Houk Group’s Quantum Mechanical Design
of Active Sites Leads to Successful Creation
of Enzymes for Non-natural Reactions in
Collaboration with David Baker’s Group
The work on two successful enzyme designs has been published in Nature and
Science. These papers describe breakthroughs made in a three-year
collaboration between Houk and Baker groups and others involved in a program
supported by DARPA:
Kemp Elimination Catalysts by Computational Enzyme Design.
Rothlisberger, D.; Khersonsky, O.; Wollacott, A. M.; Jiang, J.; DeChancie, J.;
Betker, J.; Gallaher, J. L.; Althoff, E. A.; Zanghellini, A.; Dym, O.; Albeck, S.;
Houk, K. N.; Tawfik, D. S.; Baker. D. 2008 Nature, AOP.
http://www.nature.com/nature/journal/vaop/ncurrent/full/nature06879.html
De Novo Computational Design of Retro-Aldol Enzymes.
Jiang, L.; Althoff, E. A; Clemente, F. R.; Doyle, L.; Rothlisberger, D.; Zanghellini,
A; Gallaher, J. L.; Betker, J. L.; Tanka, F.; Barbas, C. F. III; Hilvert, D.; Houk, K.
N.; Stoddard, B.; Baker, D. 2008 Science, 319, 1387.
http://www.sciencemag.org/cgi/content/abstract/319/5868/1387
For UCLA’s press release on these papers, see:
http://newsroom.ucla.edu/portal/ucla/designer-enzymes-created-by-ucla-46985.aspx
INSIDE-OUT DESIGN OF NEW ENZYME CATALYSTS
The success of inverse protein folding reached a milestone with the design and successful
experimental proof of structure of a 93-residue / protein called Top7.1 The next great
challenge of protein design is to predict and create a functional protein for a reaction for which no
natural enzymes are known.
The first successful response to this challenge was achieved in a collaborative effort between our
group and that of David Baker (University of Washington) protocol for enzyme design. The
inside-out approach we developed starts with the design of an active site with the appropriate
functionality for catalysis using quantum mechanical calculations. We call this a theozyme, for
theoretical enzyme. The advantage of this computational method is the ability to tune optimal
functionality that a protein can use to bind and stabilize the transition state.
Baker’s group then designs a protein that is predicted to fold with the calculated active site
structure. Design efforts involve splicing the active site into existing protein scaffolds, selected
based on the ability of the fold to orient the functional groups necessary for proficient catalysis.
Rosetta design energy functions and minimization methods are subsequently employed to
redesign the vicinity of the active site with side-chains that will stabilize the protein and maintain
the geometry of the catalytic site. Finally, the designed proteins are expressed in E. coli from the
corresponding DNA, and activities are assayed.
Theozymes serve as a blueprint for the construction of active sites within a protein and are used
for the prediction of activation barriers of catalyzed reactions in the theozyme relative to the
uncatalyzed reaction in aqueous solution. Building an effective theozyme active depends on
knowledge about the target reactions and how they can be catalyzed.
For the Kemp elimination, our group has previously shown that proper base (aspartate or
glutamate) orientation and nonpolar environment are paramount to achieve rate acceleration. 2 It
was also determined that hydrogen-bonding to the developing oxyanion could potentially lead to
rate acceleration.2
A covalent mechanism via enamine formation derived from a nucleophilic lysine side-chain was
designed for the retro-aldolase reaction. This covalent mechanism was applied because it has
been shown that, in general, proficiencies higher than 1011 M-1 can only be achieved through
partially covalent catalysis, including covalent intermediate formation, general acid-base, and
metal binding.3 Additional side-chains (Lys and Asp) were placed to act as a general acid/base.
The catalytic importance of these residues are supported by calculations carried out by the Houk
group4 and Wu et al.5 that point out the importance of stabilizing the negative charge of the
developing alkoxide in the transition state in order to lower the energy barrier of the C-C bond
forming step. This can be done either by proton transfer (acid catalysis) 6-10 or by good
coordination with multiple hydrogen bond donors.5
Active site designs for the Kemp elimination and the retro-aldol reaction are shown above.
Additional References:
1.
Kuhlman, B.; Dantas, G.; Ireton, G. C.; Varani, G.; Stoddard, B. L.; Baker, D., Science 2003, 302,
1364-1368.
2.
Na, J.; Houk, K. N.; Hilvert, D. J. Am. Chem. Soc. 1996, 118, 6462-6471.
3.
Zhang, X.; Houk, K. N., Acc. Chem. Res. 2005, 38, 379-385.
4.
5.
6.
7.
8.
9.
10.
Wu, Y.; Li, Y.; Na, J.; Houk, K. N., J. Org. Chem. 1993, 58, 4625-4628.
Tang, Z.; Luo-Ting, F. J.; Cui, X.; Gong, L. Z.; Mi, A. Q.; Jiang, Y. Z.; Wu, Y. D., J. Am. Chem.
Soc. 2003, 125, 5262-5263.
Bahmanyar, S.; Houk, K. N., J. Am. Chem. Soc. 2001, 123, 11273-11283.
Bahmanyar, S.; Houk, K. N., J. Am. Chem. Soc. 2001, 123, 12911-12912.
Clemente, F. R.; Houk, K. N., Angew. Chem. Int. Ed. 2004, 43, 5766-5768.
Bahmanyar, S.; Houk, K. N.; Martin, H. J.; List, B., J. Am. Chem. Soc. 2003, 125, 2475-2479.
Hoang, L.; Bahmanyar, S.; Houk, K. N.; List, B., J. Am. Chem. Soc. 2003, 125, 16-17.
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