Novel prenyl-linked benzophenone substrate analogues of mycobacterial mannosyltransferases.
PPM (polyprenol monophosphomannose) has been shown to act as a glycosyl donor in the biosynthesis of the Man (mannose)-rich mycobacterial lipoglycans LM (lipomannan) and LAM (lipoarabinomannan). The Mycobacterium tuberculosis PPM synthase (Mt-Ppm1) catalyses the transfer of Man from GDP-Man to polyprenyl phosphates. The resulting PPM then serves as a donor of Man residues leading to the formation of an alpha(1-->6)LM intermediate through a PPM-dependent alpha(1-->6)mannosyltransferase. In the present study, we prepared a series of ten novel prenyl-related photoactivatable probes based on benzophenone with lipophilic spacers replacing several internal isoprene units. These probes were excellent substrates for the recombinant PPM synthase Mt-Ppm1/D2 and, on photoactivation, several inhibited its activity in vitro. The protection of the PPM synthase activity by a 'natural' C(75) polyprenyl acceptor during phototreatment is consistent with probe-mediated photoinhibition occurring via specific covalent modification of the enzyme active site. In addition, the unique mannosylated derivatives of the photoreactive probes were all donors of Man residues, through a PPM-dependent mycobacterial alpha(1-->6)mannosyltransferase, to a synthetic Manp(1-->6)-Manp-O-C(10:1) disaccharide acceptor (where Manp stands for mannopyranose). Photoactivation of probe 7 led to striking-specific inhibition of the M. smegmatis alpha(1-->6)mannosyltransferase. The present study represents the first application of photoreactive probes to the study of mycobacterial glycosyltransferases involved in LM and LAM biosynthesis. These preliminary findings suggest that the probes will prove useful in investigating the polyprenyl-dependent steps of the complex biosynthetic pathways to the mycobacterial lipoglycans, aiding in the identification of novel glycosyltransferases.
Guy
Mark R
MR
School of Biosciences, The University of Birmingham, Edgbaston, Birmingham B15 2TT, UK.
Illarionov
Petr A
PA
Gurcha
Sudagar S
SS
Dover
Lynn G
LG
Gibson
Kevin J C
KJ
Smith
Paul W
PW
Minnikin
David E
DE
Besra
Gurdyal S
GS
D001426:Bacterial Proteins / Q000037:antagonists & inhibitors* / Q000378:metabolism; D001577:Benzophenones / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000096:biosynthesis; D008358:Mannose / Q000378:metabolism; D008364:Mannosyltransferases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D020102:Mycobacterium smegmatis / Q000201:enzymology*; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D019920:Photoaffinity Labels / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology; D010777:Photochemistry; D011106:Polyisoprenyl Phosphates / Q000138:chemical synthesis* / Q000378:metabolism / Q000494:pharmacology
D001426:Bacterial Proteins; D001577:Benzophenones; D008070:Lipopolysaccharides; D019920:Photoaffinity Labels; D011106:Polyisoprenyl Phosphates; C050016:lipoarabinomannan; C064351:lipomannan; D008364:Mannosyltransferases; C479135:polyprenyl monophosphomannose synthase, bacteria; D008358:Mannose