Complexity mini- project proposal: 2008
Supervisor 1: Professor Chris Dowson, Biological Sciences, C149.2 c.g.dowson@warwick.ac.uk,
02476 523534 Supervisor 2: Dr David Roper, Biological Sciences
Project title: Comparative flux control through the cytoplasmic stages of cell wall biosynthesis
and perturbation by inhibitors: focus upon the essentiality of MurB
Project outline: Peptidoglycan is an essential component of the bacterial cell wall and its synthesis the
target of numerous important antibiotics and concomitantly the pathway where antibiotic resistance has
evolved in important bacterial pathogens including MRSA, VISA, VRE and streptococci[1-10]. This is a
three-phase process involving the cytoplasm, intracellular- and finally extracellular-face of the
membrane. The cytoplasmic phase (Figure 1) is initiated by murA-murF, with murI contributing DGlu, ddl D-Ala-D-Ala and alanine reacemase D-Ala.
A knowledge of the fluxes of
intermediates
through
the
cytoplasmic enzymatic steps in a
range of organisms is essential
especially
as
pharmaceutical
Figure 1. Cytoplasmic stages of
determining potential efficacy of
peptidoglycan biosynthesis
drugs that target this process.
Recently drug companies have
investigated
developing
an
inhibitor of MurB.
Presence of a functional murB gene is apparently essential- as deletions of the gene need to be
complemented by an extra-chromosomal copy of the gene in order for the mutant bug to live. So,
why might MurB inhibitors not work?
We have found a possible explanation in the following – the unreduced product of Mur A could be
used by MurC by-passing MurB.
Tool Box. The biochemical tools required to reconstruct the cytoplasmic pathway of peptidoglycan
synthesis from a wide range of bacteria are available. We have already purified murA, murB, murC,
murD, murE, murF, DDL, murI, DAPF and alanine racemase from Pseudomonas aeruginosa and assays
to measure their steady state and pre-steady state kinetics and great technical support to assist students
learn these assays.
Proposed Research Programme Using these tools we intend to carry out:
Kinetics and Flux through the pathway will be measured in vitro as a function of easily determined
spectrophotometric signals, such as the ADP generation by the mur ligases, phosphate generated by
murA, C, D, E, F, (and I in a coupled assay) and the turnover of NADPH by murB, for which continuous
real time assays are available[14, AJL unpublished] . All 5 enzymes (A-F) can be run together in a single
reaction tube and the role of Mur B investigated by simply omitting this enzyme. You will also monitor
levels of UDP-linked intermediates by discontinuous sampling and FPLC analysis. Product inhibition
will also be investigated.
Perturbation. This in vitro (A-F) system will be ‘challenged’ to mimic the action of specific inhibitors
by altering the stochiometric ratio of these enzymes to mimic inhibition, and alterations to the flux
through this pathway will be determined. These results can then be compared to those generated in
silico (see Kirkillianos – linked theoretical project), to provide a validation of the in silico model and to
provide an understanding of why some small molecule inhibitors of this pathway are vastly more
effective as antimicrobial agents than others.
References: [1]Bugg TDH (1999) Comprehensive natural products Chemistry 3, 241; [2]Lloyd, A.J., Huyton, T.,
Turkenberg, and Roper, D.I. (2004). Acta Cryst. D60, 397; [3]Ho, H.T., Falk, P.J. and Ervin, K.M. et al. (1995)
Biochemistry 34, 2464; [4]Schneider, T., Senn, M.M. and Berger-Bächi, B. et al. (2004). Mol. Microbiol. 53, 675.
[5]
Walsh C.T. 2000 Nature 406, 65; [6]Mato, R., Campanile, F. and Stefani, S. et al. ( 2004). Microb Drug Resist. 10,
106; [7]El Zoeiby, A., Sanschagrin, F. and Levesque, R.C. (2003). Mol. Microbiol. 47, 1; [8]Sanders, W.E. Jr. and
Sanders C.C. (1979) Ann. Rev. Pharmacol.Toxicol. 19, 53; [9]de Dios, A., Prieto, L. and Martin, J.A. et al. (2002) J.
Med. Chem. 45, 4559; [10]Bronson, J.J., DenBleyker, K.L. and Falk, P.J. et al. (2003). Bioorganic Med. Chem. Lett. 13,
5873; [11]Gui Gu, Y., Florjancic, A.S. and Clark, R.F. et al. (2004). Bioorganic Med Chem Lett 14, 267; [12] Horton,
J.R., Bostock, J.M. and Chopra, I. et al. (2003). Bioorganic Med. Chem. Lett. 13 1557; [14]El Zoeiby A., Sanschagrin,
F. and Lamoureux J. et al. (2000). FEMS Microbiol. Lett.183 281, 288