Download Evolving Theories of Enzyme Evolution

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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
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