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Barrels in pieces?
© 2001 Nature Publishing Group http://structbio.nature.com
John A. Gerlt and Patricia C. Babbitt
Imidazole glycerol phosphate synthase (HisF) has been proposed to be the product of duplication of a gene
encoding a (β/α)4-half barrel followed by fusion to encode the complete (β/α)8-barrel. In support of this
evolutionary scenario, the N- and C-terminal (β/α)4-half barrels of HisF have been separately expressed and
purified. Each assumes a stable, soluble homodimeric structure, but neither is catalytically active; when expressed
together, a functional heterodimer is formed.
An important outcome of postgenomic
biology is the potential for elucidating the
evolutionary history of contemporary
proteins. Understanding Nature’s strategies and mechanisms for enzyme evolution may provide insights for the rational
design of proteins with novel biological
functions. Because the (β/α)8-barrel fold
is the most common active site scaffold,
much attention has been devoted to
understanding the phylogeny of proteins
sharing this fold. In particular, the question of whether the (β/α)8-barrel evolved
once (divergent evolution) or multiple
times (convergent evolution) remains
unresolved1–3.
Previous crystallographic studies by
Wilmanns and Sterner4 confirmed a proposal based on sequence comparisons5
that the structures of N-[(5′-phosphoribosyl)formimino]-5-aminoimidazole4-carboxamide ribonucleotide isomerase
(ProFAR isomerase or HisA) and imidazole glycerol phosphate synthase (HisF)
are structurally homologous (β/α)8barrels; HisA and HisF catalyze successive
steps in histidine biosynthesis (Fig. 1) .
The structures also confirmed that the
first and second (β/α)4-half barrels of both
enzymes are related by a two-fold axis of
symmetry5, supporting the hypothesis that
the genes encoding both (β/α)8-barrels are
the result of duplication of a single gene
encoding a (β/α)4-half barrel, subsequent
fusion, and modest divergent evolution.
Now, on page 32 of this issue of Nature
Structural Biology, Sterner and coworkers6
provide experimental evidence in support
of this hypothesis by demonstrating that
the N- and C-terminal (β/α)4-half barrels
of HisF (HisF-N and HisF-C) can be
expressed separately as stable, folded proteins that form soluble but inactive
homodimers. When these half barrels are
coexpressed, a catalytically active heterodimer results. These data support the
model that a single (β/α)4-half barrel was
the precursor of the HisF (β/α)8-barrel
and, by inference, suggest that other
Fig. 1 Reactions catalyzed by N-[(5′-phosphoribosyl)formimino]-5-aminoimidazole-4-carboxamide ribonucleotide isomerase (HisA) and imidazole glycerol phosphate synthase (HisF). The reacting portions of the substrates/products are highlighted in red (HisA) and blue (HisF).
(β/α)8-barrel enzymes may have been the substrate by duplication of the gene
assembled from this and/or other members encoding the original enzyme followed by
divergent evolution. Although the
of a library of (β/α)4-half barrel modules.
sequences of HisA and HisF share only
Substrate/product binding directing 24% identity, database searches reveal that
evolution
they are the closest homologs of each
HisA and HisF share the ability to bind other.
ProFAR (Fig. 1): HisA catalyzes the formaProFAR is an unusual metabolite with a
tion of ProFAR, and HisF catalyzes an pseudo two-fold axis of symmetry
ammonia-dependent reaction in which (Fig. 1). One half contains a ribose
ProFAR is converted to imidazole glycerol 5-phosphate moiety and the other an isophosphate and 5-aminoimidazole-4-car- merized ribose 5-phosphate. Thus, when
boxamide ribonucleotide (AICAR), an Nature required a binding site for ProFAR
intermediate in purine biosynthesis (Fig. for the HisF reaction, one with a two-fold
1). So, like phosphoribosyl anthranilate symmetry axis was an appropriate choice.
isomerase (TrpF) and indole glycerol Evidently, the design solution to this probphosphate synthase (TrpC), which cat- lem was the simplest one — selection of a
alyze successive reactions in the trypto- single, small, progenitor domain capable
phan biosynthetic pathway7, HisA and of binding ribose 5-phosphate followed by
HisF can be considered as additional duplication and fusion of the gene to proexamples of retrograde evolution of duce the desired binding site. According to
enzymes in a biosynthetic pathway as pro- Horowitz’s hypothesis, the two-fold symposed by Horowitz8,9. According to this metrical binding site in HisF would be
hypothesis, when the substrate for an preserved when HisA evolved to catalyze
enzyme in a biosynthetic pathway is the preceding reaction, introducing
depleted from the organism’s nutritive restrictions on both the substrate for HisA
niche, a new enzyme can evolve to supply and the chemistry needed to produce
nature structural biology • volume 8 number 1 • january 2001
5
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Fig. 2 Superposition of HisF-N and HisF-C. The
color key for the β-sheets, α-helices, and connecting segments is shown. The phosphate
anions associated with HisF-N and HisF-C are
shown in yellow and orange, respectively.
ProFAR. Not surprisingly, the substrate
for HisA contains two ribose 5-phosphate
moieties, and the HisA-catalyzed reaction
retains both parts of the substrate.
The (β/α)4-half barrels are stable,
folded proteins
The structures of the first (HisF-N) and
second (HisF-C) (β/α)4-half barrels of HisF
are superimposed in Fig. 2. This superposition includes two phosphate anions present
in the active site HisF, each presumably
occupying the position of the phosphate
groups of ProFAR. In HisF-N the binding
site is formed by hydrogen donors contributed by residues at the C-terminal end
of the third β-strand and the N-terminal
end of the fourth α-helix. In HisF-C the
binding site is formed in an analogous
manner by hydrogen donors contributed
by residues at the C-terminal end of the
seventh β-strand and the N-terminal end
of the eighth α-helix. The extent of the
overall structural similarity is remarkable,
both for the β-strands and the α-helices.
Although a recapitulation of the evolutionary events that produced HisF in the
laboratory is unlikely, Sterner and his
coworkers6 tested the hypothesis that
Nature selected a stable progenitor with a
single ribose 5-phosphate binding site by
the separate expression of HisF-N and
HisF-C. They found that both could be
expressed in Escherichia coli, albeit as
inclusion bodies. Each could then be separately denatured in guanidinium
hydrochloride and refolded to yield soluble, stable proteins. The far UV circular
dichroic spectra of these proteins showed
that their α-helix and β-sheet content was
consistent with that observed in the X-ray
structure of intact HisF. Neither HisF-N
nor HisF-C catalyze the HisF reaction.
Undoubtedly, the (β/α)4-half barrels
assumed specialized roles as they evolved
6
to efficiently catalyze the HisF reaction
within the context of the complete
(β/α)8-barrel.
When HisF-N and HisF-C were
expressed together in E. coli, a soluble
complex (HisF-NC) could be isolated
from cell extracts. Alternatively, a soluble
complex was obtained when the HisF-N
and HisF-C inclusion bodies were separately expressed, denatured, and mixed
prior to renaturing. The complex exhibits
catalytic constants similar to those for
wild type HisF when exogenous ammonia
is supplied as the cosubstrate. However,
the complex apparently is unable to form
a complex with the HisH glutaminase, so
it cannot utilize ammonia derived from
glutamine as wild type HisF does.
The molecular weights of the fragments
and intact HisF as evaluated by gel filtration and analytical ultracentrifugation are
consistent with concentration-dependent
equilibration of different aggregation
states: HisF-N and HisF-C equilibrate
between homodimers (HisF-N)2 and
(HisF-C)2, and homotetramers (HisF-N)4
and (HisF-C)4; HisF-NC equilibrates
between heterodimers (HisF-NC)2, and
heterotetramers (HisF-NC)4; and intact
HisF equilibrates between monomers and
dimers.
Taken together, the simplest interpretation of these observations is that HisF-N
and HisF-C each self-associate to form a
closed noncovalent (β/α)8-barrel, thereby
shielding the exposed hydrophobic surface of the (β/α)4-half barrels from solvent. Although the structures of (HisF-N)2
and (HisF-C)2 and the heterodimer have
not been determined, plausible models are
shown in Fig. 3.
More evidence for modular
construction
Structural and sequence data for other
enzymes support the hypothesis that
(β/α)8-barrels have been formed from
smaller progenitors. A large number of
(β/α)8-barrel proteins contain phosphate
binding motifs homologous to those found
in HisA and HisF (as deduced from comparisons of sequence and/or structure),
including the homologous dihydroorotate
dehydrogenase, flavocytochrome b2, and
glycolate oxidase (which bind FMN), tryptophan synthase (which binds indoleglycerol phosphate), and ThiE (which
assembles thiamin phosphate from phosphorylated pyrimidine and thiazole precursors). In each of these, a phosphate
binding motif is formed by the C-terminal
end of the seventh β-strand and the N-terminal end of the eighth α-helix in the barrel10,11. A priori, no geometric feature of a
(β/α)8-barrel can explain why this position
for a phosphate binding motif should be
conserved in divergently related enzymes
catalyzing different reactions, unless these
were derived from a common module.
Positional retention of catalytic groups
derived from a conserved (β/α)4-half barrel module would provide additional evidence for the modular construction of
(β/α)8-barrels from smaller progenitors.
Several examples of this are now known.
The catalytic residues in bacterial and
eukaryotic phospholipases C are located
on homologous scaffolds in the first half
of their (β/α)8-barrel domains, but the
specificity elements located in the second
half of the barrel do not share detectable
structural homology12. In addition, we
(J.A.G. and P.C.B., unpublished results)
Fig. 3 Hypothetical structures for HisF-NC (HisF-N)2, and (HisF-C)2 in which the HisF-N and HisF-C
(β/α)4-half barrels are assembled to form parallel β-sheet structures; each is viewed from the C-terminal ends of the β-sheets. The phosphate binding sites located in HisF-N and HisF-C are indicated.
The color key is the same as in Fig. 2.
nature structural biology • volume 8 number 1 • january 2001
© 2001 Nature Publishing Group http://structbio.nature.com
© 2001 Nature Publishing Group http://structbio.nature.com
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and others13 have discovered a homologous group of enzymes that catalyze different reactions but share conserved
functional groups at the ends of the
β-strands in the first (β/α)4-half barrel of
their structures. The divergently related
enzymes include orotidine 5′-phosphate
(OMP) decarboxylase and (D)-arabinohex-3-ulose 6-phosphate synthase that
catalyze different reactions, the former
nominally involving the formation of a
highly unstable vinyl anion and the second an enolate anion. Although the substrates for these enzymes also contain
phosphate groups and both the
decarboxylase and synthase contain the
conserved phosphate binding motif
found in HisF, other members of this
homologous group utilize the same modular catalytic groups in similar reactions
that do not utilize a phosphate ester as
substrate. Understanding how Nature can
devise a structural strategy for the assembly of functional chimera is important:
one strategy, predicted by the structure of
HisF4,6, would be the mixing and matching of fractional (β/α)8-barrels, for example, (β/α)4-half barrels, although other
(β/α)n-modules are possible.
Implications for evolution
The real possibility that (β/α)8-barrels can
be derived from mixing and matching of
(β/α)4-half barrels as well from other
(β/α)8-barrels by divergent evolution
introduces a new level of complexity in
analyses of the phylogeny of (β/α)8-barrels. Perhaps, ‘in the beginning’ Nature
first assembled chimeric barrels from a
library of modular structures. Then, ‘new’
enzymes evolved by duplication of genes
encoding various (β/α)8-barrels and
divergent evolution, as has been proposed.
Such evolution would give rise to a limited
number of evolutionarily distinct groups
of enzymes. These would include mechanistically diverse superfamilies whose
members catalyze reactions that share elements of mechanism as well as enzymes in
the same anabolic pathway related by
retrograde evolution. Thus, the initial
assembly from independent modular
structures would give rise to distinct lineages of (β/α)8-barrels, although some
would be related by a common (β/α)4-half
barrel ‘parent.’
The most recent attempt to develop a
comprehensive phylogeny for (β/α)8barrels reached the conclusion that catalytically and metabolically diverse
(β/α)8-barrels evolved by recruitment of
enzymes catalyzing different reactions in
different pathways3. Nature undoubtedly
is opportunistic. Given the observations
described by Sterner, Wilmanns and
their coworkers4,6, assembly of independent modular structures provides an
alternate and, perhaps, more reasonable
explanation. If the construction of
(β/α)8-barrels from smaller progenitors
is correct, phylogenetic analyses must
include the consequences of such modular complexity.
Versatility of the (β/α)8-barrel
The structure of the (β/α)8-barrel is particularly well-suited for evolution of function once an active site architecture has
been selected to catalyze a particular type
of reaction. For example, in diverse enolization reactions catalyzed by members of
the enolase superfamily, the three conserved ligands for the essential divalent
metal ion that stabilized an enolate anion
intermediate are located at the ends of
three successive and, therefore, spatially
contiguous β-sheets. The acid/base catalysts are located independently at the ends
of other β-sheets so that these can evolve
independently to accommodate either different facial selectivities for enolization
and different mechanisms for the conversion of the enolate anion intermediates to
products14.
The results obtained by Sterner and
coworkers6 illustrate another evolutionary
advantage of the topologically closed
(β/α)8-barrel that surrounds the active
site: this construction ought to allow
assembly of the complete (β/α)8-barrel
from smaller (β/α)n-modules15, each providing a scaffold for one or more func-
nature structural biology • volume 8 number 1 • january 2001
tional groups that can be utilized in different catalytic capacities.
The demonstration that the (β/α)8-barrel of HisF and, by analogy, HisA, can be
assembled from smaller components
introduces an unexpected but fascinating
complexity into understanding the
process by which enzymes evolved.
Whether human protein engineers will be
able to follow Nature’s lead by designing
and mixing (β/α)4-half barrels to create
new enzymes is unknown. However, the
structural symmetry of HisF reinforces
the growing body of evidence that the
structural features of (β/α)8-barrels are
uniquely suited for both the design and
evolution of catalysts.
John A. Gerlt is in the Departments of
Biochemistry and Chemistry, University of
Illinois, 600 South Mathews Avenue,
Urbana, Illlinios 61801, USA, and Patricia
C. Babbitt is in the Departments of
Biopharmaceutical
Science
and
Pharmaceutical Chemistry, School of
Pharmacy, University of California, San
Francisco, California 94143, USA.
Correspondence should be addressed to
J.A.G. email: [email protected] or P.C.B.
email: [email protected]
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