Effect of mutation at positively charged residues (K329 and R330) in a flexible region of firefly luciferase on structure and kinetic properties.

Firefly luciferase as a bioluminescent enzyme has many applications in various fields from scientific research to commercial goals. This enzyme is relatively unstable with low functional capacity due to rapid inactivation in physiological temperature, low in vitro stability and high susceptibility to proteolytic degradation. Based on previous studies, two regions 206-220 and 329-341 on N-domain of Photinus pyralis luciferase are known accessible and flexible. Flexible regions may lead to protein instability. Here, the effect of mutation at positively charged residues Lys(K)329 and Arg(R)330 on the stability of luciferase was studied. Furthermore, the role of these mutations on the structure and function was evaluated. Introducing of these point mutations did not affect the orientation of critical residues in bioluminescence color determination. The kinetic studies showed that thermostability and Km value for luciferin in both mutants were decreased as compared to wild type. However, optimum pH and optimum temperature showed no significant changes in both mutants. Moreover, the structural data revealed an increase in tryptophan fluorescence intensity and secondary structure content for R330Q in compared with wild type, while intrinsic fluorescence and far-UV CD intensity in K329I mutant was decreased.

Mutation of conserved residues K329 and R330 on the surface of firefly luciferase: Effect on proteolytic degradation.

Firefly luciferase is highly susceptible to proteolytic digestion that reduces its half-life and leads to loss in sensitivity. Due to the protease contamination in most in vitro and in vivo environments, it has interest to generate some mutations that may lead to improved susceptibility to digestion. Some important conserved residues (including K206, R213, R218, K329, R330 and R337) on accessible and flexible regions on the surface of Photinus pyralis luciferase have been suggested that susceptible to trypsinolysis. In current study, two mutants (K329I and R330Q) are designed to investigate the impact of these conserved sites on the protease stability and flexibility. This study showed that these mutations did not cause resistance against trypsin digestion. K329I mutant was more susceptible to trypsin, but no difference in the digestion pattern was observed. This point mutation brought about structural flexibility, which revealed by quenching and extrinsic fluorescence. The experimental and theoretical studies demonstrated that R330Q mutagenesis didn't have any noticeable effect on the tryptic sites and flexibility. Moreover, the results of proteolysis experiment showed that the primary sites for trypsin digestion are still exposed after both mutations.