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

Nur Shidaa Mohd Ali,Raja Noor Zaliha Raja Abd Rahman,Thean Chor Leow,Mohd Shukuri Mohamad Ali,Abu Bakar Salleh

doi:10.3390/toxins12010027

Nur Shidaa Mohd Ali, Raja Noor Zaliha Raja Abd Rahman + Show 3 more

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https://doi.org/10.3390/toxins12010027

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Journal: Toxins	Publication Date: Jan 1, 2020
Citations: 5	License type: CC BY 4.0

Affiliation: Universiti Putra Malaysia

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.

Highlights

Repeat in toxin (RTX) proteins represent a broad and diverse family of proteins produced byGram-negative bacteria
In order to further analyze the presence of Ca2+ ions in the AMS8 lipase predicted structure, we used the database to explore the calcium-binding site of each of the Ca2+ ions
AMS8 lipase from Pseudomonas fluorescence belongs to the repeat in toxin (RTX) lipases group since it contains an RTX parallel β-roll motif repeat structure at the C-terminal

Summary

Introduction

Repeat in toxin (RTX) proteins represent a broad and diverse family of proteins produced byGram-negative bacteria. Repeat in toxin (RTX) proteins represent a broad and diverse family of proteins produced by. RTX proteins can be divided into several classes including RTX lipases, RTX proteases, RTX cytotoxins and Multifunctional-autoprocessing repeats-in-toxin (MARTX) [1]. The first characteristic is the appearance of nonapeptide sequences (GGXGXDXUX) (consisting of glycine (G), aspartate (D), any amino acid (X). The nonapeptide sequences are responsible for creating specific calcium-binding positions and after calcium binding, the parallel β-roll motif repeat structure of the RTX was formed [2]. The RTX parallel β-roll motif repeat structure comprised nonapeptide sequences and Ca2+ ions [3]. The RTX parallel β-roll motif repeat structure was involved in the process of exporting the passenger protein (including RTX proteins) out of the bacterial cell through

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