screening were used to increase the thermalstability6 and alter the preferred mechanisticpathway7 of several enzymes (reviewed in ref.8). But these studies have focused onimproving or altering existing propertiesrather than on the creation of totally newactivities or the transmutation of oneenzyme activity into another. To accomplishthat, Altamirano et al.1 have looked atnature’s methods for generating novel pro-tein functions.

It has been suggested that new enzymeactivities evolve primarily by the recruit-ment and modification of pre-existingenzymes that catalyse a similar basic chemi-cal step9. Some low level of specificity for thenew substrate must exist in the target geneproduct, and evolution will improve theselectivity. There are many examples of thismechanism. It is also possible that certainsteps in some metabolic pathways evolved by recruiting the enzyme that produced thesubstrate, or used the product of the newreaction to be catalysed — this route ensuresspecificity but the chemistry must evolve10.Examples of this mechanism are not com-mon, but there is one dramatic case: in anumber of metabolic pathways, two consec-utive, but different, transformations arecatalysed by proteins with similar three-dimensional structures. These enzymes allhave the so-called TIM-barrel fold, namedafter the structure of the glycolytic enzyme,triosephosphate isomerase, in which it wasfirst discovered11 (Fig. 1).

The TIM barrel is perhaps the most com-mon polypeptide-chain fold in biology —about 10% of all enzymes have at least onedomain that adopts this structure. The fold is an especially robust one, tolerating loopreplacement12 and even the insertion of entiredomains into the loop regions without dis-ruption. It also has another characteristicthat drew the attention of Altamirano et al.The residues that bind the substrate and sodetermine specificity are for the most partcontributed by the barrel itself, whereas theresidues that carry out the chemical transfor-mation are located primarily in the loops thatconnect the helices and b-strands (Fig. 1). Sothe TIM-barrel fold may represent an idealscaffold for the creation of new enzyme activ-ities because the chemistry catalysed by theprotein can perhaps be altered without loss ofspecificity. To demonstrate this, Altamiranoet al. set out to convert indole glycerol phos-phate synthase (IGPS) into phosphoribosylanthranilate isomerase (PRAI).

To be fair, their chosen target has a largebull’s-eye. PRAI and IGPS catalyse consecu-tive reactions in the biosynthesis of trypto-phan. The product of the PRAI reaction isthe substrate for IGPS, although no cross-reaction exists between the two naturalenzymes, and their active sites differ consid-erably. With the aid of the known crystalstructures of the two enzymes, Altamirano

to sleep deprivation should be monotonousand dull7.

Many organs in the body can rest andrecover during relaxed wakefulness — to asimilar extent to that achieved during sleep— but the cerebral cortex seems unable to dothis. Even when we lie relaxed but awake in a dark, silent room, the cortex remains in‘quiet readiness’, prepared to respond imme-diately. Only sleep seems to provide real restfor the cortex. This ‘rest’ is indicated by thecharacteristically large, slow (delta) wavesseen in the electroencephalogram of deep,non-dreaming sleep. As Drummond et al.point out, this delta activity is most intense inthe prefrontal cortex8 when there is also a par-ticularly low rate of blood flow in this area9.

What really happens in the human cortexduring sleep is still a mystery. Althoughmuch work has been done in rats, there is little sign of brain pathology, even when theanimals are sleep deprived until death10.

However, we must bear in mind that ratshave a relatively poorly developed prefrontalcortex, so the function of sleep for much of the human cortex may differ from thefunction of sleep in the rat. ■

Jim Horne is at the Sleep Research Centre,Department of Human Sciences, LoughboroughUniversity, Loughborough LE11 3TU, UK.e-mail: j.a.horne@lboro.ac.uk1. Dinges, D. F. & Kribbs, N. B. in Sleep, Sleepiness and Performance

(ed. Monk, T. H.) 97–128 (Wiley, Chichester, 1991).

2. Drummond, S. P. A. et al. Nature 403, 655–657 (2000).

3. Horne, J. A. Br. J. Psychiat. 162, 413–419 (1993).

4. Harrison, Y. & Horne, J. A. J. Sleep Res. 7, 95–100 (1998).

5. Smith, E. E. & Jonides, J. Science 283, 1657–1661 (1999).

6. Harrison, Y. & Horne, J. A. Organis. Behav. Hum. Decision

Process. 78, 128–145 (1999).

7. Wilkinson, R. T. in Sleep, Arousal and Performance (eds

Broughton, R. J. & Ogilvie, R. D.) 254–265 (Birkhauser, Boston,

Massachusetts, 1992).

8. Werth, E., Achermann, P. & Borbély, A. A. NeuroReport 8,

123–127 (1996).

9. Maquet, P. et al. J. Neurosci. 17, 2807–2812 (1997).

10.Cirelli, C., Shaw, P. J., Rechtschaffen, A. & Tononi, G. Brain Res.

840, 184–193 (1999).

of the enzyme triosephosphate isomerase torandom mutagenesis followed by selectionfor improved catalytic potency. A number ofsubstitutions at sites other than that of theoriginal mutation were found, suggestingthat there are several solutions to the prob-lem of increasing efficiency. Multiple cyclesof random mutagenesis, recombination and

“The designs of his bright imagina-tion were never etched by the sharpfumes of necessity”, commented

the English poet Francis Thompson in theDublin Review of 1908. The poetry of PercyBysshe Shelley may indeed, as Thompsonconcluded, have been designed in the fire ofhis imagination. Protein structure, on theother hand, is etched entirely by the sharpfumes of necessity, through the process ofnatural selection. On page 617 of this issue,Altamirano et al.1 show that a combinationof design and necessity can rapidly evolveone enzyme in a biosynthetic pathway intoanother. As well as providing a possiblemodel for the natural evolution of newenzyme activities from an existing scaffold,the success of this procedure suggests thatthe particular scaffold they use may haveadvantages in the directed evolution of newbiocatalysts.

The first efforts to design proteins de novoaimed to create sequences, which adopt aparticular fold that is stable at ordinary tem-peratures2,3. Parallel efforts to engineer newfunctions into natural proteins began byaltering specificity using site-directed muta-genesis4. Despite some success with both, the creation of totally new enzyme activity by either of these ‘rational’ approaches hasproven to be extremely difficult. No surprise,then, that the latest attempts have borroweda leaf or two from nature’s book.

In an early example of what has beentermed the ‘directed evolution’ approach,Blacklow et al.5 subjected a sluggish mutant

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606 NATURE | VOL 403 | 10 FEBRUARY 2000 | www.nature.com

Enzyme evolution

Design by necessityGregory A. Petsko

Figure 1 The folded polypeptide chain oftriosephosphate isomerase, the archetypal TIM-barrel enzyme. There are eight a-helices(pink) and eight parallel b-strands (blue) in analternating pattern. The first and eighth b-strands hydrogen-bond to each other, creatinga cylinder; the a-helices line the outside of thisbarrel. The connecting loops are grey. The activesite of TIM – and all TIM-barrel enzymes – is inthe mouth of the barrel. Using directed evolution,Altamirano et al.1 have converted one TIM-barrelenzyme into another with a different function.

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et al. identified two active-site loops in IGPSthat differed most from those in PRAI and sowere targeted for replacement. Deletion ofone loop followed by insertion of a shorter,random-loop library produced an ensembleof altered enzymes. The second loops ofthese enzymes were also modified. The mixture of mutant IGPS enzymes was thenselected for PRAI activity by using them to transform a PRAI-deficient strain ofEscherichia coli that does not grow in theabsence of tryptophan. Around 500 out of30,000 transformants were able to grow inlow concentrations of tryptophan.

The second round of selection wasdesigned to improve the function of theseproteins. It involved in vitro sexual recombi-nation13 with pools of genes from the clonesselected above. Eighty colonies out of400,000 bacteria were found to grow on verylow concentrations of tryptophan; one ofthem could grow in its absence. Anotherround of DNA shuffling among the 80 clonesproduced 360 colonies capable of growingwithout tryptophan. Sequencing 30 of theserevealed that only eight different sequenceshad been generated. Characterization of oneof these proteins showed it to be soluble, stably folded and highly active.

Interestingly, although this evolved‘PRAI’ enzyme has PRAI activity six timeshigher than that of natural E. coli PRAI, itlacks its original IGPS activity entirely. Theimprovement over the wild-type enzymearises primarily from better binding of thesubstrate phosphoribosyl anthranilate1. Thesequence of the evolved ‘PRAI’ does notresemble natural PRAI, and there is evidencethat the substrate is not bound in preciselythe same way. So, converting one TIM-barrelenzyme into another by altering the loopregions has led to a different solution to theproblem of catalysing the PRAI reaction.

Experiments of this kind should providevaluable insights into the machinery ofdivergent evolution. They also show that,with the aid of recombination, new catalyticactivities can evolve quite rapidly. Similarcombinations of structure-guided and selec-tion-driven strategies are likely to be themethod of choice for new protein ‘design’ inthe near future. One can expect productionof many proteins with novel functions forapplications in medicine, biotechnology,organic synthesis and bioremediation at afaster rate than we could make them by‘rational’ design alone. Samuel Johnson,who died eight years before Shelley was born,would have approved. In Rasselas (ChapterXVI), he offered advice that would seemappropriate for would-be protein engineers:“Many things difficult to design prove easy toperformance.” ■

Gregory A. Petsko is at the Rosenstiel Basic MedicalSciences Research Center, Brandeis University, 415South Street, Waltham, Massachusetts 02454-9110,USA.

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e-mail: petsko@brandeis.edu1. Altamirano, M. M., Blackburn, J. M., Aguayo, C. & Fersht, A. R.

Nature 403, 617–622 (2000).

2. Regan, L. & DeGrado, W. F. Science 241, 976–978 (1988).

3. Hecht, M. H., Richardson, J. S., Richardson, D. C. & Ogden,

R. C. Science 249, 884–891 (1990); erratum, Science 249, 973

(1990).

4. Cronin, C. N. & Kirsch, J. F. Biochemistry 27, 4572–4579

(1988).

5. Blacklow, S. C., Liu, K. D. & Knowles, J. R. Biochemistry 30,

8470–8476 (1991).

6. Zhao, H. & Arnold, F. H. Protein Eng. 12, 47–53 (1999).

7. Joo, H., Lin, Z. & Arnold, F. H. Nature 399, 670–673 (1999).

8. Arnold, F. H. & Volkov, A. A. Curr. Opin. Chem. Biol. 3, 54–59

(1999).

9. Petsko, G. A., Kenyon, G. L., Gerlt, J. A., Ringe, D. & Kozarich,

J. W. Trends Biochem. Sci. 18, 372–376 (1993).

10.Farber, G. K. & Petsko, G. A. Trends Biochem. Sci. 15, 228–234

(1990).

11.Banner, D. W. et al. Nature 255, 609–614 (1975).

12.Thanki, N. et al. Protein Eng. 10, 159–167 (1997).

13.Stemmer, W. P. Nature 370, 389–391 (1994).

swapping the protein-coding sequences oftwo Hox genes, they show that, even thoughtheir sequences are very different and thegenes have distinct biological functions,their protein products are functionallyequivalent. Greer et al. conclude that, when it comes to Hox proteins, what matters isquantity rather than quality — a provocativestatement, which may have important developmental implications.

Mammalian Hox genes come in 13groups, most of which have three or four paralogous genes. Expression analyses of thegenes in group 3, Hoxa3, Hoxb3 and Hoxd3(Fig. 1), revealed largely overlapping tran-script domains, but targeted inactivationshowed that Hoxa3 and Hoxd3 have uniquefunctions4,5. For example, mice lacking

In the course of evolution, the vertebrategenome experienced several rounds ofduplication. As a consequence, for each

gene described in flies or in worms, three orfour copies of closely related (or paralogous)counterparts are routinely found in mam-mals. The duplicated genes may have takenon new functions by being differently regu-lated, but the persistence of ancestral func-tions amongst duplicates also generatedredundancy1,2. Evidence for these principlesarose from the study of the mammalian Hoxfamily, a set of 39 developmental controlgenes, located in four complexes derivedfrom duplications of a single original cluster.

On page 661 of this issue3, Greer et al.report the analysis of functional relation-ships between paralogous Hox genes. By

Developmental genetics

A Hox by any other nameDenis Duboule

Hoxd3A3

Hoxa3D3

Hoxa

HoxA3/HoxD3

Hoxb

Hoxc

Hoxd

1236 5 478910111213

a

b

Figure 1 The Hoxa3 and Hoxd3 genes and their proteins. a, Comparison between the HoxA3 andHoxD3 protein sequences. Each vertical bar represents a pair of identical amino-acid residues at aconserved position. The grey boxes indicate segments of sequences that are more than 50% identicalto each other. White boxes highlight the highly conserved ‘pentapeptide’ motif (top) andhomeodomains (middle). Overall, sequence conservation is less than 50%. b, Scheme of the four Hox complexes with the 13 paralogous groups. Genes are indicated by the orange boxes. Group 3 ishighlighted and the arrows indicate the swap between the Hoxa3 and Hoxd3 coding sequencescarried out by Greer et al.3, leading to the two novel alleles Hoxa3D3 and Hoxd3A3.

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