DECODING THE Sxt GENE CLUSTER: STRUCTURAL CHARACTERIZATION OF CORE ENZYMES FOR SAXITOXIN INHIBITION

Saxitoxin (STX) is a potent neurotoxin produced by cyanobacteria and marine dinoflagellates that causes severe cases of paralytic intoxication, representing a significant threat to public health, food safety, and biological defense. STX is synthesized through a highly complex, multi-step enzymatic pathway regulated by the sxt gene cluster. Within this intricate pathway, a highly conserved set of core genes—specifically encoding the SxtA, SxtG, SxtI, and SxtX proteins—is essential for the initial assembly and structural backbone of the toxin. However, the shortage of experimentally resolved three-dimensional structures for these core enzymes severely limits a deeper molecular understanding of their catalytic mechanisms. To address this gap, this study aimed to structurally characterize these core proteins (SxtA, SxtG, SxtI, and SxtX) using structural bioinformatics tools. Amino acid sequences were obtained from the National Center for Biotechnology Information (NCBI), and structural coordinates for SxtA (PDB ID: 7UCL) and SxtG (PDB ID: 6U1R) were retrieved from the Protein Data Bank (PDB). For targets lacking experimentally determined structures, AlphaFold-predicted models were utilized. The models were validated using PROCHECK and ERRAT to evaluate their stereochemical and atomic quality. Additionally, multiple sequence alignments were performed to identify conserved residues and functional domains within the proteins. Active-site prediction will be carried out using the FTMap and CavityPlus servers [1, 2] to support subsequent molecular docking analyses via the DockThor server. The AlphaFold structural models of SxtI and SxtX exhibited high confidence and topological consistency. ERRAT validation yielded overall quality factors of 98.17% and 93.97% for SxtI and SxtX, respectively, exceeding stringent quality thresholds and supporting their viability as molecular receptors. The identification of conserved catalytic residues and the mapping of potentially ligand-binding (druggable) cavities within these core structures will provide fundamental theoretical insights for the rational design of targeted enzyme inhibitors. Consequently, by inhibiting these essential core proteins, the entire biosynthetic pathway would be disrupted, effectively preventing the formation of saxitoxin and strengthening biological defense strategies.