Pyridoxal-5’-phosphate (PLP) is a widely utilized cofactor that aids a diverse range of enzyme-catalyzed chemistry, including transaminations, decarboxylations, and aldolase reactions. PLP-dependent enzymes perform regioselective and stereospecific reactions in mild conditions to create nonproteinogenic amino acids for direct application or further downstream chemical transformations. Recently, we discovered the biosynthetic pathway for guanitoxin (GNT), a potent irreversible acetylcholinesterase inhibitor from a freshwater cyanobacterium. We were interested in the PLP-dependent enzyme GntC, which catalyzes a stereospecific intramolecular cyclization of gamma-hydroxylated arginine to L-enduracididine, within the GNT biosynthetic pathway. L-enduracididine and its epimers are found in other natural products with potent antibiotic activities, such as teixobactin, mannopeptimycin, and guadinomine B, which are effective against pathogenic bacteria like methicillin-resistant <i>Staphylococcus aureus </i>(MRSA) and <i>Yersinia sp</i>. Previously characterized biosynthetic pathways use multiple enzymes to synthesize L-enduracididine, making GntC an unusually efficient and notable enzyme of study. Herein, we investigate the mechanism of GntC using isotopic mass spectrometric studies through the synthesis of stable isotope labeled substrates and deuterium oxide enzymatic assays to determine the mechanism. We also employed site-directed mutagenesis on GntC to study the structural and functional features of the enzyme. Through these studies, we found that PLP has local and longer distance impacts on the substrate, facilitating deprotonations on both the alpha and beta carbons positions, allowing for hydroxyl group elimination and guanidine cyclization to occur. We are currently aiming to apply these mechanistic and structural data to expand the substrate scope of GntC through synthesis of diversified forms of the substrate. Future research will investigate the scalability of GntC for the biocatalytic application of L-enduracididine production.

Elucidating the PLP-dependent cyclase mechanism of GntC from the guanitoxin biosynthetic pathway

ACS Spring 2022

The official archive and repository for all ACS Meetings & Expositions abstracts and select posters or presentations.