Calcium-Induced Activity and Folding of a Repeat in Toxin Lipase from Antarctic Pseudomonas fluorescens Strain AMS8

Abstract

It is hypothesized that the Ca2+ ions were involved in the activity, folding and stabilization of many protein structures. Many of these proteins contain repeat in toxin (RTX) motifs. AMS8 lipase from Antarctic Pseudomonas fluorescens strain AMS8 was found to have three RTX motifs. So, this research aimed to examine the influence of Ca2+ ion towards the activity and folding of AMS8 lipase through various biophysical characterizations. The results showed that CaCl2 increased lipase activity. The far-UV circular dichroism (CD) and Fourier-transform infrared (FTIR) analysis suggested that the secondary structure content was improved with the addition of CaCl2. Fluorescence spectroscopy analysis showed that the presence of CaCl2 increased protein folding and compactness. Dynamic light scattering (DLS) analysis suggested that AMS8 lipase became aggregated at a high concentration of CaCl2.The binding constant (Kd) value from the isothermal titration calorimetry (ITC) analysis proved that the Ca2+ ion was tightly bound to the AMS8 lipase. In conclusion, Ca2+ ions play crucial roles in the activity and folding of the AMS8 lipase. Calcium binding to RTX nonapeptide repeats sequences will induced the formation and folding of the RTX parallel β-roll motif repeat structure.

The Influence of Calcium toward Order/Disorder Conformation of Repeat-in-Toxin (RTX) Structure of Family I.3 Lipase from Pseudomonas fluorescens AMS8

“…Experimental data on the effect of CaCl 2 on the AMS8 lipase activity supported the idea that Ca 2+ ions contributed to the enzyme structural stability [14]. Far-UV CD spectra revealed that the presence of CaCl 2 improved the secondary structure of AMS8 lipase.…”

“…The lower the value of the k D , the higher the ligand's binding affinity to AMS8 lipase. If the k D value was high, the ligand would be weakly bound to the protein (other target molecules) [14]. Even though the unit measurement was used in the laboratory experiment (ITC) and the in silico study (molecular docking) was different due to the different modes of analysis, both analyses gave the same target, which focused on the binding affinity between the ligand (Ca 2+ ) and protein structure (AMS8 lipase).…”

“…Ca1 and Ca2 were located at the catalytic domain, while Ca3, Ca4, and Ca5 were bound at the RTX parallel β -roll motif repeats structure (RTX domain) in the non-catalytic domain with Ca6 [ 14 ]. Ali et al (2020) revealed that the AMS8 lipase consists of three RTX nonapeptide repeats sequence that built up the RTX parallel β -roll motif repeats structure (residue 373–405) and three of the Ca 2+ ions (Ca3, Ca4, and Ca5) were bound to the RTX parallel β -roll motif repeats structure [ 14 ]. The role of Ca 2+ ions toward the activity and folding of AMS8 lipase has been investigated using various well established biophysical tools.…”

RTX-Toxins

“…On the other hand, it is clear that calcium-mediated conformation changes of the repeats play an important role in target cell recognition and in membrane-activity of certain RTX toxins [13,17]. This is not only the case for pore formation of cytolytic RTX toxins but also for the AMS8 lipase from the Antarctic Pseudomonas fluorescens strain AMS8 [23]. The three RTX motifs of this protein react to the presence of calcium ions.…”

“…The three RTX motifs of this protein react to the presence of calcium ions. The tightly bound calcium results in an increase of lipase activity together with changes in its secondary structure [23].…”