Microviridins are a class of ribosomally produced and post-translationally modified peptides identified as serine-type protease inhibitors and produced mostly by Cyanobacteria. The ribosomally produced and post-translationally modified peptide (RIPPs) family includes many small molecules with useful therapeutic properties that fall within distinct structural classes. Featuring unique, cage-like structures, their stabilities against both chemical and enzymatic degradation is remarkable compared with linear ribosomally synthesized peptides. In a canonical Microviridin biosynthetic pathway, two macrocyclases, one acyl-transferase and one C39-peptidase-fused ABC transporter are required for precursor peptide modification. Here in this project, we investigate the binding and reaction mechanisms of important post-translationally modification enzymes through X-ray crystallography study, more specifically, by crystalizing enzymes such as macrocyclases and acetyl-transferases from both Microcystis aeruginosa and Anabaena sp. PCC 7012 pathways, we will be able to provide novel insights into the understanding, discovery and engineering of RIPPs pathways. Meanwhile, binding measurements such as isothermal titration calorimetry and mutagenesis will also be performed as efficient tools to help us understand the interaction between substrates and enzymes. Disulfide bond linkage approach was also used to capture a propeptide/cyclase co-complex structure. With a more complete picture of the Microviridin biosynthetic pathway in hand, genomic engineering such as reducing the specificity of post-translation enzymes, inserting desired core peptide sequence, including multiple core peptide regions in one precursor peptide will be performed to expand the macrocyclic peptides library. The resultant natural products will ultimately be screened for analogs as the inhibitors of therapeutically relevant serine proteases. The results will provide a solid foundation toward the development of potent, selective serine protease inhibitors with novel scaffolds, providing guidance for creating synthetic biology platforms to produce other families of RiPPs analogs.

Investigation of substrate recognition and biosynthesis in microviridin pathways

2019 Fall National Meeting

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