Figure 1.
Action of recombinant SicTox PLD enzyme αIB2bi on palmitoyl lysophosphatidylcholine (16∶0 LPC).
(A) No palmitoyl lysophosphatidic acid (16∶0 LPA) is detected, but palmitoyl cyclic phosphatidic acid (16∶0 CPA) can be detected as a product by NMR and confirmed by MS/MS based on known ion fragmentation patterns of 16∶0 CPA [21], [22]. (B) Degradation of 16∶0 LPC by SicTox enzyme αIB2bi as measured by 31P-NMR. The only observed product resonance (+17.2 ppm) is characteristic of a cyclic phosphate species with a five-membered ring, matches the chemical shift of commercially available 16∶0 CPA, and is inconsistent with the chemical shift of 16∶0 LPA (see Figure S2). After 40 h, nearly all the LPC substrate is consumed, but the putative CPA resonance remains weak, presumably due to poor solubility. Trimethyl phosphate (TMP; 1 mM) was added as a chemical shift and concentration standard (see Materials and Methods). (C) Mass spectrum of 16∶0 CPA standard showing [M-H]- monomer at m/z = 391 as well as the [2M-H]− dimer at m/z = 783. Inset shows the MS/MS fragmentation of the m/z 391 species, yielding the daughter ions depicted in (A). (D) Mass spectrum of extracted reaction mixture of palmitoyl LPC substrate treated with αIB2bi enzyme showing the same [M-H]- and [2M-H]- species as in (C). Inset shows the MS/MS fragmentation of m/z 391 which yields the same daughter ions as the 16∶0 CPA standard in (C).
Figure 2.
Recombinant SicTox enzyme αIB2bi generates cyclic phosphatidic acid from octanoyl lysophosphatidylcholine (8∶0 LPC).
(A) Degradation of 8∶0 LPC by SicTox enzyme αIB2bi as measured by 31P-NMR. Two isomers of LPC (1 and 2) are observed (see text) and the only observed product upon enzyme addition is a far downfield chemical shift. Trimethyl phosphate (TMP) is an internal chemical shift and concentration standard. (B) LC-MS characterization of the NMR sample from (A), before (top) and after (bottom) addition of enzyme. Chromatograms represent total ion count (TIC) from reverse phase LC-MS as a function of retention time. Insets show time-averaged negative-ion mode mass spectra of substrate (red) and product peak (blue).
Figure 3.
Diverse Loxosceles whole venoms cleanly convert 8∶0 LPC substrate to 8∶0 CPA product.
Relative concentrations of 8∶0 LPC substrate (squares) and 8∶0 CPA product (circles) as a function of time at 298 K were measured by integration of 31P-NMR signals relative to a trimethyl phosphate standard (see Materials and Methods), and reported as percentages of the LPC concentration at time zero. Data points for the recombinant enzyme αIB2bi were derived from the experiment shown in Figure 2A, with the H47N variant shown as a negative control. Data for the three venoms were derived from similar experiments, but with addition of whole venom rather than purified recombinant enzyme.
Figure 4.
SicTox PLD enzymes from Loxosceles venoms convert sphingomyelin to cyclic ceramide phosphates.
(A) Sphingomyelin (SM) is not converted not to ceramide-1-phosphate (C1P) but to a cyclic six membered ceramide(1,3)phosphate [CC(1,3)P]. (B) 31P-NMR spectra of 4 mM hexanoyl SM (HexSM) micelles, before (top) and 30 min after (bottom) addition of L. arizonica venom. The cyclic product persisted for over 12 h, and no monoester product (i.e. C1P) was detected. Analogous results were observed with purified SicTox αIB2bi enzyme. (C) LC-MS characterization of the product from the NMR sample in (B), showing results analogous to those shown in Figure 2B for conversion of LPC to CPA.