A high-throughput solid-phase extraction assay capable of measuring diverse polyprenyl phosphate: sugar-1-phosphate transferases as exemplified by the WecA, MraY, and MurG proteins.
The bacterial proteins WecA and MraY are members of the polyprenyl phosphate:N-acetylhexosamine-1-phosphate transferase family, each of which catalyzes the transfer of a specific hexosamine 1-P from a soluble UDP-hexosamine substrate to a bactoprenyl phosphate carrier at the membrane surface. Currently, assays designed to quantitate the activity of these enzymes rely on paper chromatography or liquid-liquid extractions or are specialized to a few members of the family. We describe a generalizable, high-throughput, one-pot assay for these activities that uses a solid-liquid bead-based separation system to selectively adsorb the highly hydrophobic products of reaction. By judicious choice of radiolabeled UDP-hexosamine precursor, the same format can be used to quantitate not only diverse members of this transferase family, but also enzymes that catalyze the further modification of these transferase products. This possibility is exemplified by the MurG protein of bacterial cell wall synthesis, which catalyzes the addition of an N-acetylglucosamine residue to the product of the MraY reaction. Thus, the use of this flexible assay tool will allow a critical biochemical and enzymologic analysis of many such membrane-bound transferases in a similar setting.
Hyland
Sheryl A
SA
Department of Atherosclerosis and Endocrinology, Merck Research Laboratories, P.O. Box 2000, Rahway, NJ 07065, USA.
Anderson
Matt S
MS
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D029968:Escherichia coli Proteins; D013058:Mass Spectrometry / Q000379:methods*; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000378:metabolism; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D013997:Time Factors; D014166:Transferases / Q000032:analysis* / Q000235:genetics / Q000378:metabolism; D017855:Transferases (Other Substituted Phosphate Groups) / Q000235:genetics / Q000378:metabolism; D014539:Uridine Diphosphate Sugars / Q000378:metabolism
D001425:Bacterial Outer Membrane Proteins; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D011106:Polyisoprenyl Phosphates; D014539:Uridine Diphosphate Sugars; D014166:Transferases; D017351:N-Acetylglucosaminyltransferases; C064057:UDP-N-acetylglucosamine-N-acetylmuramyl-(pentapeptide)pyrophosphoryl-undecaprenol N-acetylglucosamine transferase; D017855:Transferases (Other Substituted Phosphate Groups); C083617:wecA protein, E coli; C079803:mraY protein, Bacteria