300
- ISSN
- 0304-4165
- DOI
- 10.1016/j.bbagen.2017.05.015
- language
- English
- LU publication?
- yes
- id
- 3f971d71-a9a5-4956-90fc-e18191146000
- date added to LUP
- 2017-06-29 08:55:22
- date last changed
- 2025-01-07 16:16:53
@article{3f971d71-a9a5-4956-90fc-e18191146000,
abstract = {{<p>Background A 12-lipoxygenase in zebra fish (zf12-LOX) was found to be required for normal embryonic development and LOXs are of great interest for targeted drug designing. In this study, we investigate the structural-functional aspects of zf12-LOX in response to calcium. Methods A soluble version of zf12-LOX was created by mutagenesis. Based on multiple sequence alignment, we mutated the putative calcium-responsive amino acids in N-PLAT domain of soluble zf12-LOX. Using a series of biophysical methods, we ascertained the oligomeric state, stability, structural integrity and conformational changes of zf12-LOX in response to calcium. We also compared the biophysical properties of soluble zf12-LOX with the mutant in the absence and presence of calcium. Results Here we provide a detailed characterization of soluble zf12-LOX and the mutant. Both proteins exist as compact monomers in solution, however the enzyme activity of soluble zf12-LOX is significantly increased in presence of calcium. We find that the stimulatory effect of calcium on zf12-LOX is related to a change in protein structure as observed by SAXS, adopting an open-state. In contrast, enzyme with a mutated calcium regulatory site has reduced activity-response to calcium and restricted large re-modeling, suggesting that it retains a closed-state in response to calcium. Taken together, our study suggests that Ca<sup>2 +</sup>-dependent regulation is associated with different domain conformation(s) that might change the accessibility to substrate-binding site in response to calcium. General significance The study can be broadly implicated in better understanding the mode(s) of action of LOXs, and the enzymes regulated by calcium in general.</p>}},
author = {{Mittal, Monica and Hasan, Mahmudul and Balagunaseelan, Navisraj and Fauland, Alexander and Wheelock, Craig E. and Rådmark, Olof and Haeggström, Jesper Z and Rinaldo-Matthis, Agnes}},
issn = {{0304-4165}},
keywords = {{Activity; Calcium; Dynamic light scattering; Eicosanoids; Lipoxygenases; SAXS}},
language = {{eng}},
month = {{08}},
number = {{8}},
pages = {{2099--2111}},
publisher = {{Elsevier}},
series = {{Biochimica et Biophysica Acta - General Subjects}},
title = {{Investigation of calcium-dependent activity and conformational dynamics of zebra fish 12-lipoxygenase}},
url = {{http://dx.doi.org/10.1016/j.bbagen.2017.05.015}},
doi = {{10.1016/j.bbagen.2017.05.015}},
volume = {{1861}},
year = {{2017}},
}