A biosynthetic pathway for ornithine lipid formation dependent on a GH3 (Gretchen Hagen 3)-like enzyme in planctomycetes

Amino lipids with acyloxyacyl structures, particularly ornithine lipids (OLs), are widespread in bacteria, but are absent from archaea and eukaryotes. In these lipids, an α-amino acid is N-acylated with a 3-hydroxy fatty acyl residue, and a secondary fatty acid is ester-bound to the hydroxyl group of the first fatty acid. Based on the presence of genes encoding the fatty acid transferases OlsBA or OlsF, involved in ornithine lipid (OL) synthesis, it has been estimated that approximately 50% of sequenced bacterial species can form OL. Here, based on genomic context, structure-based comparisons, and activity essays, we identified a novel pathway for OL synthesis in the planctomycete Singulisphaera acidiphila. This pathway initiates with OlsH, a GH3 (Gretchen Hagen 3)-like enzyme, that catalyzes the ATP-dependent formation of N-acyl ornithine. The α/β-hydrolase OlsI catalyzes the O-acyltransferase step. In the presence of OlsH and OlsI, we could reconstitute OL synthesis in a cell-free Escherichia coli protein extract. Structural modelling of OlsH revealed conserved GH3 nucleotide-binding motifs, and mutagenesis of serine residues validated their functional importance. Enzymatic assays demonstrated substrate specificity for ornithine and diverse fatty acids, and preference for Mg²⁺as cofactor. A phylogenomic analysis showed that bacterial GH3-homologues are widely distributed across diverse bacterial phyla. We conclude that bacterial GH3-like enzymes, such as OlsH described here, catalyze the initial step in OL synthesis in the planctomycete S. acidiphila and likely in other bacteria as well. These findings expand our understanding of bacterial lipid diversity and suggest evolutionary convergence in OL biosynthesis.