Where do new enzymes come from?

Biochemists often rave about the great wonders of enzymes, lavishing praise on the prodigious rate enhancements they produce, and their exquisite positioning of functional groups. One can quite reasonably ask how such magnificently useful proteins came into being. One accurate answer, of course, is that after a couple hundred million years evolution can get almost anything right. Another answer is that most enzymes come from other proteins, via a process called gene duplication. The genetic changes that follow one of these duplications turn two copies of one protein into two completely different proteins with diverse activities.

Gene duplication events are infrequent errors of DNA replication or repair. Diploid eukaryotes such as ourselves carry two copies (or near-copies) of most genes as a matter of course, but gene duplications produce extra copies beyond that. In theory, the presence of these extra copies of a gene means that one of them can mutate freely, without the pressure of carrying out its normal job. When it drifts into a useful function, selective pressure is again applied, causing a refinement of the active site to maximize the efficiency of the new activity. The overall scheme looks something like this:

Duplication → Divergence → Refinement

It may seem incredible that a vast diversity of protein structures and activities can arise simply by making copies, even imperfect copies. However, certain quirks of the translation machinery mean that small changes in DNA can amount to enormous changes in a protein's topology. For instance, an insertion or deletion of a single base can cause a frameshift mutation, producing a protein that bears no resemblance to its progenitor despite having only 1 different base pair. Many DNA triplets that normally encode amino acids are only a single base-pair mutation away from becoming a stop codon, truncating a protein and likely changing its structure significantly. Similarly, stop codons can be easily eliminated, producing much larger proteins. In eukaryotes, point mutations near the borders between introns and exons can cause new regions of DNA to be translated into protein. Of course, drastic changes like these mostly just produce useless junk, but occasionally a novel fold or function arises.

More conservative alterations of a gene sequence can still produce significant changes. As I've mentioned before on this blog, some members of the Cro family of proteins have very high sequence identity and yet possess different structures. I also have not yet tired of reminding you that the chemokine lymphotactin has two different structures with a single sequence, either of which can be stabilized into an exclusive fold by a point mutation.

Additionally, research from the lab of John Orban shows that a mere 7 mutations are required to convert the engineered protein GA88 (PDB) into a completely different structure, GB88 (PDB) (1). These proteins were previously shown to have different folds and functions, but the contrast between the high resolution structures (shamelessly stolen figure on the right) is striking. Moreover, the Orban lab has refined this system so that the structural conversion can be effected with only three mutations, rather than seven. What all this research indicates is that the transitions that convert a sequence from one fold into another may be sharper than previously realized; even a relatively small number of fairly conservative mutations may be able to completely transform a protein's structure.

For all that, most new enzymes arising via gene duplication resemble their ancestors in identifiable ways. Often the two proteins perform the same chemical steps, and the novel function amounts to a different substrate specificity. This suggests the possibility of an alternate mechanism of gene duplication, in that a protein could evolve a novel specificity while retaining its original function. Diversifying its activities in this way would probably limit an enzyme's catalytic effect in both reactions, but a subsequent gene duplication event would allow each copy to refine its particular reaction. The scheme would look like this:

Diversification → Duplication → Refinement

The advantage of this model, from an adaptationist's perspective, is that it brings selective pressure to bear at every step. Once a new function has evolved in response to environmental conditions, duplicating the gene may provide an organism a concrete advantage. After duplication, the advantage of separately refining the two activities is obvious.

The two models are not as different as they might seem at first glance, because nearly every enzyme catalyzes two reactions anyway, that is, the forward and reverse reactions of an equilibrium. A "new" activity for a given enzyme can therefore result from something as simple as being targeted to a different cellular compartment or a change in specificity that involves an oppositely-oriented equilibrium.

The most obvious objection to the latter model is that during the period of gene sharing prior to duplication, neither protein function will be very efficient. As a matter of fact, the appearance of a new activity does not always impair an enzyme's ability to do its original job (and indeed can even enhance that activity). Still, because of the exquisite tuning of enzyme active sites we can expect that many modifications to this region will reduce catalytic power. That being the case, how might an organism survive or thrive during the gene-sharing period? The answer, which always seems obvious in retrospect, is to make more of the less efficient enzyme, as was demonstrated in a recent paper by Sean Yu McLoughlin and Shelley Copley (2).

McLoughlin and Copley took a strain of E. coli that lacked an enzyme, ArgC, that is critical for glucose metabolism. They treated these bacteria with a strong mutagen and then picked a colony that grew well on uncomplemented glucose. After showing that these bacteria had developed a novel activity equivalent to ArgC, they isolated the "new" enzyme and found that it was actually an existing enzyme, ProA, which performs similar chemistry. This enzyme had gained the ability to take over the tasks of the missing ArgC, enhancing the rate of that reaction 12-fold. The actual chemistry of these reactions was quite similar, but in gaining the ability to operate on ArgC's substrate, the activity of ProA towards its own substrate was reduced 2800-fold. The bacteria compensated for this by upregulating the production of the enzyme. A second mutation in the promoter region of the gene was helpful, but not necessary, in this respect.

Because enzymes are catalysts, a small increase in protein concentration can result in a significant increase in the availability of the reaction products. Biochemists often say, seeing a 3000-fold reduction in activity, that an enzyme is dead. The reality is that it's just slower, and a living thing can compensate for that in ways not available to an isolated reaction in a test tube. Organisms have shown that they have ways to survive what an enzymologist might see as fatal.

Of course, modern bacteria benefit from a number of well-tuned regulatory and feedback mechanisms that allow them to sense when particular metabolites are running low and to increase the production of proteins that can replenish them. Earlier, more primitive organisms might not have had these expedients available. Could they have survived gene sharing?

Too little is known about early life forms to answer such a question definitively. However, it is interesting to note that one method of making more protein is to make more of the gene. That is, the concentration of a deficient enzyme can be increased via gene duplication. By a fortuitous coincidence, a single mechanism could both enable an organism to tolerate reduced enzymatic efficiency and allow the evolutionary process to independently refine its activities.

It is also worth bearing in mind that just as ancient organisms did not necessarily resemble modern ones, ancient proteins might not have resembled the modern item. The exquisite positioning of functional groups that characterizes modern enzymes requires a rigid fold and contributes significantly to the rate accelerations they produce. However, substantial rate enhancements can still be achieved in the absence of a stiff native state.

One occasional result of mutations is the formation of a molten globule, a protein that lacks a stable fold but still exists in a collapsed state with something resembling a hydrophobic core. Although that doesn't sound particularly useful, many molten globules have enzymatic or other functional activities. Recent computational studies on a molten-globule mutant of Methanococcus jannaschii chorismate mutase suggest that realistically low energy barriers can be achieved by a broader array of structural states in these proteins (3).

Researchers from the lab of Arieh Warshel used a simplified model to sample the conformational space available to the molten globule enzyme (mMjCM) and a stably folded form of the enzyme (EcCM). As you might expect, the lowest-energy conformations are much more diverse for mMjCM than for EcCM. Roca et al. then computed the energy barrier for catalysis for conformations that closely resembled the ideal structure (region I), conformations which had most of the groups in the right general position but were significantly removed from the ideal (region II), and conformations that did not resemble the ideal at all (region III). For EcCM, only structures in region I had energy barriers low enough to plausibly allow catalysis. The molten globule, however, had energy barriers that would allow catalysis in region I and region II. You can see this in the figure below, which I shamelessly stole from their paper: the dotted orange line corresponds to a 16 kcal/mol energy barrier, what they felt to be the largest barrier reasonable for a catalyst. The results for mMjCM are on the left, EcCM on the right.



The upshot of this is that molten globules may be able to maintain catalytic power in the face of structural diversity that causes folded proteins to fail. While the stable fold produces greater rate enhancements (note that EcCM has lower energy barriers), the molten globule tolerates a wider array of structural conditions. Consequently, proteins of this kind may be much more amenable to the addition of new functions. So long as an appropriate orientation of functional groups is reasonably likely, a protein without a rigid conformation can still achieve impressive rate enhancements.

Conceivably, an early molten globule enzyme could have the ability to catalyze several different reactions, switching between the required conformations as needed, without a significant loss of catalytic power to any of them. Duplication of a multi-functional molten globule like this would allow each chemical function to be refined independently, with additional duplications and refinements giving rise to substrate specificity.

The different models of gene duplication each have their own explanatory advantages, and the available evidence suggests that new proteins and enzymatic activities have evolved (even within the last century) using both routes. As this is one of nature's favored methods of generating novel activities, so it is becoming ours. The artificial enzymes recently produced by David Baker's lab were designed onto an existing protein scaffold in what could be taken as a computational mimicry of the gene duplication process.

1. Y. He, Y. Chen, P. Alexander, P. N. Bryan, J. Orban (2008). NMR structures of two designed proteins with high sequence identity but different fold and function Proceedings of the National Academy of Sciences, 105 (38), 14412-14417 DOI: 10.1073/pnas.0805857105

2. S. Y. McLoughlin, S. D. Copley (2008). A compromise required by gene sharing enables survival: Implications for evolution of new enzyme activities Proceedings of the National Academy of Sciences, 105 (36), 13497-13502 DOI: 10.1073/pnas.0804804105

3. M. Roca, B. Messer, D. Hilvert, A. Warshel (2008). On the relationship between folding and chemical landscapes in enzyme catalysis Proceedings of the National Academy of Sciences, 105 (37), 13877-13882 DOI: 10.1073/pnas.0803405105

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Microwave Pfu CelB 5 minutes for highest activity

Like many bachelors, I regularly eat meals heated up in a microwave oven. I'd like to think I eat a somewhat lower percentage of frozen dinners than others in my situation, but even when it comes to food I cooked myself I usually don't have the time or patience to cook a recipe for one every night. That means I'm often eating reheated leftovers from the trusty Radar Range. The microwave oven works using dielectric heating, a process in which the movements of dipolar bonds are coupled to an oscillating external field. Water, for instance, is a molecule with a large dipole moment, and when you cook something in a microwave a lot of the heating occurs by the accelerated movements of water molecules. In principle this kind of excitation should also occur for other kinds of polar molecules, and thus we come to an interesting study from the lab of Alex Dieters, in which microwave radiation was used to activate a cellulase from a hyperthermophile.

Protein backbones consist of series of peptide bonds which include a carbonyl group, a classic example of a polar bond. Naturally, one might expect that the motion of these groups would be excited by microwave radiation. However, it does not directly follow that additional motion of the peptide backbone will actually accelerate chemical reactions, because this motion may be chaotic or unproductive. Moreover, from the fact that microwaves cook things (like eggs), we know that microwave radiation does a good job of denaturing proteins, sometimes at lower temperatures than we expect. Both of these problems can conceivably be avoided by studying a hyperthermophilic protein.

Proteins from hyperthermophiles such as Pyrococcus furiosus tend to be stable and optimally active at very high temperatures, at or even exceeding the boiling point of water. At lower temperatures, they retain their stability, but tend to become inactive. In many cases this reduction in activity appears to result from squelching internal motions that may be necessary to bind or properly orient substrates. Young et al. decided to study the β-glucosidase CelB from P. furiosus as a way of understanding whether microwaves might enhance enzymatic catalysis. Because CelB has optimal activity at 110° C it should be possible to see a significant difference in activity if microwave activation works. The stability of this protein at high temperatures also suggests that you will not accidentally cook it.

Sharp readers will have noticed an obvious problem with this idea—because heat activates this protein, and microwaves heat aqueous solutions, we must incorporate some kind of control in order to determine the pure effect of the radiation as opposed to the temperature. Young et al. resolve this problem by monitoring the heating of the sample during microwave irradiation, and then using a normal thermal apparatus to match this temperature profile (Figure 2). When a reaction reached 40° C using either heating method, it was quenched by the addition of a basic solution and the concentration of products was measured. Simply heating the Pfu CelB reaction to 40° C produced negligible activity, but microwaving it increased the activity by 4 orders of magnitude (i.e. a factor of 10,000). Less dramatic, but still significant, effects were observed for two other hyperthermophilic enzymes, but an enzyme from a mesophilic organism (the almond) was not activated by microwave irradiation.

That the microwaves caused increased backbone motion was supported by the finding that irradiating Pfu CelB at 75° C caused it to denature; this temperature is well below the normal melting temperature of this enzyme (115° C). The authors attribute the differences in activation between CelB and the other hyperthermophiles to their lower optimal activity temperatures, but it is also possible that the particular motions enhanced by microwaves are simply not as productive in those molecules. Although all the dipoles should be affected in similar ways by microwaves, they are all oriented differently with respect to each other in the protein molecule. As a result, the induced motion may be chaotic, perhaps specifically so, and therefore the activation of a particular thermophile may depend on the nature of the motions needed for its catalytic cycle. Enzymes that require large ensemble motions of subdomains, such as adenylate kinase, might not be activated as much as a protein that simply needs to be melted a little. Examining the differences in structural dynamics of enzymes differentially activated by microwaves may be an interesting area of future study.

While microwave activation is unlikely to revolutionize some of the more common uses of hyperthermophilic proteins (i.e. PCR), it does have promise. In ligations, for instance, hyperthermophilic enzymes can not be used at present because many DNA inserts denature at the optimal active temperature. With microwave activation, it may be possible to employ extremophilic ligases in these reactions, gaining the benefits of their speed and durability without having to worry about accidentally melting your DNA. Depending on the enzymes available, this technique may also prove valuable in improving mobile medical laboratories and developing novel diagnostic tools for field work.

1. Young, D.D., Nichols, J., Kelly, R.M., Deiters, A. (2008). Microwave Activation of Enzymatic Catalysis. Journal of the American Chemical Society DOI: 10.1021/ja802404g

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Enzymes almost as good as Ma Nature used to make

Biological 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 ~5x10³ - 2x10⁵ -fold increases in rate over the spontaneous reaction in a very slightly basic solution. This amounted to actual k_cat (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 k_cat 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 k_cat of 1.37 s^-1. While this is still slow for an enzyme, it represents a rate enhancement of ~1x10⁶ 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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