Investigation of calcium-dependent activity and conformational dynamics of zebra fish 12-lipoxygenase

Biochim Biophys Acta Gen Subj. 2017 Aug;1861(8):2099-2111. doi: 10.1016/j.bbagen.2017.05.015. Epub 2017 May 19.


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.

Keywords: Activity; Calcium; Dynamic light scattering; Eicosanoids; Lipoxygenases; SAXS.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Arachidonate 12-Lipoxygenase / chemistry
  • Arachidonate 12-Lipoxygenase / metabolism*
  • Binding Sites
  • Calcium / pharmacology*
  • Humans
  • Models, Molecular
  • Protein Structure, Tertiary
  • Sequence Homology, Amino Acid
  • Zebrafish / metabolism*


  • Arachidonate 12-Lipoxygenase
  • Calcium