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Structure of GntC, a PLP-dependent enzyme catalyzing L-enduracididine biosynthesis from (S)-4-hydroxy-L-arginine, with the substrate bound
Structural highlights
FunctionPublication Abstract from PubMedCyclic arginine noncanonical amino acids (ncAAs) are found in several actinobacterial peptide natural products with therapeutically useful antibacterial properties. The preparation of ncAAs like enduracididine and capreomycidine currently takes multiple biosynthetic or chemosynthetic steps, thus limiting the commercial availability and applicability of these cyclic guanidine-containing amino acids. We recently discovered and characterized the biosynthetic pathway of guanitoxin, a potent freshwater cya-nobacterial neurotoxin, that contains an arginine-derived cyclic guanidine phosphate within its highly polar structure. The ncAA L-enduracididine is an early intermediate in guanitoxin biosynthesis and is produced by GntC, a unique pyridoxal-5'-phosphate (PLP)-dependent enzyme. GntC catalyzes a cyclodehydration from a stereoselectively gamma-hydroxylated L-arginine precursor via a reaction that functionally and mechanistically diverges from previously established actinobacterial cyclic arginine ncAA pathways. Herein, we interrogate L-enduracididine biosynthesis from the cyanobacterium Sphaerospermopsis torques-reginae ITEP-024 using spectroscopic, stable isotope labeling techniques, and X-ray crystal structure-guided site-directed mutagenesis. GntC initially facilitates the reversible deprotonations of the alpha- and beta-positions of its substrate prior to catalyzing an irreversible diastereoselective dehydration and subsequent intramolecular cyclization. The comparison of holo- and substrate bound GntC structures and activity assays on sitespecific mutants further identified amino acid residues that contribute to the overall catalytic mechanism. These interdisciplinary efforts at structurally and functionally characterizing GntC enables an improved understanding of how Nature divergently produces cyclic arginine ncAAs and generates additional tools for their biocatalytic production and downstream biological applications. Mechanistic and structural insights into a divergent PLP-dependent L-enduracididine cyclase from a toxic cyanobacterium.,Cordoza JL, Chen PY, Blaustein LR, Lima ST, Fiore MF, Chekan JR, Moore BS, McKinnie SMK bioRxiv. 2023 Mar 21:2023.03.21.533663. doi: 10.1101/2023.03.21.533663. Preprint. PMID:36993528[1] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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