Abstract

Cyanophycin is a biopolymer consisting of a poly-aspartate backbone and arginines linked to each Asp side chain though isopeptide bonds. Used in bacteria for storage of fixed nitrogen, carbon and energy, chains of cyanophycin of 80-400 β-Asp-Arg dipeptide residues coalesce into inert, membrane-less granules which can occupy most of the volume of a cell. Cyanophycin has a variety of potential green industrial and biomedical applications, which could be potentiated by the understanding and biochemical engineering of the enzymes involved in its metabolism. Cyanophycin is made by cyanophycin synthetase 1 or 2 through ATP -dependent polymerization of Asp and Arg, or β-Asp-Arg, respectively. It is degraded into dipeptides by specialized exo-cyanophycinases, and these dipeptidase are hydrolyzed into free amino acids by nonspecific isoaspartyl dipeptidases. I will share highlights of our structural and functional studies of cyanophycin biosynthesis and degradation, which led to surprising discoveries: Our structures and biochemical assays of the cyanophycin synthetase 1 revealed it to be a remarkable, multi-domain, multi-functional biosynthetic machine and uncovered a hidden hydrolytic active site that is crucial for rapid biosynthesis. We also showed that cyanophycin synthetase 2 can assume several elegant architectures that influence its synthetic activity. Further, we discovered and characterized a novel, large family of isoaspartyl dipeptidases dedicated to cyanophycin metabolism, which allows the human pathogen Pseudomonas aeruginosa to use β-Asp-Arg as a sole carbon source, and as good a sole nitrogen source as ammonium. Bioinformatics results underscore how common it is for bacteria to be cyanophycin producers or scavengers, much more so than currently appreciated.

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