In past Zebra fish Danio rerio

In past, Zebra fish (Danio rerio) has emerged as a suitable model for early vertebrate development, and a number of targeted mutations in the zebrafish genome led to phenotypic alterations that resemble human diseases [34]. A 12-LOX in Zebra fish has been cloned and knock down of this zf12-LOX enzyme showed its indispensability for embryonic development [35], [36]. This makes it a very good candidate to study how zf12-LOX behaves in solution and how presence of calcium and/or ARM1 of putative calcium binding residues alter the biophysical and functional properties of enzyme. Here we aimed to characterize zf12-LOX and created a soluble and more stable version of the enzyme. The solution structure of zf12-LOX has been investigated. Then we describe that Ca activates the oxygenase activity of soluble zf12-LOX and this is related to a conformational change of the protein structure as observed by SAXS and other biophysical techniques. In comparison to the homologous crystal structures, zf12-LOX is suggested to display an outward shift of the N-terminal PLAT domain w.r.t. catalytic domain, thus adopting an open-state. The region near the active site also remodels, where all these changes may provide an easy transit to the incoming substrate into active site. Further, the role for putative calcium regulatory site (site-2) has been discussed and the site-2 mutant is shown to have reduction in calcium induced enzyme activity and adopts a closed state in solution. This is a detailed investigation that links Ca2 + binding with the activity of LOX and can be extended to other enzymes as well.
Materials and methods
Results
Discussion Among several factors that can regulate LOXs, transfer from cytosol to membranes is a common property for most of the enzymes in the family [8], [18], [56]. Ca has been a pre-requisite for membrane binding and thus activity, as described in many cases both in vitro and in vivo. However, very few studies have addressed the effects of Ca on LOX physical properties and the conformational changes that the enzyme may undergo upon Ca-binding. In this study, we found that the oxygenase activity of zf12-LOX is up-regulated by Ca in presence of phosphatidylcholine. We generated a stabilized form of zf12-LOX protein by exchange of four residues (F74G, I75S, S241A, C558A), which was also activated by Ca. On comparing the sequence alignments for known Ca activated LOXs, zf12-LOX shows a ‘site-2’ stretch containing putative Ca binding residues. Mutagenesis of site-2 residues rendered the enzyme less active in presence of Ca, thus indicating a potential role in Ca regulation of enzyme catalysis. The KD for binding of Ca to soluble zf12-LOX was estimated as 10±2μM, similar to values reported for 5(S)-LOX [16]. The observation that the site-2 mutant is less active than its soluble counterpart is intriguing. There was no change in secondary structure (CD spectroscopy), or the oligomeric state as seen earlier for some LOXs earlier [21]. However, the reduced maximum activity of the site-2 mutant suggests ‘action at a distance’ behavior. Our in-solution studies provide an evidence for the above effect. Although the Rh and Rg values are similar for soluble zf12(S)-LOX and the site-2 mutant proteins, the pair distribution P(r) profiles and ab-initio modeling clearly indicate the differential mass distributions in space. The observed differences in solution envelopes could be compared with the probable movements that the enzyme experiences w.r.t. the template. The Ca-induced movements are more pronounced in soluble zf12-LOX, which was also supported by the evident decrease in transition temperature (thermostability assay) in response to calcium. Mutation of site-2 residues shows a rather more rigid state of mutant protein, thus suggesting that this might govern the re-positioning of N-PLAT and catalytic domains, directly or indirectly through calcium. The Ca induced movements of the N-PLAT and catalytic domain regions are very different in the model structures of soluble zf12-LOX and the site-2 mutant, resulting in distinct ‘open’ and ‘closed’ states in the absence and presence of Ca. Additionally the remodeling dynamics has also been observed in the center region of the solution envelopes of zf12-LOX that corresponds to the active site of the enzyme (Fig. 8). Previous studies have suggested that the α2-helix region near the active site acts as a “lid” in an otherwise open active site [26], [57], [58]. This suggests that calcium binding to the N-domain may allow zf12-LOX to adopt a state more accessible for the substrate (AA) into the active site in C-catalytic domain. Fiddling with the site-2 residues renders the protein trapped in a more ‘closed state’, resistant to Ca induced transitions, also seen in case of decrease in calcium dependent activity of site-2 mutant. Thus, our study supports the concept of multiple conformations as reviewed for many other LOXs [59], [60]. We also provide an explanation for the possible allosteric connection between the N- and C-terminal domains of zf12-LOX region in response to calcium that can be extended to the exposure of putative substrate entrance channels and altered enzyme activity.