Enhancing the catalytic activity of cyanobacterial chlorophyllase from Oscillatoria acuminata PCC 6304 through rational site-directed mutagenesis

Abstract

In our previous study, a novel cyanobacterial chlorophyllase from Oscillatoria acuminata (OaCLH) was successfully overexpressed and biochemically characterized in Escherichia coli BL21(DE3). Recombinant OaCLH has higher heterologous expression in E. coli than plant chlorophyllases. However, catalytic efficiency (kcat/Km) of the recombinant OaCLH is significantly lower than those of several reported algae and plant chlorophyllases from Chlamydomonas reinhardtii (CrCLH), Triticum aestivum (TaCLH), and Chenopodium album (CaCLH). In this study, three single-site mutant enzymes (W160R, V228C, and D224 N) were constructed by site-directed mutagenesis to improve the catalytic efficiency of OaCLH using rational design based on multiple sequence alignment and homology modeling. Trp160 was found to be important in substrate access and binding, while Val228 was found to be involved in substrate recognition. Mutants W160R and V228C significantly increased the enzyme affinity and catalytic activity against all substrates. Asp224 played an important role in substrate specificity. Mutant D224 N changed its preferred substrate from chlorophyll b to chlorophyll a. According to the circular dichroism spectra, the introduced mutations had no significant effect on the overall secondary structures of three mutant enzymes. When compared to mutant W160R, double mutant W160RV228C showed a slight improvement in enzyme affinity and catalytic activity. These mutation sites are reported for the first time in OaCLH, and provided information about the structural basis of catalytic activity and substrate specificity of OaCLH. Mutant enzymes (W160R, V228C, D224 N, and W160RV228C) with improved catalytic efficiency may be good candidates for further engineering of OaCLH for potential industrial applications.