Biology Reference
In-Depth Information
Jiang et al. 51 succeeded in designing proteins catalyzing a retro-aldol reaction, which is a
multistep transformation capable of breaking carbon
carbon bonds. Several natural
enzymes exist that accelerate the complementary reverse reaction. The most critical active
site element of these natural aldolases is a strategically placed lysine that carries out a
nucleophilic attack on a ketone moiety of the substrate to form a Schiff-base covalent
adduct and thus initiate the reaction. Several other protic residues act in concert with the
lysine to carry out a number of proton shuffling steps necessary to complete the reaction.
Consequently, Jiang et al. picked this lysine as the central element of the theozyme used for
the retroaldolase designs. The authors used three variations of the theozyme: one featuring
the lysine as the only amino acid but with explicit water molecules to carry out the proton
transfers; the other two more similar to the natural active sites, with different combinations
of protic side-chains supporting the lysine. Out of a total of 72 designs that were
experimentally characterized, 32 showed activity, albeit at far lower efficiencies than natural
enzymes. Somewhat surprisingly, designs based on the first, simple theozyme showed a
higher rate of success than those featuring the more complicated theozyme.
Siegel et al. presented the first example of an enzyme catalyzing a nonnatural reaction, in
designing the first biocatalyst for a Diels-Alder reaction, 52 which is a cycloaddition reaction
that simultaneously forms two carbon
carbon bonds and four stereocenters, and is thus
highly useful in organic synthesis. This study demonstrates that, in principle, the catalytic
repertoire of proteins is not limited to the types of reactions observed in nature, and thus
suggests new possibilities for the biotechnological application of enzymes, such as the
incorporation of novel steps into biosynthesis routes. In their study, the authors employed
quantum-mechanical methods to compute the relative orientation of the two reacting
molecules in the transition state and to devise the placement for protein functional groups
to lower the activation barrier of the cycloaddition. Out of a total of 82 designs, two
showed low activity, and for one of them, the activity could be improved 100-fold through
five point mutations. In addition, the resulting variant showed high selectivity for the
designed stereo-configuration of the product.
116
In summary, a number of impressive breakthroughs have been achieved with computational
enzyme design over the last few years, suggesting that this method might play an important
role in future synthetic biology applications. What is important to understand however, is
that the current computational methods still lack an accurate modeling of the quantum-
mechanical aspects of reactivity, and thus computational design of catalytic activity requires
an exquisite understanding of the reaction of interest. For a successful project, the user needs
to determine which aspects of a designed active site to enforce and which parts to allow
CPD to determine. A future avenue of research is the incorporation of more advanced
quantum-mechanical modeling protocols into the currently used design codes. Another
caveat is that for successful design of catalytic activity, i.e. discrimination between ground
and transition states, placement of the active site functional groups with sub-angstrom
precision is required, whereas in the design of a binding interface, somewhat more
wiggle
'
room
is allowed. For these two reasons, de novo enzyme design will remain a challenging
endeavor in the foreseeable future.
'
PROTEIN THERMOSTABILIZATION BY COMPUTATIONAL DESIGN
The earliest application of CPD was the design of mutations to increase the stability of a
protein of interest. As side-chain placement algorithms were developed, their ability to
predict stabilizing mutations in naturally occurring proteins was used as the first
experimental verification of the methodology. 53 It should be noted that, like for other CPD
applications discussed so far, an experimental structure of the protein of interest needs to be
available in order to stabilize it by CPD. If this is the case, CPD can be used as a valuable
tool to create thermostable variants of the protein of interest for synthetic biology
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