β-Ketoacyl Synthase II Homologs from a Ladderane-Producing Organism Form a Ketosynthase/Chain Length Factor-like functional heterodimer

Ladderanes are extraordinary, highly strained chemical structures consisting of linearly concatenated cyclobutane rings. Ladderane-containing fatty acids are found in the lipids of anaerobic ammonium oxidizing (anammox) bacteria, reducing the proton permeability of their membranes to support their unique metabolism. Almost nothing is known about the biosynthesis of ladderanes, but a gene cluster unique to ladderane-producing bacteria is likely to be involved. This cluster encodes radical SAM enzymes as well as homologs of enzymes known from fatty acid biosynthesis. Amongst these are two homologs of FabF, the enzyme performing chain elongation in canonical fatty acid synthesis. The presence of two chain-elongating enzymes is unexpected; a single one would be expected to suffice, and the fact that one of the two homologs has a mutated, nonfunctional catalytic triad deepens the mystery. Here we present an in-depth characterization of the FabF homologs from anammox organisms, using biochemistry, enzymology, structural biology and computational biology. We show that the two homologs form a heterodimer analogous to the ketosynthase/chain length factor complexes known from polyketide synthases. This heterodimer is capable of performing the decarboxylation of malonate-loaded acyl carrier protein, thus initiating fatty acid biosynthesis. The crystal structure of the heterodimer explains how homodimer formation is avoided, and shows the details of the substrate binding tunnel. Mechanism-based crosslinking studies of wild-type and mutant heterodimers show the influence of residues on both subunits on substrate preference (which differs from canonical FabFs), and, together with computational studies, the crystal structure of the heterodimer in complex with substrate-loaded ACP helps explain its preference for ladderane-specific ACP. The results clearly refute an early proposal for a ladderane biosynthetic mechanism and greatly expand our current knowledge on how anammox bacteria produce their extraordinary lipids.