Abstract
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.
Highlights
Dictyostelium discoideum secretes hydrolytic enzymes that degrade bacterial cell walls, enabling it to utilize soil-borne bacteria as a source of nutrition
The activity of purified FAA II has been assessed on metabolically radiolabeled Nod factors from R. fredii USDA257, and the results indicate that these become de-N-acylated
After treatment of N-palmitoyl[1-14C]glucosamine with FAA II in aqueous Buffer B and the addition of sodium chloride, selective counting of the aqueous and organic phases is an indication of the reaction progress
Summary
Materials—D. discoideum strain Ax-3 (ATCC 28368) was obtained from American Type Culture Collection (Rockville, MD). Following precipitation with ammonium sulfate (70%), and centrifugation (15,000 ϫ g, 15 min, 4 °C), the protein pellet was redissolved in Buffer B (3 ml) and dialyzed overnight against Buffer B. It was further fractionated on a DEAE-cellulose column (20 ϫ 1 cm) as follows: 1) washed with Buffer B (5 bed volumes) and 2) eluted with a gradient of NaCl (150 –500 mM) in Buffer B. After 18 h, the bacteria were removed by centrifugation (10,000 ϫ g, 4 °C), and radiolabeled Nod factors were recovered from the culture supernatant by solid-phase extraction with C18-functionalized silica (Waters) [18]. After 2 days further growth, the radiolabeled Nod factors were isolated as described above
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