Abstract
Sulfonolipids are unusual lipids found in the outer membranes of Gram-negative bacteria in the phylum Bacteroidetes. Sulfonolipid and its deacylated derivative, capnine, are sulfur analogs of ceramide-1-phosphate and sphingosine-1-phosphate, respectively; thus, sulfonolipid biosynthesis is postulated to be similar to the sphingolipid biosynthetic pathway. Here, we identify the first enzyme in sulfonolipid synthesis in Alistipes finegoldii as the product of the alfi_1224 gene, cysteate acyl-acyl carrier protein (ACP) transferase (SulA). We show SulA catalyzes the condensation of acyl-ACP and cysteate (3-sulfo-alanine) to form 3-ketocapnine. Acyl-CoA is a poor substrate. We show SulA has a bound pyridoxal phosphate (PLP) cofactor that undergoes a spectral redshift in the presence of cysteate, consistent with the transition of the lysine–aldimine complex to a substrate–aldimine complex. Furthermore, the SulA crystal structure shows the same prototypical fold found in bacterial serine palmitoyltransferases (Spts), enveloping the PLP cofactor bound to Lys251. We observed the SulA and Spt active sites are identical except for Lys281 in SulA, which is an alanine in Spt. Additionally, SulA(K281A) is catalytically inactive but binds cysteate and forms the external aldimine normally, highlighting the structural role of the Lys281 side chain in walling off the active site from bulk solvent. Finally, the electropositive groove on the protein surface adjacent to the active site entrance provides a landing pad for the electronegative acyl-ACP surface. Taken together, these data identify the substrates, products, and mechanism of SulA, the PLP-dependent condensing enzyme that catalyzes the first step in sulfonolipid synthesis in a gut commensal bacterium.
Original language | English |
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Article number | 102195 |
Journal | Journal of Biological Chemistry |
Volume | 298 |
Issue number | 8 |
DOIs | |
State | Published - Aug 2022 |
Bibliographical note
Funding Information:We thank Karen Miller for technical assistance with protein purification, Jayaraman Seetharaman (St Jude Children’s Research Hospital (SJCRH) Structural Biology X-ray Center) for crystallography support, Dr Amanda Nourse (SJCRH Molecular Interactions Analysis Shared Resource) for sample analysis by analytical ultracentrifugation, and the SJCRH Hartwell Center DNA Sequencing Shared Resource for DNA sequencing. This work was supported by the Cancer Center Support Grant CA21765 and the American Lebanese Syrian Associated Charities, United States . The FMX (17-ID-2) beamline of the National Synchrotron Light Source II is a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory under Contract No. DE-SC0012704. FMX is part of the Center for BioMolecular Structure (CBMS) that is primarily supported by the National Institutes of Health Center Core P30 Grant ( P30 GM133893 ) and by the DOE Office of Biological and Environmental Research, United States ( KP 1605010 ).
Funding Information:
We thank Karen Miller for technical assistance with protein purification, Jayaraman Seetharaman (St Jude Children's Research Hospital (SJCRH) Structural Biology X-ray Center) for crystallography support, Dr Amanda Nourse (SJCRH Molecular Interactions Analysis Shared Resource) for sample analysis by analytical ultracentrifugation, and the SJCRH Hartwell Center DNA Sequencing Shared Resource for DNA sequencing. This work was supported by the Cancer Center Support Grant CA21765 and the American Lebanese Syrian Associated Charities, United States. The FMX (17-ID-2) beamline of the National Synchrotron Light Source II is a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by Brookhaven National Laboratory under Contract No. DE-SC0012704. FMX is part of the Center for BioMolecular Structure (CBMS) that is primarily supported by the National Institutes of Health Center Core P30 Grant (P30 GM133893) and by the DOE Office of Biological and Environmental Research, United States (KP 1605010). C. D. R. and C. O. R. methodology; C. D. R. D. J. M. M. W. F. and C. O. R. investigation; C. D. R. and C. O. R. formal analysis; C. D. R. writing–original draft; C. D. R. D. J. M. M. W. F. and C. O. R. writing–review and editing. This work was supported by National Institutes of Health, United States grants 1K99AI166116 (C. D. R.) and GM034496 (C. O. R.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Funding Information:
This work was supported by National Institutes of Health, United States grants 1K99AI166116 (C. D. R.) and GM034496 (C. O. R.). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.
Publisher Copyright:
© 2022 The Authors
Keywords
- 3-ketocapnine
- Alistipes finegoldii
- Bacteroidetes
- condensing enzyme
- crystal structure
- fatty acid
- gut microbiome
- sulfonolipid biosynthesis
ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
- Cell Biology