Date Published: July 3, 2017
Publisher: Public Library of Science
Author(s): Fatemeh S. Jazayeri, Mehriar Amininasab, Saman Hosseinkhani, Eugene A. Permyakov.
Luciferase is the key component of light production in bioluminescence process. Extensive and advantageous application of this enzyme in biotechnology is restricted due to its low thermal stability. Here we report the effect of heating up above Tm on the structure and dynamical properties of luciferase enzyme compared to temperature at 298 K. In this way we demonstrate that the number of hydrogen bonds between N- and C-domain is increased for the free enzyme at 325 K. Increased inter domain hydrogen bonds by three at 325 K suggests that inter domain contact is strengthened. The appearance of simultaneous strong salt bridge and hydrogen bond between K529 and D422 and increased existence probability between R533 and E389 could mechanistically explain stronger contact between N- and C-domain. Mutagenesis studies demonstrated the importance of K529 and D422 experimentally. Also the significant reduction in SASA for experimentally important residues K529, D422 and T343 which are involved in active site region was observed. Principle component analysis (PCA) in our study shows that the dynamical behavior of the enzyme is changed upon heating up which mainly originated from the change of motion modes and associated extent of those motions with respect to 298 K. These findings could explain why heating up of the enzyme or thermal fluctuation of protein conformation reduces luciferase activity in course of time as a possible mechanism of thermal functional inactivation. According to these results we proposed two strategies to improve thermal stability of functional luciferase.
The process of light emission through chemical reaction in living organisms is called bioluminescence. Bioluminescent organisms are a diverse group including bacteria, fungi, algae, fish, squid, shrimp, and insects . Luciferase is a general name of the enzymes that catalyze the oxidative reaction of light production. Luciferase structure, its substrate and cofactors varies in these organisms and it seems the bioluminescent systems have developed independently from different evolutionary origins .
Experimental studies including random mutagenesis and rational design have been used extensively to explain and improve thermal stability and activity of firefly luciferase. These factors are the most serious problematic parameters which limit luciferase usage. In spite of many outcomes, the approach of these methods was based on crystal structure of the enzyme. In this regard, the dynamic nature of protein and its impact on enzyme behavior (structure dynamic relationship) is disregarded. The dynamic behavior of proteins is difficult to probe experimentally, but the molecular dynamic simulation provides an atomistic approach in this regard.