AREAS RELATED TO ENZYME CATALYSIS - New Trends in Enzyme Catalysis and Biomimetic Chemical Reactions - page 170

Biological redesign uses recent achievements in the recombinant DNA method, site-
directed mutageneses, and growth of the databases of protein structures and sequences
(Arnold, 2001). The main problem with this approach is the requirement of a detailed
understanding of the structure and mechanisms of potential enzyme and its connection
with the protein sequence. An example of such an approach came from work on
dehalogenase (Kiang at al., 1999). The enzyme active site consists of a portion that
provide 2-enoyl-coenzyme A (CoA) binding, an oxyanion pocket, and stations at which
the enzyme binding and functional groups are in a position suitable for the catalylitic
process. The site-directed diversification of eight amino-acid groups in 4-chlorobenzoyl-
CoA-dehalogenase has led to the new ability to catalyze the hydration of crotonyl-CoA.
Another approach to breeding new catalysts is the use random, mutagenesis, gene,
recombinaton and screening in vitro conditions (Arnold, 2000, Ness et al., 2000). By
such a method an enzyme desaturase, which normally introduces double bonds into
phytoene was converted to a biocatalyst with the ability to produce other carotenoids
containing double bonds at various positions. Nonheme chemistry was used as an
exemplar for the emergence of superoxide dismutase, Fenton-like and dioxygen reductin
functions in Escherichia coli thioredoxin lacking iron and oxygen bindingsites (Benson
et al., 2000).
Two approaches have been suggested for an alteration of large segments of protein
sequence (domain swapping) (Penning and Jetz, 2000 and references therein). One of
them can be used when two enzymes share common restricted sites in their DNAs, In
thr second method, a target protein is composed from a series of synthetic or
biosynthetic fragments.
Site directed mutagenesis, based on knowledge of three-dimensional structures and
amino-acid sequences, has been successful in redesigning the substrate specificity of
many enzymes including dehydrogenases, acetylholinesterase, proteases,
aminotransferases, restriction enzymes, etc. (Fersht, 1992 and references therein) Malate
dehydrogenase, which catalyzes lactate to pyruvate, was converted into malate
dehydrogenase, which converts malate to oxaloacetate . Mutation of three residues in the
area of the enzyme substrate pocket resulted in a 107-fold increase for the malate
dehydrogenase reaction. Tripple mutation of a pyridoxal phosphate dependent enzyme,
L-aspartate amino aminotransferase drastically altered the partitioning of the covalent
intermediate aldimine: the ratio of b-decarboxylase activity to transaminase activity
increased 25 million-fold.
It was experimentally shown that the indol-3-glycerol-phosphate synthase (IGPS)
could switch its activity to that of phosphoribosylanthranilate isomerase (PRAI)
(Altamarino et al., 2000: Fersht and Altamarino, 2001). Both classes of enzymes share
similar Asn protein served as an scaffold for introducing a new
function. The PRAI function was evolved using the combined approach of rational
design, in vivo mutation, recombination and in vivo selection. The new enzymes exhibit
catalytic activity which is similar to the activity of native enzymes.
The authors suggested the strategy of enzymes loop replacing may be of wider
application.
The current status of the problem of de novo design of proteins and prospects in the
area including energy landscape theory of protein folding, atomistic and minimal