Structural and Biochemical characterization of a class III sirtuin OsCobB in Oryza sativa indica and its association to stress response in plants

Abstract

Sirtuins have attained recognition in the sphere of epigenetic regulation, owing to its affiliation with numerous biological processes including metabolism and genomic stability. Many post translational modifications (PTMs) are controlled by these sirtuins, playing critical roles in escaping abiotic and biotic stresses, although their pattern in plants have been poorly understood. In our investigation, a novel class III sirtuin has been discovered in Oryza sativa var indica with a high sequence identity with bacterial CobB. No class III sirtuins have been reported in plants till this study. Correlating molecular docking analysis with in-vitro and in-vivo deacylation assays helped in the understanding of various aspects of enzymatic mechanisms of OsCobB. We identified the important motifs for substrate and NAD+ binding like GAGISA, FGE, YXXR and TQNID, which were unique to class III sirtuin family. Although all modifications could be accommodated at the catalytic site, its selectivity also depended on the orientation of the peptide backbone and its interaction (bonded and non-bonded) with the sirtuin. OsCobB had a preference for the negatively charged lysine modifications like malonyl, succinyl and glutaryl through H-bonds with conserved YXXR motif. OsCobB can also accommodate the longer acyl modifications like myristoyl by a possible shift of its and#945;4 helix. It was also established that an initial attachment of its co-substrate, NAD+ was mandatory to facilitate a conformational change for the correct binding of the peptide. In contrast to some class III members, OsCobB didnot exhibit any ADP-ribosyl transferase activity. We have identified this sirtuin to be majorly localized in the mitochondria with trace amounts in the nucleus. However, the preferential localization of this protein in the nucleus in response to low temperature and dehydration conditions could be related to its functioning. Histones (H3 and H4) as well as ACS were identified as the nuclear targets for this enzyme. Though OsCobB was incapable of ef