Chemistry Reference
In-Depth Information
and/or for some unusual “building blocks” might be difficult to deduce from
the structure. In such cases, a series of putative precursors labeled with either
13
Cor
15
N are used in so-called “feeding” experiments. In essence, the labeled
precursors are added to the growing culture of antibiotic-producing organism,
which allows for their full or partial incorporation into the antibiotic molecule in
the course of the biosynthesis. Antibiotic is then extracted, purified, and inves-
tigated with regard to incorporation of the labeled precursors (or parts thereof)
by means of LC-MS/MS or NMR (48, 49). In some cases, stereochemically
labeled precursors shall be used to determine the biosynthetic origin of certain
“building blocks” (50). Besides determining the origin of the building blocks of
antibiotic molecules, feeding studies using unnatural substrates for enzymes that
participate in the biosynthesis can be performed. In such studies, the antibiotic-
producing organism is fed a series of chemically synthesized substrates for a
particular antibiotic biosynthesis enzyme, and the secondary metabolites pro-
duced are analyzed (51). Results of these experiments can reveal the flexibility
of certain antibiotic biosynthesis enzymes and complete pathways in terms of
unnatural substrates and their incorporation into antibiotic molecules. Such stud-
ies are important for engineered biosynthesis that may provide novel antibiotics
with improved properties.
11.3.2 Mutational Analysis and Enzymatic Assays
A method frequently used for determination or confirmation of antibiotic biosyn-
thetic pathways takes advantage of the availability of antibiotic biosynthesis
genes. If these genes can be manipulated in either an original or heterologous
host, then in many cases the exact function of the genes can be established
through their inactivation and subsequent analysis of metabolite profiles in the
resulting mutants. This method in itself is mostly used for experimental confir-
mation of a certain enzyme activity in antibiotic biosynthetic pathway that has
been suggested from bioinformatics analysis. For example, involvement of spe-
cific glycosyltransferase or P450 monooxygenase in antibiotic biosynthesis can
be confirmed through inactivation of respective genes in the producing organ-
isms and identification of the resulting deglycosyl- or deoxy-intermediates (52,
53). However, this method relies heavily on analytical methods (LC-MS/MS,
LC-MS-TOF, NMR) for identification of antibiotic biosynthesis intermediate(s)
produced as a result of pathway interruption. In certain cases, these intermediates
are either chemically unstable, scarcely available, or undergo extensive conver-
sion by noncognate enzymes, which makes exact placement of a step performed
by inactivated enzyme on a biosynthetic pathway rather difficult.
Providing that antibiotic biosynthesis intermediate can be purified, a more
comprehensive analysis of a biochemical reaction performed by an enzyme
responsible for the next biosynthetic step may be achieved. This result can
be performed by expression of the enzyme either in the original host or
heterologously, and by performing
in vitro
enzyme assays (54). Individually
expressed and purified antibiotic biosynthesis enzymes can also be used in
