Structural and Functional Characterization of Cysteate Fatty Acyltransferases Involved in Bacterial Sulfonolipid Biosynthesis.
6 affiliations
- 1International Cancer Center, School of Pharmacy, Guangdong Key Laboratory of Genome Instability and Human Disease Prevention, Shenzhen University Medical School, Shenzhen 518055, China.
- 2Institute of Chemical Biology, Shenzhen Bay Laboratory, Shenzhen 518132, China.
- 3Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China.
- 4State Key Laboratory of Quantitative Synthetic Biology, Shenzhen Institute of Synthetic Biology, Shenzhen Institutes of Advanced Technology, Chinese Academy of Sciences, Shenzhen 518055, China.
- 5School of Food Science and Pharmaceutical Engineering, Nanjing Normal University, Nanjing 210023, China.
- 6Shenzhen Medical Academy of Research and Translation, Shenzhen 518107, China.
Abstract
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.
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BibTeX
@article{pang2026structural,
title = {{Structural and Functional Characterization of Cysteate Fatty Acyltransferases Involved in Bacterial Sulfonolipid Biosynthesis.}},
author = {Cuiping Pang and Meihan Liu and Yang Gu and Jiahai Zhou and Xiaoyu Tang},
journal = {ACS bio \& med chem Au},
year = {2026},
month = aug,
doi = {10.1021/acsbiomedchemau.6c00154},
url = {https://europepmc.org/article/MED/42921287},
note = {CC BY-NC-ND},
}
RIS
TY - JOUR
TI - Structural and Functional Characterization of Cysteate Fatty Acyltransferases Involved in Bacterial Sulfonolipid Biosynthesis.
AU - Cuiping Pang
AU - Meihan Liu
AU - Yang Gu
AU - Jiahai Zhou
AU - Xiaoyu Tang
PY - 2026
DA - 2026/08/21
JO - ACS bio & med chem Au
DO - 10.1021/acsbiomedchemau.6c00154
UR - https://europepmc.org/article/MED/42921287
AB - 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.
SN - 2694-2437
ER -