Structure of cysteate acyltransferase, the condensation enzyme that initiates sulfonolipid biosynthesis by inhabitants of the gut microbiome

Abstract

Sulfonolipids (SL) are unusual lipids found in the outer membranes of Gram‐negative bacteria in the phylum Bacteroidetes. SL and its deacylated derivative, capnine, are sulfur analogs of ceramide‐1‐phosphate and sphingosine‐1‐phosphate, respectively. The structural similarity to key mammalian signaling molecules led us to investigate the biochemistry and distribution of SL production in inhabitants of the gut microbiome. Alistipes finegoldii, an anaerobic gut commensal contains two genes related to the bacterial serine palmitoyl transferase (Spt) identified in Sphingobacteriia. The two proteins were purified and the Afl_1224 gene product catalyzed the condensation of [14C]acyl‐acyl carrier protein and cysteate (3‐sulfo‐alanine). The product of cysteate acyltransferase (AfCat) was identified as 3‐ketocapnine by mass spectrometry. AfCat has a bound pyridoxal phosphate cofactor (PLP) that undergoes a spectral blue shift when exposed to cysteate consistent with the transition of a lysine‐aldimine complex to a substrate‐aldimine complex. We solved the crystal structure of dimeric AfCat using SAD phasing. AfCat has the prototypical fold enveloping the PLP cofactor bound to Lys‐251 found in Spt and has an electrostatic surface patch for ACP binding. AfCat is distinguished from Spt by its carboxy terminal domain that extends to interact with the amino terminus, an enlarged substrate binding site to accommodate and stabilize the negatively charged sulfono head group of cysteate, and the HAS motif in Spt is HAC in AfCat. These unique structural elements were used to identify SL producing organisms in metagenomic sequencing data from the gut microbiome that are responsible for SL and capnine in mammalian stool samples. Supported by GM034496.