Schouten, James A., Bagga, Sangeev, Lloyd, Adrian J., De Pascale, Gianfranco, Dowson, Christopher G., Roper, David I. and Bugg, Timothy D. H. (2006) Fluorescent reagents for in vitro studies of lipid-linked steps of bacterial peptidoglycan biosynthesis: derivatives of UDPMurNAc-pentapeptide containing d-cysteine at position 4 or 5. Molecular BioSystems, 2 (10). pp. 484-491. doi:10.1039/b607908c ISSN 1742-206X.

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Abstract

UDPMurNAc-L-Ala-gamma-D-Glu-X-D-Ala-DAla (X = L-Lys or m-DAP) is the cytoplasmic precursor for the lipid-linked cycle of bacterial peptidoglycan biosynthesis, consisting of at least four enzymatic reactions, which are targets for antibacterial agents. Fluorescent derivatives of the UDPMurNAc-pentapeptide labelled at the 3rd, 4th, and 5th position of the peptide chain were prepared chemoenzymatically, in order to study the reactions catalysed by enzymes in this cycle. Derivatives labelled on the epsilon-amino group of the 3rd amino acid (N-dansyl, N-fluorescamine and N-phthalaldehyde) were prepared by chemical modification. Two methods were developed for preparation of analogues of UDPMurNAc-pentapeptide containing D-cysteine at position 4 or 5: either by MurF-catalysed ligation of the UDPMurNAc-tripeptide to synthetic D-Ala-D-Cys or D-Cys-D-Ala dipeptides; or by enzymatic synthesis of D-Ala-D-Cys by ligase VanD. D-Cys-containing UDPMurNAc-pentapeptides were labelled with pyrene maleimide, to give 4-pyrene and 5-pyrene labelled derivatives. The fluorescent UDPMurNAc-pentapeptides were processed as substrates by Escherichia coli MraY or E. coli membranes, giving 1.5-150-fold changes in fluorescence upon transformation to lipid intermediate I. Subsequent processing to lipid intermediate II gave rise only to small changes in fluorescence. Pyrene-labelled lipid intermediates I and II can be generated using Micrococcus flavus membranes, enabling the study of the later lipid-linked steps.

Item Type:	Journal Article
Divisions:	Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- )
Journal or Publication Title:	Molecular BioSystems
Publisher:	Royal Society of Chemistry
ISSN:	1742-206X
Official Date:	October 2006
Dates:	Date Event October 2006 Published 7 September 2006 Available 18 August 2006 Accepted 5 June 2006 Submitted
Volume:	2
Number:	10
Page Range:	pp. 484-491
DOI:	10.1039/b607908c
Status:	Peer Reviewed
Publication Status:	Published
Access rights to Published version:	Restricted or Subscription Access
Date of first compliant deposit:	3 October 2022

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