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148 Part 2: Biotechnology and EnzymologyFigure 7.17.A generalised schematic for the prediction of protein three-dimensional structure.- Genetic manipulation of the wild-type nucleotide
sequence. A combination of previously published
experimental literature and sequence/structure analysis
information is usually necessary for the identification
of functionally important sites in the protein. Once an
adequate three-dimensional structural model of the protein
of interest has been constructed, manipulation of the gene
of interest is necessary for the construction of mutants.
Polymerase chain reaction (PCR) mutagenesis is the
basic tool for the genetic manipulation of the nucleotide
sequences. The genetically redesigned proteins are
engineered by the following:
a. Site-directed mutagenesis: alteration of specific amino
acid residues. There are a number of experimental
approaches designed for this purpose. The basic
principle involves the use of synthetic oligonucleotides
(oligonucleotide-directed mutagenesis) that are com-
plementary to the cloned gene of interest but contain
a single (or sometimes multiple) mismatched base(s)
(Balland et al. 1985, Garvey and Matthews 1990,
Wagner and Benkovic 1990). The cloned gene is
either carried by a single-stranded vector (M13
oligonucleotide-directed mutagenesis) or a plasmid
that is later denatured by alkali (plasmid DNA
oligonucleotide-directed mutagenesis) or heat
(PCR-amplified oligonucleotide-directed mutagenesis)
in order for the mismatched oligonucleotide to anneal.
The latter then serves as a primer for DNA synthesis
catalysed by externally added DNA polymerase for the
creation of a copy of the entire vector, carrying, how-
ever, a mutated base. PCR mutagenesis is the most fre-
quently used mutagenesis method (Fig. 7.18). For ex-
ample, substitution of specific amino acid positions by
site-directed mutagenesis (S67D/H68D) successfully
converted the coenzyme specificity of the short-chain
carbonyl reductase from NADP(H) to NAD(H) as well
as the product enantioselectivity without disturbing
enzyme stability (Zhang et al. 2009). In another exam-
ple, engineering of the maize GSTF1–1 by mutating
selected G-site residues resulted in substantial changes
in the pH-dependence of kinetic parameters of the
enzyme (Labrou et al. 2004a). Mutation of a key
residue in the H-site of the same enzyme (Ile118Phe)
led to a fourfold improved specificity of the en-
zyme towards the herbicide alachlor (Labrou et al.
2005).
So far, substitution of a specific amino acid by an-
other has been limited by the availability of only 20
naturally occurring amino acids. However, it is chemi-
cally possible to construct hundreds of designer-made
amino acids. Incorporation of these novel protein
building blocks could help shed new light into the
cellular and protein functions (Wang and Schultz 2002,
Chin et al. 2003, Deiters et al. 2003, Arnold 2009).