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Copyright 0 1989 by the Genetics Societyof America Perspectives Anecdotal, Historical and Critical Commentaries on Genetics Edited by James F. Crow and William F. Dove EVOLVINGTHEORIES OF ENZYMEEVOLUTION F IFTEEN years ago in GENETICS, BARRY HALL and I published a paper on the evolved @-galactosidase in Escherichiacoli (HALLand HARTL1974). Thanks and his colto previous work by JOHN H. CAMPBELL laborators at the University of California in Los Angeles, this system for experimental enzyme evolution seemed especially promising for exploring the evolution of a novel catalytic activity using an organism with a well developed system for genetic manipulation. It seemed a way out of the dilemma that many of the deepest processes in evolutionary biology appeared almost inaccessible to direct experimental investigation. These included the origin of life itselfand the evolution of new enzyme functions. Although experiments were possible, they were often indirect andtheirrelevance speculative. Against this background, the prospect of the experimental evolution of @-galactosidaseseemed to provide a great opportunity both to determine theevolutionary potentialof a specific enzyme in a well adapted and well studied organism and to define the nature of these potentials at the molecular level. We assumed that understanding the molecular basis of enzyme adaptation to utilize novel substrates would also provide some insight into the fundamental processes by which new enzyme activities can arise in the course of evolution. The evolution of novel catalytic activities was well recognized as paradoxical. As expressed by EDEN ( 1 967) in the Wistar Symposium on Mathematical Challenges to the Neo-Darwinian Interpretation of Evolution, the problem was in the infinitesimal probability that catalytically useful proteins containing hundreds of amino acids could result from the random assembly of their amino acid subunits. For example, the probability that a particular sequence of 100 amino acids in a functional polypeptide would occur by chance combination is only 20"0° or 10"30 which, even allowing for some freedom in the amino acids that can occupy individual positions, is pretty small. While the probability paradox was not emphasized in evolutionarythinking, it remained unresolved in the major theories of enzyme evolution, including the classical Genetics 1 2 2 1-6 (May, 1989) theory of duplication and divergence in which new catalytic activities were supposed to evolve by random amino acid changes resulting from nucleotide substitutions in duplicate copies of preexisting genes. If the acquisition of a new enzyme function requires more than a few substitutions, then it is extremely improbable to occur by chance. The dilemma was that "either functionally useful proteins are very common . . . so that almost any polypeptide [of random amino acid sequence] . . . has a useful function to perform, or else . . . there exist certain strong regularitiesfor finding useful paths [of protein evolution]" (EDEN1967, p. 7). Of course, polypeptides are not assembled at random but are selected step by step from preexisting ones, and in the same book WRIGHT(1967) likened natural selection to thegame of Twenty Questions, in which it is possible to arrive at the correct sequence of 100 amino acids in a polypeptideby a series of 500 questions answered yes or no. Nevertheless, there is a paradox in that acquisition of new enzyme functions may require multiple amino acid substitutions and therefore may not be selectable step by step. The evolved @galactosidase was first discovered by the microbial evolutionary biologist JOHN H. CAMPBELL, who had noticed that colonies of E. coli with a lac2 deletion mutation often gave rise to lactose-fermenting papillae when the plates were incubated for two weeks or so. By contrast, typical lac2 missense mutations usually yielded Lac+ revertant papillae within a few days. Overcoming considerable skepticism in the orthodox microbial genetics community, CAMPBELL and collaborators showed that the papillae in the aged plates grew from mutants containing a novel @-galactosidaseactivity coded by a gene designated ebg (evolved beta galactosidase), currently positioned at 67.5 min on the geneticmap,notquite directly across the chromosome from lac2 at 8.0 min (CAMPBELL, LENGYEL and LANGRIDCE 1973). BARRY HALL and I were both quite takenwith these observations and were all the moreimpressed after an all-night brainstorming session in CAMPBELL'S room at a Lake Arrowhead Meeting in February of 1972 2 D. L. Hart1 when CAMPBELL laid out thedetails and oureyes were opened to the experimentalpossibilities. We resolved to exploit the system to study experimental enzyme evolution.InOctober 1972, CAMPBELL visited my laboratory at Minnesota to help get things startedand in June 1973, BARRYtransferred his NIH Postdoctoral Fellowship there and thework began in earnest. The first discovery was that ebg mutants were very easy to get. Although the 0-galactosidase enzyme in the evolved strains was indistinguishable fromthat discovered by CAMPBELL, our mutants fell into two types according to whether their0-galactosidase activity wasconstitutive or inducible by lactose. T h e lactose inducibility suggested that the normal product of the ebg structural gene is a lactose-inducible P-galactosidase, butonethat doesnot hydrolyze lactose well enough to allow growth on lactose as the sole carbon source. The problem was to prove it, because Laccells failed to grow on lactose and Lac+ cells growing on lactose produced so much ZacZ 0-galactosidase that any ebg enzyme produced could not be detected.The experimental trick was to grow ZacZ- cells on a very poor carbon source in the presence of excess lactose, plus isopropyl thiogalactoside (IPTG) to induce the lacy permease; out of context the experiment makes no sense, but under these conditions the ebg enzyme was induced sufficiently that its presence could be detected by its ability to hydrolyze the chromogenic 0-galactoside ONPG (HARTLand HALL 1974). The ability of the ebg @galactosidase to hydrolyze lactose, or a number of other 0-galactosides that BARRYstudied later (HALL1977, 1981), evidently resulted from just one or a small number of amino acid replacements, and this was something of a disappointment at the time. After a year of happy collaboration, BARRYmoved to Newfoundland to pursue ebg and I moved to Purdue andsoon became interested in naturally occurring enzyme polymorphisms in E. coli. BARRYshowed that the ebg strains we had selected for growth on lactose also containedregulatorymutations in the closely linked gene ebgR encoding a repressor that regulates transcription of the ebg structural gene (HALLand CLARKE1977). Comparisons of the amountof enzyme synthesized in the wild-type andmutant strains showed that all mutant strains synthesized more molecules of enzyme per cell than did the wild type. Some strains had become constitutive for ebg but most were still regulated by a mutant repressor moresensitive to lactose as inducer. Mutations in the repressor were necessary for growth on lactose because none of the mutant enzymes had sufficient lactase activity to permit growth unless the level ofexpression was increased above the normal level of induction. A fully induced wild-type ebg operon produces only about 5% as much enzyme as an ebgR-constitutive strain (HALL1983). BARRYalso showed that the ebg &galactosidase had a remarkable potential for acquiring new substrate specificities according torather specific rules.For example,strainsthat constitutively synthesized the wild-type ebg enzyme gave rise to two distinct types of mutants capable of growth on lactose. T h e mutant enzymes were designated class I and class 11. The class I enzyme enabled good growth on lactose but not on lactulose (galactosyl-fructose), whereas the class I1 enzyme enabled good growthon lactulose but moderate growth on lactose. The kinetic properties and substrate specificities of the class I and class I1 enzymes were different and correlated well with the growth characteristics of the strains (HALL 1981).Genetic studies demonstratedthatthe class I and class I1 enzymes differed from the wild type by mutations at opposite ends of the structural gene(HALLand ZUZEL 1980), which has since beenconfirmed directly by DNA sequencing. Remarkably, when boththe class I and class I1 mutations in the structural ebg gene were brought together in the same gene, the doubly mutant enzyme, designated class IV, exhibited two new substrate specificities (HALLand ZUZEL 1980). The first was the ability to allow growthon galactosyl-arabinose. Although thewild-type ebg enzyme exhibits some activity toward galactosyl-arabinose (as well as toward lactose and lactulose), these wild-type activities were too feeble to allow growth on any of these substrates. The class IV enzyme also exhibited a novel activity that could not be demonstratedin the wild type or in class I or class I1 mutants. This was the ability to hydrolyze lactobionic acid. While the lactobionic acid activity was too weak to allow growth, it could be increased by a third mutation so that growth on this substrate became possible. The class IV enzyme could also do something else that neither of the others could-it could produce allolactose as a side product of lactose hydrolysis (HALL1982a). Allolactose is the in vivo inducer of the lac operonand it is normally produced as a side reaction by the ZacZ enzyme. Growth of most ebg mutants on lactose requires the presence of IPTG or some other gratuitous (nonmetabolized) inducer to enable synthesis of the lacy permease.However, strains with the class IV enzyme produceenough allolactose to induce the lac operon on theirown. The finale was the production of a strain with the two structural mutationsin class IV plus a mutation in the ebg repressor increasing the level of enzyme induced by lactose (HALL1982b). This strain was able to grow on lactose alone without added gratuitous inducerthe lactose induced the class IV enzyme, which in turn produced enough allolactose to induce the lacy permease. One of the important findings of the ebg work was Perspectives that identical enzymes occurred repeatedly in replicate experiments, suggestingthat selection for growth on novel substrates favored only a very few of the large numberof possible amino acid replacements. In addition, the different amino acid replacements had different evolutionary potentials as defined by their abilities to sustain growth on different substrates, and some mutationsthatenhanced activity toward one substratehad no detectable effect toward another substrate. Although the ebg mutations occurred in the laboratory, different potentials for selection also occur among naturally occurring aminoacid polymorphisms in a variety of enzymes. ROGERMILKMAN(1 973) had shown that electrophoretic variation in enzymes among E. coli isolates was widespread-more so, in fact, than in eukaryotes. If single amino acid replacements could have such profound effects on ebg function, then it seemed possible that natural variants of enzymes might also have important functionaleffects. T h e experimental system to study natural enzyme variants made use of the power of E. coli genetics to create isogenic pairs of strains differing only in the enzyme gene of interest. These pairs were placed in chemostats in strong competition for substrates that require the target enzyme in their metabolism. DANIEL DYKHUIZEN was the chemostat expert who made this system work. The main finding was that naturally occurring enzyme variants usually produced no detectable effects on growth rate under growth conditions usually considered optimal for E. coli but that many did produce significant effects on growth rate when the conditions were altered, for example under competition for an unusual substrate (DYKHUIZEN and HARTL1980; HARTLand DYKHUIZEN 1981). This situation was in many ways analogous to the various forms of ebg. T h e interpretation was that naturally occurring genetic variants, many of whichmay be selectively neutral or nearly neutral under the prevailing mosaic of environments, may nevertheless have a latent potential for selection that can be expressed under alternativeenvironmentalconditions (HARTLand DYKHUIZEN 1984, 1985). T h e implications of this principle for general evolution have been discussed by STEBBINSand HARTL(1988)and by KIMURA(1989). Even though the selective effects of many naturally occurring enzyme polymorphisms are often too small to detect in chemostats, their effects under natural conditions can nevertheless be estimated from DNA sequences. This approach was made possible through SAWYER, an importanttheoretical analysis by STANLEY who analyzed the sample configurations (number of occurrences of each possible nucleotide) at 768 homologous nucleotide positions within the DNA coding for 6-phosphogluconate dehydrogenase in seven natural isolates (SAWYER, DYKHUIZEN and HARTL1987). 3 T h e sequenced regions included 12 amino acid polymorphisms and 78 silent nucleotide polymorphisms. On thehypothesis that aminoacid polymorphisms are as weakly selected as are thesilent-site polymorphisms in the samegenes,their sampling configurations among the genes should be the same as at the silent sites. This hypothesis could be rejected, and indeed it could be asserted with 95% confidence that no more than half of the amino acid polymorphisms in this enzyme are selectively neutral. The data are also consistent with a model in which all of the observed amino acid polymorphisms are mildly deleterious with an estimatedaverage selection coefficient of 1.6 X lo”. This is smaller by several orders of magnitude than the minimum amount of selection detectable in chemostats. (I know of no experimental systemin which selection of such small magnitudecouldbe detected directly.) The finding that many naturally occurring enzyme variants have very small effects on fitness makes considerable sense in light of metabolic control theory, an approach to understanding integrated metabolic systems that was pioneered by KACSER and BURNS (1973). Metabolic control theory also helps to explain why all of the ebg variants so far isolated exhibit only a small fraction of the lactase activity found in strains that contain the 8-galactosidase coded by lacZ. In their mathematical analysis of the flux across a metabolic pathway at steady state, KACSERand BURNS (1973, 1979) developed several important principles with wide applicability to complex metabolic systems, and their metabolic control theory has been widely cited because of its straightforwardness and intuitive appeal (KACSER and PORTEOUS 1987; HARTL, DYKHUIZEN and DEAN1985; DEAN, DYKHUIZEN and HARTL 1988a; HARTL1989). Some of the evolutionary implications of metabolic control theory have been discussed by HARTL, DYKHUIZEN and DEAN(1985) and STEBBINSand HARTL(1988). The KACSER-BURNS analysis demonstrated that the control of metabolic flux through a pathway is not usually through a single rate-limiting enzyme, but instead is shared among all enzymes in the pathway through control coefficients that are functions of the kinetic parameters. As the activity of any enzyme in the pathway increases, the flux becomes less sensitive to small perturbations in the activity and the controlcoefficient of the enzyme decreases. This is the familiar diminishing-returns or saturation phenomenon encountered in many complex systems with interactingcomponents. Under rather general conditionsthe summation of all control coefficients in a pathway must equalunity, which implies that large controlcoefficients of some enzymes must be accompanied by small control coefficients of others. In the example of E. coli growing on lactose, the controlcoefficient of the 8-galactosidase permease 4 D. L. Hart1 with respect to growth rate is greater than thatof the &galactosidase by a factor of approximately 30 (DYKHUIZEN, DEANand HARTL1987). This implies that cells with as little as 5% of wild-type lac2 activity can grow on lactose almost as well as does the wild type (DEAN,DYKHUIZENand HARTL 1986), so thatthe relatively low P-galactosidase activities that occur in ebg strains are nevertheless compatible with virtually normal growth. The relatively large control coefficient of the 0galactoside permease also helps to explain an unexpected result obtained by ANTHONYDEANin his studies of the effects of spontaneous amino acid replacements in the lacZ P-galactosidase (DEAN, DYKHUIZEN and HARTL1988b). Theseessentially random replacements were obtained in the laboratory as revertants of nonsense codons that restored enzyme synthesis but altered its electrophoretic mobility or thermostability as compared with wild type. The unexpected result was that most of the amino acid replacements produced effects too small to be detected in chemostats. Among 25 amino acid replacements occurring in 17 codonsdistributedapproximately uniformly along the gene, only three produced selective effects large enough to be statistically significant. The remaining 22 produced effects that could not be detected under conditions in which the limit of resolution was a selection coefficient of approximately 0.4% per generation. It seemed reasonable to assume that many ofthe aminoacid replacements actually resulted in small differences in enzyme activity but that these gave undetectable effects in chemostats owing to the relatively small control coefficient of P-galactosidase with respect to fitness (DEAN, DYKHUIZEN and HARTL 1986). The experiments with ebg helped todefinethe alterations in substrate specificity resulting from simple amino acid replacements. As noted at the outset, we had assumed that these studies would also provide some insight intothe mechanisms bywhich novel enzyme activities are created during the course of evolution.However,asthings turned out, the ebg system proved to be a better model for the refinement of enzyme activity than for the evolution of catalytic novelty. Indeed, it now appears that novel catalytic activities are often acquired by a process quite beyond anything that we, or anyone else, had imagined at the time. It now appears that enzymes with truly novel functions, rather than being derived from single amino acid replacements, are often assembled piecemeal from smaller functional units. One indication that protein evolution may involve a combinatorialprocess came fromthe finding of similar folding domainswith similar functions in otherwise unrelatedproteins (PHILLIPS,STERNBERG and SUTTON 1983). Another indication came from the discovery that many genes in eukaryotes are split into exons and introns, often with a correlation between exons and protein-folding domains suggestive of piecemeal assembly from smaller units capable of somewhat autonomous folding and function (GILBERT1978). The powerful combinatorial possibilities help to overcometheodds against protein evolution by random amino acid replacement. Strong support for this model of protein evolution came from the finding that the low-density lipoproteinreceptorgene contains exonsthat are clearly paralogous (homologous because of gene duplication} with exons in genes for componentsof complement, blood-clotting factors and epidermal growth factor (SUDHOFet al. 1985). A combinatorial mechanismof protein evolution provides a resolution of EDEN’Sprobability paradoxmentionedearlier, because piecemeal combination gives “strong regularities for finding useful paths” of protein evolution. The genes-in-pieces mechanism of protein evolution has been further elaborated by BRENNER(1988) in a principle of localfunctionality, accordingto which the folding ofsmall segments of a polypeptide is determined mainly by local interactions within each segment. Brenner has argued that the principle of local functionality allowed discrete functions of individual peptides to continue undisturbed in composite molecules and eventually resulted in the formation of present-day exons. One implication of local functionality is that certain polypeptide segments of proteins may be interchangeable with segments of comparable local structure from totally unrelated proteins. This prediction is subject to direct experimental test using oligonucleotide sitedirected mutagenesis to interchange comparable local segments of proteins whose three-dimensional structure is well determined. ROBERTDUBOSEhas succeeded in doing this with a-helical segments in the alkaline phosphatase of E. coli, each seven amino acids in length. The helical segments were obtained either from different domains within the alkaline phosphatase or from a helical segment within the bacteriophage T 4 Iysozyme. Three such replacements were carried out, and in all three cases the activity of the alkaline phosphatase was retained,although it was different from the wild-type enzyme (R. DUBOSEand D. L. HARTL,unpublished results). This result provides strong support for the principle of local functionality, at least with regard to helical segments. If the principle can be demonstrated more generally, then it supports the hypothesis that combinations of units with novel functions can be assembled piecemeal and theiractivity and specificity can later be improved and refined by individual amino acid replacements. In addition to the evolutionary implications of ebg, the locus also presents some interesting molecular Perspectives biology. At the protein level, HALL andcollaborators have demonstrated that the e6g /3-galactosidase contains multiple subunits of two polypeptides coded by the partially overlapping cistrons e6gA and e6gC. The major polypeptide is the e6gA gene product of 1032 amino acids (STOKES, BETTS and HALL 1985) and the minor polypeptide is the e6gC gene product of 173 amino acids. Expression of the e6gA and e6gC cistrons is negatively regulated by the e6g repressor produced from the tightly linked e6gR gene which codes for a polypeptide of 328 amino acids. The mutations responsible for the class I and class I1 e6g enzymes have also been identified at thenucleotide level. Class I mutations in e6g include the e6gAP and e6gA4 alleles described in the1974paper. As noted earlier, they significantly increase the activity of the enzyme toward lactose but not towardlactulose. The dramatic change in substrate specificityof the class I alleles results from a single amino acid replacement of asparagine for aspartic acid at position 92. In the class I1enzymes, which significantly increase activity toward lactulose as well as lactose, one mutation always results in the replacement of a cysteine for a tryptophan at position 977, and in some alleles there is also a serine-to-glycine replacement at position 978. The positions of these replacementshave no apparent proximity to the region implicated in the active site of theenzyme, which shouldperhapsnotbetoo surprising because refinement of enzyme function may often involve long-rangeinteractions between residues in different domains. Selection may result in a molecular coadaptation that promotesthe particular domains to function well in combination. This principle supervenes thatof local functionality for thefine tuning of enzyme function. As RILEY,SOLOMON and ZIPKAS (1978) predicted from the relative mappositions of the genes in the E. coli chromosome, e6gA is paralogous with lacZ and e6gR is paralogous with lacl. At the amino acid level, the sequences of the e6gA and lacZ polypeptides are about 34% identical and those of the e6gR and l a c l polypeptides are about 25% identical. The relatively large divergence suggests that the duplication of the common ancestors of these genes was ancient. The e6gA gene has, however, retained its @-galactosidase activity and sufficient potential for lactose hydrolysis that direct selection for growth onlactose is successful. Interestingly, E. coli strainscontainingdeletions of both e6gA and lacZ do not give rise to a third Pgalactosidase, even after heavy mutagen treatment and prolonged selection on lactose (unpublished results of D. L. HARTLand of B. G. HALL).Comparisons of gene sequences and functions around the E. coli chromosome suggest that genesparalogous to lacZ and ebg should exist midway between them on both sides of the chromosome (RILEY, SOLOMON and ZIPKAS 5 1978) and perhapseven at 7-min intervals (KUNISAWA and OTSUKA 1988). Evidently, these other paralogous genes are so divergent that no single or small number of amino acid replacements are sufficient to generate a @-galactosidasewith enough lactose hydrolysis to survive direct selection for growth on lactose. The results of experiments with e6g vindicate JOHN CAMPBELL’S perspicacity in pursuing the unexpected papillae thatemergedfromdecrepit Lac- colonies after prolonged incubationon lactose indicator plates. Anyone who has studied old bacterial plates knows that a lot of strange things can appear, most of them grungy, many of them uninteresting, some unanalyzable and a few downright hazardous to one’s research career if not to one’s health. While CALVINBRIDGE’S admonition to treasure your exceptions is often quoted toencouragestudentsnottoignore unexpected observations that might conceivably be significant, the image of an old bacterial plate may signify the important corollary that it takes a specific kind of genius to foresee which exceptions should be treasured among the many that are dross. I thank BARRY G. HALL forhis important contributions to this Perspective and ROBERTDUBOSEfor his many helpful comments. I am also grateful to the collaborators mentioned in the text who have made the scientific odyssey from ebg not only possible but also very agreeable. BARRY G. HALLwas typically generous in permitting ebg sequences. This work is presently the use of unpublished data on supported by National Institutes of Health grants GM30201 and M40322. DANIELL. HARTL Department of Genetics Washington University School of Medicine St. Louis, Missouri 631 10-1095 LITERATURE CITED BRENNER, S., 1988 A tale of two serines. 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