Chemistry Reference
In-Depth Information
coupled
in vitro
assays to determine the order of events in the biosynthetic
pathway (42).
11.3.3 Studies on Enzyme-Bound Substrates and Intermediates
Another method used extensively for studying molecular details of antibiotic
biosynthesis involves investigations into particular biosynthetic enzymes with
bound substrates or intermediates. The method allows investigation of substrate
tolerance and specificity, timing and order of reactions, transfer of substrates
and biosynthetic intermediates from one enzyme to another. Early examples of
this method relied completely on radioactively labeled substrates, which were
mixed with purified antibiotic biosynthesis enzymes, and substrate/intermediate
protein complexes were separated by chromatographic methods. Purified com-
plexes were then studied with respect to the amount of the substrate/intermediate
bound, as well as the identity of the intermediates. The latter was achieved
after denaturation of the complexes and identification of released intermedi-
ates by analytical methods (55, 56). Recent development of efficient protein
expression systems and new analytical methods greatly enhanced this technique,
which allows deeper mechanistic insights into the mode of action of enzymes
involved in antibiotic biosynthesis. One new method involves electrospray ion-
ization Fourier-Transform mass spectrometry (ESI-FTMS), which is particularly
suitable for studies on modular biosynthetic enzyme systems, such as PKS and
NRPS (57). ESI-FTMS offers high-resolution power in determining masses of
proteins with bound intermediates in complex mixtures, which are produced as
a result of limited proteolysis of the enzyme-substrate complexes. Using this
method, it is possible to determine relative ratios of intermediates at specific
sites on antibiotic biosynthesis enzymes and to identify particular intermediates
directly.
Another recently introduced method involves crystallographic trapping
of potential substrate in purified antibiotic biosynthesis enzyme that allows
determination of enzymatic mechanism (58). In this method, crystals of a
biosynthetic enzyme are soaked in potential substrate mimics, and subsequent
crystallographic analysis allows identification of a true substrate, even in the
mixture of apparently unstable intermediates.
REFERENCES
1. Chain E, Florey HW, Adelaide MB, Gardner AD, Heatley NG, Jennings MA, Orr-
Ewing J, Sanders AG. Penicillin as a chemotherapeutic agent. Lancet 1940;236:
226 - 228.
2. Yim G, Wang HH, Davies J. Antibiotics as signalling molecules. Philos. Trans. R.
Soc. Lond. B. Biol. Sci. 2007;362:1195 - 1200.
3. McDaniel R, Welch M, Hutchinson CR. Genetic approaches to polyketide antibiotics.
1. Chem. Rev. 2005;105:543 - 558.
