Structural and Functional Characterization of Cysteate Fatty Acyltransferases Involved in Bacterial Sulfonolipid...

Sulfonolipids are bacterial sphingolipid-like lipids featuring a sulfonated long-chain base derived from cysteate. Although several cysteate-utilizing enzymes have been implicated in sulfonolipid biosynthesis, their phylogenetic relationships and the structural basis of cysteate utilization remain poorly understood. Here, we combined phylogenetic analysis, biochemical characterization, X-ray crystallography, and mutagenesis to investigate cysteate fatty acyltransferase (CFAT) homologues involved in bacterial sulfonolipid biosynthesis. Phylogenetic analysis revealed that previously characterized cysteate-utilizing enzymes form a distinct branch within the broader serine palmitoyltransferase (SPT)-related pyridoxal 5'-phosphate (PLP)-dependent enzyme superfamily. Biochemical assay on six representatives confirmed a conserved cysteate-dependent long-chain-base synthase activity and a strong preference for cysteate over serine, glycine, and alanine. To elucidate this specificity, we solved crystal structures of a representative CFAT from Capnocytophaga ochracea in both PLP-bound and PLP-cysteate-bound states, capturing a PLP-cysteate external aldimine intermediate. This structure reveals a composite active-site pocket formed across the dimer interface, where the conserved PLP-dependent catalytic scaffold is retained but the substrate-binding pocket is significantly remodeled compared to canonical SPTs. Structure-guided mutagenesis identified key residues important for cysteate-dependent catalysis but failed to generate serine activity. Together, these findings establish CFAT homologues as a functionally coherent family of cysteate-utilizing enzymes and provide critical molecular insights into bacterial sulfonolipid biosynthesis.