Structure of CphA2 from Stanieria sp.; insights into oligomeric behaviour and substrate binding - Minor Research Project

Abstract

Cyanophycin is a natural polymer found in a wide range of bacteria, where it functions as a reservoir for fixed nitrogen. Additionally, cyanophycin has various commercial application in bio-industry and biomedicine. Cyanophycin consists of a poly-aspartate backbone with arginines attached to their sidechains. The polymer is synthesized by cyanophycin synthetase 1 and 2 (CphA1 and CphA2). While CphA1 uses aspartate and arginine as substrate, CphA2 uses the dipeptide β-Asp-Arg. Although the structure of dimeric CphA2 from G. citriformis has been resolved in a previous study, questions about oligomeric behaviour and substrate binding in CphA2 remained unanswered. In this study, I resolved the structure of the hexamer-forming CphA2 homolog from Stanieria sp. to 2.76 Å by cryo-EM. The hexamer form is stable and is important for enzyme activity. Structures of Stanieria sp. CphA2 with substrates obtained by cryo-EM at resolutions of 2.6 Å and 2.7 Å show the binding of cyanophycin and ATP or ATP analog, revealing similarities to and differences from substrate binding in CphA1. Moreover, mutagenesis experiments provided insights into the binding site of β-Asp-Arg. The structures and biochemical assay performed in this study assist in understanding of the reaction mechanism of cyanophycin formation by CphA2.