title: The first nonradioactive fluorescence assay for phosphatidylglycerol:prolipoprotein diacylglyceryl transferase that initiates bacterial lipoprotein biosynthesis.
abstract: The unique and physiologically vital bacterial enzyme, prolipoprotein diacylglyceryl transferase (Lgt), which catalyzes the committed first step in the posttranslational transfer of diacylglyceryl group from phosphatidylglycerol to the prospective N-terminal cysteine of prolipoproteins, remains to be characterized for want of a simpler but equally sensitive nonradioactive assay. We, for the first time, report a coupled enzymatic fluorescence assay for Lgt using the de novo synthetic peptide substrate MKATKSAVGSTLAGCSSHHHHHH. The assay is based on the conversion of the by-product, glycerol-1-phosphate, to dihydroxyacetone using an alkaline phosphatase-glycerol dehydrogenase combination and estimating the fluorescence of the coupled reduction of resazurin to resorufin. The minimum amount of glycerol-1-phosphate, and hence the modified peptide, detected by this method is approximately 20pmol, thereby making this assay a promising alternative to the radioactive assays. The assay is rapid, more convenient, less laborious, and suitable for purification and characterization of Lgt.
authors.affiliation: Centre for Biotechnology, Anna University, Chennai 600 025, India.
authors.forename: Srividhya
authors.initials: S
authors.lastname: Sundaram
authors.forename: Sanchari
authors.initials: S
authors.lastname: Banerjee
authors.forename: Krishnan
authors.initials: K
authors.lastname: Sankaran
mesh_terms: D000595:Amino Acid Sequence; D001419:Bacteria / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D057075:Enzyme Assays / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry*; D007700:Kinetics; D008074:Lipoproteins / Q000096:biosynthesis*; D008969:Molecular Sequence Data; D010078:Oxazines / Q000737:chemistry; D010455:Peptides / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D013379:Substrate Specificity; D014166:Transferases / Q000378:metabolism*; D014966:Xanthenes / Q000737:chemistry
chemical_list: D005456:Fluorescent Dyes; D008074:Lipoproteins; D010078:Oxazines; D010455:Peptides; D014966:Xanthenes; C005843:resazurin; D014166:Transferases; C042308:phosphatidylglycerol glyceryl transferase