J Biol Chem, Vol. 273, Issue 8, 4459-4464, February 20, 1998

Dictyostelium discoideum Fatty-acyl Amidase II Has Deacylase Activity on Rhizobium Nodulation Factors

Peggy J. Sutherland, Andrea E. Tobin, Charles L. Rutherford^¶, and Neil P. J. Price

From the  Department of Chemistry, College of Environmental Science and Forestry, State University of New York, Syracuse, New York 13210 and the ^¶ Department of Biology, Virginia Polytechnic Institute, Blacksburg, Virginia 24061

Dictyostelium discoideum (Amoebidae) secretes cell-lysing enzymes: esterases, amidases, and glycosylases, many of which degrade soil bacteria to provide a source of nutrients. Two of these enzymes, fatty-acyl amidases FAA I and FAA II, act sequentially on the N-linked long chain acyl groups of lipid A, the lipid anchor of Gram-negative bacterial lipopolysaccharide. FAA I selectively hydrolyzes the 3-hydroxymyristoyl group N-linked to the proximal glucosamine residue of de-O-acylated lipid A. Substrate specificity for FAA II is less selective, but does require prior de-N-acylation of the proximal sugar, i.e. bis-N-acylated lipid A is not a substrate. We have synthesized a ¹⁴C-labeled substrate analog for FAA II and used this in a novel assay to monitor its purification. Inhibitory studies indicate that FAA II is not a serine protease, but may have a catalytic mechanism similar to metalloprotein de-N-acetylases such as LpxC. Interestingly, rhizobial Nod factor signal oligosaccharides that induce root nodules on leguminous plants have many of the structural requirements for substrate recognition by FAA II. In vitro evidence indicates that Rhizobium fredii Nod factors are selectively de-N-acylated by purified FAA II, suggesting that the enzyme may reduce the N₂-fixing efficiency of Rhizobium-legume symbioses. In contrast, N-methylated Nod factors from transgenic R. fredii carrying the rhizobial nodS gene were resistant to FAA II, suggesting a mechanism by which Nod factors may be protected from enzymatic de-N-acylation. Since FAA II and Nod factors are both secreted, and Nod factors that lack the N-acyl group are unable to induce nodules, dictyostelial FAA II may decrease the efficiency of symbiotic nitrogen fixation in the environment by reducing the available biologically active nodule inducer signal.