Evolutionary shift in catalytic function of OsGLYI-10 from a glyoxalase homolog to a glutathione-s-transferase in rice.

Glyoxalase I (GLYI) constitute the first enzyme of the glyoxalase pathway which is a two-step reaction to convert methylglyoxal (MG), an inherent cytotoxin to D-lactate. In plants, multiple members of the glyoxalase pathway genes have been reported. However, not all exhibit glyoxalase activity. OsGLYI-10 is one such member from rice GLYI family which we report here, to lack GLYI enzymatic activity. Instead, OsGLYI-10 shows high structural homology to glutathione-S-transferase (GST) proteins and exhibits GST activity. Further, we found OsGLYI-10 to be highly expressed in seeds, its expression starting at the milk stage, reaching its maximum in the mature seed and finally disappearing after four days of imbibition. Importantly, through molecular docking and site-directed mutagenesis studies, we showed that GLYI activity can be reinstated to some extent via the introduction of a 10 amino acid stretch as well as substitution of certain amino acids in OsGLYI-10. Further increase in GLYI activity could be achieved through the substitution of Met with Tyr at 55th position, restoring 35% activity in OsGLYI-10 relative to a functionally active and highly efficient GLYI, OsGLYI-8 enzyme from rice. Our findings therefore, suggest OsGLYI-10 to be a reminiscent of GLYI enzyme that has diverged in its catalytic function over the course of evolution to adopt newer activities and roles in cellular physiology.