H ow Proteins Adapt: Lessons from Directed Evolution

F.H.ARNOLD Division of Chemistry and Chemical Engineering, California Institute ofTechnology, Pasadena, California 91125 Correspondence: [email protected] u

Applying artificial selection to create new proteins has allowed us to explore fundamental processes of molecular evolution. These "'directed evolution" experiments have shown that proteins can readily adapt to new functions or environments via sim­ ple adaptive walks involving small numbers of mutations. With the entire ··fossil record·· available for detailed study. these experiments have provided new insight into adaptive mechanisms aod the effects of mutation and recombination. Directed evolution has also shown how mutations that are functionally neutral can set the stage for further adaptation. Watching adap­ tation in real time helps one to appreciate the power of the evolutionary design algorithm.

In making his case for the role of natural selection in Darwin· s great insight long predated any understand­ evolution, Darwin started by pointing to the enom1ous ing of the molecular mechanisms of inheritance and evo­ phenotypic variation that could be achieved in just a few lution. How DNA-coding changes alter protein function generations of artificial selection. In Darwin's day, the is new information that di rected evolution experiments importance of good breeding practices in detemlining the contribute to the evolution story. With access to the entire productivity and quality of a farmer's stock or the crop "fossil record" of an evolution experiment, we can deter­ size of a pigeon fancier's prize bird was clear to all, and mine precisely how gene sequences change during adap­ Darwin' s artificial selection arguments provided a power­ tation and can connect specific mutations to specific ful foundation for his idea that competition for limited acquired trait . During the last 20 years, directed evolu­ resources could imilarly tailor phenotypes, and ultimately tion experiment have revealed that useful properties create new species, by selecting for beneficial traits. such as catalytic activity or stability can frequently be en­ Today. we can use artificial selection to breed not just hanced by single-amino-acid substitutions and that sig­ organisms, but also the protein products of individual nificant functional adaptation can occur by accumulation genes. By subjecting them to repeated rounds of mutation of relatively few such beneficial mutations (changing as and selection (a process usually referred to as '"directed little as 1%-2% ofthe sequence). Thi contrasts with the evolution''), we can enhance or alter specific traits and large sequence distances-frequently 50% or more-that even force a protein to acquire traits not apparent in the separate natural protein homologs, which have diverged parental molecule. And, just as in Darwin's day, these arti­ and adapted to different functions or environments. ficial selection experiments have the potential to teach us Directed evolution can identify minimal sets of adaptive a great deal about evolution, only now at the molecular mutations, but the precise mechanisms by which adapta­ level. The remarkable ease with which proteins adapt in tion occurs are still difficult to discern: The individual the face of defined selection pre ures. from acquiring the effects of beneficial mutations are usually quite small, ability to function in a nonnatural environment to degrad­ and their locations and identities are often surprising ing a new antibiotic, was largely unexpected when the first (e.g .. distant from active sites). laboratory protein evolution experiments were performed Directed evolution experiments have also elucidated a two decades ago. key feature of the fitness landscape for protein evolution. Directed evolution experiments can recapitulate differ­ A common expectation has been that mutational path­ ent adaptive scenarios that may at least partially character­ ways to new properties would be tortuous, reflecting a fit­ ize natural protein evolution. But perhaps even more ness landscape that is highly rugged. In fact, laboratory interesting is the opportunity to go where nature has not evolution experiments have demonstrated over and over necessarily gone. Under artificial selection, a protein can again that smooth mutational pathways- simple uphill evolve outside of its biological context. This allows us to walks consisting ofsingle beneficial mutations--exist and explore the acquisition of novel features, including those lead to higher fitness. Many interesting and useful proper­ that may not be useful in nature. In this way. we can dis­ ties can be manipulated by the accumulation of beneficial tinguish properties or combinations of properties that are mutations one at a time in iterative rounds of mutagenesis biologically relevant and found in the natural world from and screening or selection. others that may be physically possible but are not relevant The role of neutral mutations in protein evolution has and not easily encoded, and therefore are not encountered also been explored. Directed evolution has demonstrated in natural proteins. an important mechanism whereby mutations that are func-

Cold Spring Harbor Symposia on Quamirarire Biology. Volume LXX IV. ~ 2009 Cold Spring Harbor Laboratory Press 978-087969870-6 41 42 ARNOLD tionally neutral but stabilize the protein's three-dimen­ of environments than might be required for their biologi­ sional structure can set the stage for further adaptation by cal functions. This functional promiscuity, even if only at providing the extra stability that allows functionally some minimal level. provides the jumping-off point for important but destabilizing mutations to be accepted. By optimization toward that new goal. A good starting pro­ this mechanism, stability contributed by functionally neu­ tein for directed evolution exhibits enough of the desired tral mutations promotes evolvability. In addition, it has function that small improvements (expected from a single been demonstrated that accumulating mutations which are mutation) can be discerned reliably. If the desired behav­ neutral for one function can lead to the appearance of oth­ ior is beyond what a single mutation can confer, the prob­ ers-a kind of functional "promiscuity"-that can serve lem can be broken down into a series of smaller ones. as a handle for evolution of new functions such as the abil­ each of which can be solved by the accumulation of sin­ ity to bind a new ligand or to catalyze a reaction on a new gle mutations, for example, by gradually increasing the substrate. selection pressure or evolving against a series of interme­ The molecular diversity on which artificial selection acts diate challenges. can be created in any number ofways in order to mimic nat­ Epistatic interactions occur when the presence of one ural mutagenesis mechanisms: Directed evolution experi­ mutation affects the contribution of another. These nonad­ ments use random (point) mutagenesis of a whole gene or ditive interactions lead to curves in the fitness landscape domain, insertions, and deletions. as well as other, more and constrain evolutionary searches. Mutations that are hypothesis-driven mutagenesis chemes. Another impor­ negative in one context but become beneficial in another tant natural mutation mechanism is recombination. We are a ubiquitous feature of protein landscapes, where they have explored how recombination can contribute to mak­ create local optima that could frustrate evolutionary opti­ ing new proteins, by looking at its effects on folding and mization. Directed evolution, however, does not find all structure as well as function. Recombination of homolo­ paths to high fitness, only the most probable paths. These gous proteins is highly conservative compared to random follow one of many smooth routes and bypass the more mutation-a protein can acquire dozens of mutations by rugged, epistatic routes. Hundreds of directed evolution recombination and still fold and function, whereas similar experiments have demonstrated that such smooth paths to levels of random mutation lead to loss of function. Al­ higher fitness can be found for a wide array of protein fit­ though the mutations made by recombination are less dis­ ness definitions, including stability. ability to function in ruptive of fold and function, they can nonetheless generate nonnatural environments, ability to bind a new ligand, functional diversity. Experiments have shown that recom­ changes in substrate specificity or reactivity, and more bined, or "chimeric," proteins can acquire new properties, (Bloom and Arnold 2009). such as increased stability or the ability to accept new sub­ strates, through novel combinations of the mostly neutral mutations that accumulated during natural divergence of CYTOCHROME P450 BM3: A MODEL the homologous parent proteins. ENZYME FOR DIRECTED EVOLUTION In the remainder ofthis chapter, I describe how directed The cytochrome P450 enzyme superfamily provides a evolution experiments performed in this laboratory on a superb example of how nature can generate a whole spec­ model enzyme, a bacterial cytochrome P450, have pro­ trum of catalysts from a single shared structure and mech­ vided support for these lessons. This is a personal account, anism (Lewis and Arnold 2009). More than I 0,000 P450 and I apologize in advance fo r making no attempt to cover sequences have been identified from all kingdoms of life, the large relevant literature and contributions from other where they catalyze the oxidation of a stunning array of laboratories. organic compounds. These enzymes all recruit a cysteine­ bound iron heme cofactor responsible for this activity. The widely varying substrate specificities of the P450s are DlRECTED EVOL TION: A SIMPLE determined by their protein sequences, which accumu­ MOLECULAR OPTlMIZATION STRATEGY lated large numbers of amino acid substitutions as they Directed evolution starts with a functional protein and diverged from their common ancestor. Despite differences uses iterative rounds of mutation and selection to search in up to 90% of the amino acid sequences, the P450s all for more "fit" proteins, where fitness is defined by the share a common fold. experimenter via an assay or some other test (e.g., a P450 BM3 from Bacillus megaterium (BM3) is partic­ genetic selection). The parent gene is subjected to muta­ ularly attractive for laboratory evolution experiments. It tion, and the mutants are expressed as a library of protein is one of only a handful of known P450s in which the variants. Variants with improved fitness are identified, heme domain and the diflavin reductase domains (FMN and the process is repeated until the desired function is and FAD) required for generation of the active oxidant achieved (or not). Directed evolution usually involves the are fused in a single polypeptide chain. Furthermore, it is accumulation of beneficial mutations over multiple gen­ soluble and readily overexpressed in Escherichia coli, an erations of mutagenesis and/or recombination, in a simple excellent host for directed evolution experiments. The uphill walk on the protein fitness landscape (Romero and substrates of BM3 are largely limited to long-chain fatty Arnold 2009). acids, wh ich it hydroxylates at subterminal position at Directed evolution relies on proteins' abilities to high rates (thousands of turnovers per minute). During exhibit a wider array of functions and over a wider range the past decade, we and others investigators have used di- HOW PROTEINS ADAPT 43 rected evolution to alter the specificity of this well­ tiona! rearrangements within and among the heme and behaved bacterial P450 family member so that it can reductase domains mediate electron transfer and effi­ mimic the activities of widely different P450s, including ciently couple 8M3-catalyzed hydroxylation to consump­ some of the human enzymes. These experiments have tion of the ADPH (nicotinomide adenine dinucleotide demonstrated that dramatic changes in substrate speci­ phosphate) cofactor. When these processes are disrupted, ficity can be achieved with just a few mutations in the either by mutations or by introduction of novel substrates, catalytic (heme) domain (Landwehr et al. 2006; Rent­ catalysis is no longer coupled to cofactor consumption: meister et al. 2008; Lewis and Arnold 2009; Lewis et al. ADPH consumption instead produces reactive oxygen 2009). species that eventually cause the enzyme to self-destruct. To retune the whole system for oxidation of propane, we therefore also targeted the FMN and FAD domains of Is a P450 Propane Monooxygenase variant 35E 11 for mutagenesis (individually, but in the PhysicaUy Possible? context of the holoenzyme) and continued to screen for One of our early directed evolution goals was to gener­ increased ability to convert propane to propanol. We then ate a P450 that could hydroxylate small, gaseous alkanes combined the optimized heme, FAD, and FMN domains such as propane and ethane. In nature, these are substrates to generate P450PMo (Fasan et al. 2007, 2008). This of methane monooxygenases, enzymes that are mecha­ enzyme displayed activity on propane comparable to that nistically and evolutionarily unrelated to the cytochrome ofBM3 on fatty acids and 98% coupling ofNADPH con­ P450 enzymes. A P450 had never been reported to accept sumption to product hydroxylation. P450PMo thus became propane, ethane, or methane as a substrate. We were curi­ as good an enzyme on propane as the wild-type enzyme ous as to whether a P450 heme oxygenase was capable of is on Ia urate with a total of 23 amino acid substitutions, binding and inserting oxygen into ethane or methane, amounting to changes in less than 2.3% of its (> 1000 whose C- H bond strengths are considerably higher than amino acid holoenzyme) sequence. those of the usual natural P450 substrates. Creation ofP450PMO' a complex, multidomain enzyme P450 BM3 hydroxylates the alkyl chains of fatty acids finely tuned for activity on a substrate not accepted by the containing 12- 16 carbons and has no measurable activity wild-type enzyme, demonstrates the remarkable ability of on propane or smaller alkanes. We have never found any the cytochrome P450 to adapt to new challenges by accu­ single mutation that confers this activity. To make a ver­ mulating single beneficial mutations over multiple gener­ sion ofBM3 that hydroxylates propane, we therefore first ations. targeted activity on a longer alkane (octane), a substrate that the wild-type enzyme does accept, albeit poorly The P4SOPMO Evolutionary Trajectory (Glieder et al. 2002). We reasoned that variants of BM3 having enhanced activity on octane might eventually Studying the evolutionary intermediates along the lin­ acquire measurable activity on shorter alkanes and thus eage of P450PMO revealed interesting features of adapta­ that further mutagenesis and screening on progressively tion to propane. Activity on propane first emerged in smaller substrates could ultimately generate enzymes 139-3, a variant that is active on a wide range of sub­ with good activity on the gaseous alkanes. This reasoning strates. But by the time the enzyme became highly active assumed that the problem was mainly one of substrate and fully coupled on propane, it had lost its activity on recognition and that there is no inherent mechanistic lim­ laura te-a more than I 0 10-fold change in specificity, just itation to hydroxylation of small alkanes at the heme iron. from 139-3 to P450PMo· Thus, becoming a good propane Five generations of random mutagenesis of the berne monooxygenase in P450PMo came at the cost of the native domain, recombination of beneficial mutations, and enzyme's activity on fatty acids, even though tbis prop­ screening for activity on an octane surrogate led to BM3 erty was not included in the artificial selection pressure. variant 139-3, which contains I I amino acid substitutions This is apparently the easiest route to high activity on and is much more active on octane (Glieder et al. 2002). propane. The improved octane activity was in fact accompanied by Substrate specificity changes for selected variants measurable activity on smaller alkanes, including pro­ along the lineage to P450PMO were also investigated on pane. Further rounds of mutation and recombination of alkanes having one to 10 carbons. These activity profiles beneficial mutations further enhanced activity on pro­ revealed that intermediate variants (e.g., 139-3, 35£11) pane. Variant 35Ell, with 17 mutations relative to 8M3, acquired activity on a range of alkanes before ultimately was highly active on propane and even provided modest respecifying for propane (Fig. I) (Fa san et a!. 2008). conversion of ethane to ethanol (Meinhold et al. 2005). P450PMo is highly specific compared to its precursors: Its Breaking down the more difficult problem of obtaining activity drops precipitously on alkanes having just one activity on very small substrates by first targeting octane more or one less methylene group. Only positive selection and then propane lowered the bar for each generation and (for high activity on propane) had been used to obtain allowed the new activities to be acquired one mutation at P450P\IO; there was no selection against activity on any a time. other substrate. One can conclude that it is easier to obtain This enzyme, however, was still not as efficient at very high activity on propane than it is to have high activ­ bydroxylating the alkanes as is the wild-type enzyme with ity on a range of substrates; thus, a highly active "special­ its preferred fatty acid substrates. Finely tuned conforma- ist" is easier to find than a highly active "generalist." 44 ARNOLD

A 1/1 50,000 • Propanols ... 0 G) Ethanol > 40,000 0 c: ... 30,000 ::l ~ 20,000_ iii 0 10,000 ~- 0 WT 13~3 35E11 19A12 11 -3 1-3 7-7 P450PMO B 120 M3 (WT) P4506 1;100 139-3 1:' 100 > ~ 80 z 80 " 60 • 60 ! 40 ! 40 .,. 20 .,. 20 0 2 3 4 5 6 7 8 9 10 1 2 3 7 8 9 10 1 2 3 4 5 6 7 8 9 10 120 120 1-3 120 P450pMO 1:' 100 1:' 100 1;.. 100 ! 80 > 80 ;>., 80 ~ 60 ~ 80 . 60 ! 40 ! 40 !.,. 40 .,. 20 .,. • 20 o- 0 - 1 2 3 4 5 6 7 8 9 10 2 3 4 5 6 7 8 9 10 1 2 3 4 5 6 7 8 9 10 Figure I. (A) Total turnovers catalyzed by selected variants along the P450P"o lineage on propane and ethane. (B) Relative activities on c. (n = 1- 10) alkanes. (Reprinted with permission. from Lewis and Arnold 2009 [© Swiss Chemical Society].)

Sequencing reveals the mutations acquired in each gen­ nisms. The crystal structure of 139-3 (Fasan et al. 2008) eration of directed evolution. The 21 amino acid substitu­ revealed only small changes in the active site volume, con­ tions in the heme domain of P450PMo (two of the 23 are in sistent with its activity toward a wide range of substrates. the reductase domain) are distributed over the entire protein Modeling studies, however, indicate much more dramatic (Fig. 2). Many are distant from the active site and influence reduction in the volume accessible to substrate in P450P~to specificity and catalytic activity through unknown mecha- (C Snow, unpubl.).

Figure 2. P450 8M3 heme domain backbone, showing locations of21 of23 mutations that convert P450 8M3 to a highly active, fully coupled propane monooxygenase ( P4 50P~ 1 0). HOW PROTEINS ADAPT 45

STABILITY PROMOTES EVOL\~B ILITY: Directed evolution experiments have hown that protein A ROLE FOR NEUTRAL MUTATIONS L~ activities or functions present at a low level can often be ADAPTIVE EVOLUTION improved via an adaptive pathway of equential benefi­ cial mutations. Protein functional promiscuity thus pro­ It is useful to consider when this simple adaptive walk vides a stepping stone for generation and optimization of might fail. Of course, it will fail if the functional bar is set new functional molecules by adaptive evolution. too high- this happens when the fitness improvements Directed evolution experiments with P450 BM3 have required to pass the screen or selection are not reached by also demonstrated that promiscuous activities can emerge single mutations. It also fails when the protein is not robust on mutations that are neutral with respect to a main (bio­ to mutation (Bloom et al. 2005, 2006). At one point during logical) function (Bloom et al. 2007). We performed a the evolution ofP450PMO' in fact at mutant 35E II, we could kind of neutral evolution by random mutagenesis and find no additional mutations that further enhanced the selection for retention of catalytic activity on a fatty-acid­ enzyme's activity on propane. Upon characterizing 35Ell like substrate. The variants containing these '·neutral'" and its precursors, the reason for this became clear: The mutations were then examined for activity on several other enzyme had become so unstable that it simply could not nontarget substrates. In many cases, the neutral mutations tolerate any further destabilization and still function under had led to changes in these promiscuous activities. Neutral the expression and assay conditions. Most mutations are mutations can also set the stage for adaptation by explor­ destabilizing. and most activating mutations are also desta­ ing a varied set of evolutionary starting points. at little or bilizing. possibly more so than the average mutation. The no cost to the current biological function. process ofenha ncing P450's activity on propane had desta­ l already discussed how neutral mutations can enhance bilized it so much that 35EI1 simply could not accept any a protein's stability, thereby increasing its tolerance for further destabilizing mutations. Once we incorporated subsequent functionally beneficial but destabilizing mutations that stabilized the structure (but were neutral or mutations. eutra1 mutations can also lead to changes in nearly neutral with respect to activity), directed evolution functions that are not currently under selective pressure of activity could continue as before, and significant addi­ but can subsequently become the starting points for the tional improvements were achieved (Fasan et al. 2007). adaptive evolution of new functional proteins. A process Stabilizing the structure made it robust to further mutation that generates large numbers of mostly neutral mutations and opened up the ability to explore a whole spectrum of is recombination (of homologous proteins), which mutational paths that were previously inaccessible. exploits the genetic drift that underlies the divergence of We demonstrated this key role of functionally neutral their sequences. As we discuss below, swapping these but stabilizing mutations in adaptive evolution with mutations in the laboratory can generate proteins differ­ another experiment that directly compared the frequency ent from the parent proteins, including those that are more with which a marginally stable and a highly stable cyto­ stable or exhibit activities not present in the parents. chrome P450 enzyme could acquire activities on a set of new substrates upon random mutation (Bloom et al. 2006). A markedly higher fraction of mutants of the sta­ NOVEL PROTEINS BY RECOMBINATION ble protein were found to exhibit the new activities. This Recombination is an important mutation mechanism in increased evolvability could be traced directly to the natural protein evolution. We have studied the effects of enzyme ·s ability to tolerate catalytically beneficial but mutations made by recombination of homologous pro­ destabilizing mutations. teins (that share a three-dimensional structure but may Directed evolution has thus shown the crucial role that differ at hundreds of amino acid residues) by making and stability-based epistasis can have in adaptive evolution. A characterizing large sets of "chimeric'· proteins. The protein that has been pushed to the margins of tolerable probability that a protein retains its fold and fu nction stability may lose access to functionally beneficial but declines exponentially with the number of random muta­ destabilizing mutations. But this protein is still not stuck tions it acquires-random mutations are quite deleterious on a fitness peak, because it can regain its mutational on average. By quantifYing the retention of function with robustness and evolvability via a neutral path, by accu­ mutation level in chimeric ~-lactamases made by swapping mulating stabilizing mutations that do not directly affect sequence elements between two homologous enzymes, we function. Tn natural evolution. such a process might showed that the mutations made by recombination are require stabilizing mutations to spread by genetic drift much more conservative. presumably because they had (Bloom and Arnold 2009). already been selected for compatibility with the lacta­ mase folded structure (Drummond et al. 2005). Recom­ ADAPTIVE EVOL TION RELIES ON bination can generate proteins that have a high probability FUNCTIONAL PROMISCUITY, WHICH offolding and functioning despite having dozens of muta­ CHANGES WITH NE TRAL MUTATIONS tions compared to their parent sequences. Thus, recombi­ nation is conservative. But does it lead to new functions A well-recognized feature of proteins is their functional or traits? promiscuity. Enzymes, for example, often catalyze a We generated a large set of recombined P450 heme much wider range of reactions, or reactions on a wider domains by swapping sequence element among three nat­ range of substrates. than are biologically relevant ural P450 8M3 homologs sharing -65% sequence identity 46 ARNOLD

(Otey et al. 2006). A sampling of the functional P450s REFERENCES showed that they exhibited a range of activities, including Bloom JD. Arnold FH. 2009. In the light of directed evolution: activity on substrates not accepted by the parent enzymes Pathways of adaptive protein evolution. Proc Nat/ A cad Sci (Landwehr et aL 2007). The chimeric P450s also exhibited a 106:9995-10000. range of stabilities, with a significant fraction of them more Bloom JD, Silberg JJ. Wi lke CO, Drummond DA, Adami C. stable than any of the parent enzymes from which they were Arnold FH. 2005. Thennodynamic prediction of protein neu­ trality. Proc Nat/ Acad Sci 102: 606-611. constructed (Li et aL 2007). Like many proteins, P450s are Bloom JD, Labthavikul ST, Otey CR. Arnold FH. 2006. Protein only marginally stable, never having been selected for ther­ stability promotes evolvability. Proc Nat/ A cad Sci I 03: 5869- mostability or long-term stability. 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Drummond DA, Sawayama AM, Snow CD. Bloom JD, what biology tends to throw away. Laboratory evolution Arnold FH. 2007. A diverse family ofthennostable cytochrome experiments beautifully demonstrate that biological sys­ P450s created by recombination of stabilizing fragments. Nat Biotechno/ 25: I 051 - 1056. tems, themselves the products of millions of years of evo­ Meinhold P, Peters MW, Chen MY, Takahashi K. Arnold FH. lution, readily evolve to meet new challenges. 2005. Direct conversion of ethane to ethanol by engineered cytochrome P450 8M3. Chembiochem 6: 1765-1768. Otey CR. Landwehr M. Endelman JB, Hiraga K. Bloom JD, ACKNOWLEDGMENTS Arnold FH. 2006. Structure-guided recombination creates an artificial family of cytochromes P450. ?LoS Bioi 4: 0789- The author thanks all of her coworkers that have con­ 0798. tributed to the work described here. and especially thanks Rentmeister A, Arnold FH. Fasan R. 2008. Chemo-enzyme fluo­ Jesse D. Bloom, Phil Romero, Jared C. Lewis, and Rudi rination of unactivated organic compounds. Nat Chem Bioi 5: Fasan. Support is from the Jacobs Institute for Molecular 26-28. Romero PA, Arnold FH. 2009. Exploring protein fitness land­ Medicine, the Department of Energy, the U S. Army, scapes by directed evolution. Nat Rev Mol Cell Bioi 10: 866- DARPA, and the ationallnstitutes of Health. 876.