Lysophosphatidylethanolamine is a substrate for the short-chain alcohol dehydrogenase SocA from Myxococcus xanthus.
Short-chain alcohol dehydrogenases (SCADHs) synthesize a variety of intercellular signals and other chemically diverse products. It is difficult to predict the substrate of a SCADH on the basis of amino acid sequence homology, as the substrates are not known for most SCADHs. In Myxococcus xanthus, the SCADH CsgA is responsible for C signaling during fruiting body development, although the mechanism is unclear. Overexpression of the SCADH SocA compensates for the lack of CsgA and restores development and C signaling in csgA mutants. The potential of SocA in generating the C signal enzymatically was explored by developing a dehydrogenase assay-based screen to purify the SocA substrate(s). A SocA substrate was extracted from M. xanthus cells with acidified ethyl acetate and sequentially purified by solid-phase extraction on silica gel and by reverse-phase high-performance liquid chromatography. The fraction with the highest SocA dehydrogenase activity contained the lysophospholipid 1-acyl 2-hydroxy-sn-glycerophosphoethanolamine (lyso-PE) as indicated by the fragment ions and a phosphatidylethanolamine-specific neutral loss scan following liquid chromatography coupled to mass spectrometry. The abundant lysophospholipid with the mass m/z 450 (molecular ion [M-H]-) had a monounsaturated acyl chain with 16 carbons. SocA oxidizes lyso-PE containing either saturated or unsaturated fatty acids but exhibits poor activity on l-alpha-glycerophosphorylethanolamine, suggesting that an acyl chain is important for activity. Of the five different head groups, only ethanolamine showed appreciable activity. The apparent Km and Vmax for lyso-PE 18:1 were 116 microM and 875 micromol min(-1) mg(-1), respectively. The catalytic efficiency (k(cat)/Km) was 1 x 10(8) M(-1) s(-1). The proposed product, 1-acyloxy-3-(2-aminoethylphosphatyl) acetone was unstable, and the fragmented products were unable to rescue csgA mutant development. The active fraction from thin-layer chromatography also contained an unidentified SocA substrate that had morphogenic properties.
Avadhani
Madhavi
M
Department of Microbiology, University of Georgia, Athens, GA 30602, USA.
Geyer
Roland
R
White
David C
DC
Shimkets
Lawrence J
LJ
D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008246:Lysophospholipids / Q000302:isolation & purification / Q000378:metabolism*; D016941:Myxococcus xanthus / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D013379:Substrate Specificity
D001426:Bacterial Proteins; C063576:CsgA protein, Myxococcus xanthus; D008246:Lysophospholipids; C008301:lysophosphatidylethanolamine; D000426:Alcohol Dehydrogenase