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Cordoza, J. L.

Publications and source records attributed to Cordoza, J. L..

2 recordsLinked to original sources
bioRxiv2025

Integrated metabolomic and genomic insights into amino acid incorporation within the hybrid polyketide-alkaloid antibiotic TLN-05220

Actinobacteria are a rich source of bioactive compounds and unique biosynthetic chemistry. Micromonospora echinospora subsp. challisensis NRRL 12255 produces the aromatic polyketide TLN-05220, which exhibits nanomolar activity against antibiotic-resistant human pathogens including vancomycin-resistant Enterococcus faecalis and methicillin-resistant Staphylococcus aureus. The pentangular polyphenol core of TLN-05220 is decorated with a piperazinone moiety, yet the enzymes responsible for the construction of this uncommon modification from amino acid precursors are unknown. Synthetic piperazinone-containing molecules have diverse antimicrobial, antiviral, anticancer, and anti-inflammatory bioactivity profiles, and determining biosynthetic routes for the assembly of this heterocycle may enhance drug discovery and medicinal chemistry efforts. We identified a putative TLN-05220 biosynthetic gene cluster (BGC) in the commercially available strain M. echinospora ATCC 15837 containing both type-I and type-II polyketide synthases, two predicted asparagine synthetase-like enzymes, and two genes (tln1 and tln5) that putatively encode pyridoxal 5-phosphate (PLP)-dependent amino acid synthases. Stable isotopic feeding studies coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS), identified L-alanine, L-serine, and glycine as metabolic precursors of TLN-05220. Subsequent in vitro enzymology established that Tln1 is a PLP-dependent alanine racemase, while Tln5 performs a stereoselective {beta}-substitution reaction of O-phospho-L-serine with a preferential D-alanine nucleophile. Alanine racemization and Tln5 pseudodipeptide L-serine-C{beta}-N-D-alanine (D,L-PDP) incorporation into TLN-05220 were further supported using deuterated intermediates and mass spectrometry techniques. Establishing the enzymes that catalyze amino acid installation within TLN-05220 inspires further biosynthetic discovery and engineering while enabling the biocatalytic syntheses of novel amino acid-containing polyketide antibiotics. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/685158v1_ufig1.gif" ALT="Figure 1"> View larger version (20K): org.highwire.dtl.DTLVardef@4402e8org.highwire.dtl.DTLVardef@11af789org.highwire.dtl.DTLVardef@1717c5borg.highwire.dtl.DTLVardef@19b6529_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗
bioRxiv2023

Mechanistic and structural insights into a divergent PLP-dependent L-enduracididine cyclase from a toxic cyanobacterium

Cyclic 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 - 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.

biochemistry↗