Mar 252008
 
ResearchBlogging.orgBiological systems have the interesting property that most of the reactions enabling life processes are, when left to their own devices, exceedingly slow. To reach the timescales that we associate with “living”, these reactions must be sped up, which requires the presence of enzymes. Because they significantly enhance reaction rates under conditions that can be encountered almost anywhere, the design of artificial enzymes is an active area of research. In two papers this month, David Baker’s lab describes notable success in designing enzymes in silico to have specific activities with significant (106-fold) rate enhancement.

As a graduate student at UNC, I was fortunate to interact frequently with Richard Wolfenden, who did a great deal of work to find out just how good enzymes are at what they do (1). The fact is that there is a wide range of activities and rate enhancements. The proline isomerase cyclophilin, for instance, achieves a modest 106-fold rate increase, depending on the substrate. In contrast, arginine decarboxylase achieves an amazing rate enhancement of about 1019. Many reactions we think nothing of, such as hydrolysis of a phosphodiester bond (found in nucleic acids) would take millions of years in pure neutral water at 25° C. Of course, deviations from neutral pH and the presence of other molecules greatly enhance these rates, and obviously the same is true of changes in temperature, but this is a useful starting point for comparing enzymes to basal rates.

In the works at hand, collaborative teams involving several labs coordinated by David Baker designed enzymes to perform a novel retro-aldol reaction (2) and the Kemp elimination from 5-nitro-benzisoxazole (3) (a proton abstraction causing a ring to open). The retro-aldol paper is fascinating, particularly because of the multi-step nature of the reaction, but I’m going to focus on the Nature paper because its results are more complete, in that they implemented an appropriate wet-lab extension to the computational procedure.

The fundamental strategy of both papers is the same. For most enzymes it is believed that catalysis occurs because the transition state, the moment when the chemical reaction has the highest energy, is stabilized by the functional groups of the enzyme (see (1), among others). Using their knowledge of chemistry, the researchers of these groups predicted a transition state, and then positioned functional groups of side chains in such a way that they would stabilize this predicted state. They also placed potential bases in an appropriate geometry to attack protons as necessary. This done, they used a program based on Baker’s ROSETTA to predict sequences that would fold to produce this geometry. This required a somewhat more complicated process in the case of the retro-aldol reaction due to its multiple steps.

One interesting outcome was that TIM barrels were a popular choice of this algorithm in both papers. The final results in the Röthlisberger paper are all based on backbones identified by CATH as TIM-barrel folds (explore these scaffolds at the PDB: 1thf, 1a53, 1h61, 1jcl). As the authors note, the TIM barrel is a very common catalytic scaffold in nature, in part because the central β-strands provide a convenient way to orient side-chains towards the catalytic pocket. In both papers, the structures predicted using the ROSETTA algorithm were shown to be very close to the actual result, although they only checked successful catalysts. A comparison of the failed designs to their predicted structures may be of great use in refining the computational approach.

As the above paragraph implies, the groups did in fact succeed in designing enzymes that achieved significant rate enhancements. In the case of the Kemp elimination, the eight enzymes reported had ~5×103 – 2×105 -fold increases in rate over the spontaneous reaction in a very slightly basic solution. This amounted to actual kcat (reaction rate) values of 0.006 – 0.29 s-1, which is significantly slower than is common for enzymes.

In order to improve these results, Röthlisberger et al. turned to the process that produced our own prodigious enzymes in the first place, i.e. evolution. Using a relatively standard in vitro evolution approach, they altered one of the early successes, KE07, which had a kcat of 0.018 s-1. Keep in mind, this was not the best computational design result, just one of the first that worked. This in vitro evolution procedure, in just a few rounds, produced an enzyme with a kcat of 1.37 s-1. While this is still slow for an enzyme, it represents a rate enhancement of ~1×106 over the spontaneous reaction in solution, an acceleration comparable to that of a modest enzyme like cyclophilin.

This is nowhere near a complete journey. I’ve already mentioned that the enzymes produced in these experiments are still quite slow in comparison to the genuine article, and the rate enhancements are still modest. The specificity of the enzymes also has yet to be proven—can these proteins distinguish their targets from a sea of similar molecules, or are they promiscuous catalysts? A further dissection of the failed designs is essential to refining the computational approach employed. More careful consideration of effects beyond the secondary shell, and (as the authors note) backbone dynamics and loop positioning may prove particularly helpful in future iterations.

So, we are not all the way to the creation of a truly proficient man-made enzyme, but this is a tremendous step in that direction. The combination of wet lab and computational approaches proved to be very successful in this case. In principle, it should be possible to incorporate all that was learned in the in vitro evolution experiments into the design algorithm from the start. We will not be designing custom catalysts for biofuel production and bioremediation tomorrow or next week. These results, however, demonstrate substantial promise for the future.

In particular, the retro-aldol paper suggests that this approach will work for multi-step reactions. However, provided that the intermediates are stable and soluble this will not be strictly necessary. So long as efficient catalysts can be designed for each step, the ability of ROSETTA to design protein-protein interfaces will make it possible to assemble functional synthetic or catabolic enzyme cassettes to achieve very complex chemistry with tremendous accelerations over basal rates.

1. Wolfenden, R., Snider, M. (2001). The Depth of Chemical Time and the Power of Enzymes as Catalysts. Accounts of Chemical Research, 34 (12), 938-945. DOI: 10.1021/ar000058i

2. Jiang, L., Althoff, E.A., Clemente, F.R., Doyle, L., Rothlisberger, D., Zanghellini, A., Gallaher, J.L., Betker, J.L., Tanaka, F., Barbas, C.F., Hilvert, D., Houk, K.N., Stoddard, B.L., Baker, D. (2008). De Novo Computational Design of Retro-Aldol Enzymes. Science, 319(5868), 1387-1391. DOI: 10.1126/science.1152692

3. Röthlisberger, D., Khersonsky, O., Wollacott, A.M., Jiang, L., 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). Kemp elimination catalysts by computational enzyme design. Nature DOI: 10.1038/nature06879

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