Download Divergent Evolution of ( )8-Barrel Enzymes

Biol. Chem., Vol. 382, pp. 1315 – 1320, September 2001 · Copyright © by Walter de Gruyter · Berlin · New York
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Divergent Evolution of ()8-Barrel Enzymes
Martina Henn-Sax1,2, Birte Höcker1, Matthias
Wilmanns3 and Reinhard Sterner1,*
1 Institut
für Biochemie, Universität zu Köln, OttoFischer-Strasse 12-14, D-50674 Köln, Germany
2
Abteilung Molekulare Genetik und Präparative
Molekularbiologie, Institut für Mikrobiologie und
Genetik, Georg-August-Universität Göttingen,
Grisebachstr. 8, D-37077 Göttingen, Germany
3
European Molecular Laboratory (EMBL) Hamburg
Outstation, c/o DESY, Notkestrasse 85, D-22603
Hamburg, Germany
* Corresponding author
The ()8-barrel is the most versatile and most frequently encountered fold among enzymes. It is an
interesting question how the contemporary ()8barrels are evolutionarily related and by which mechanisms they evolved from more simple precursors.
Comprehensive comparisons of amino acid sequences and three-dimensional structures suggest
that a large fraction of the known ()8-barrels have
divergently evolved from a common ancestor. The
mutational interconversion of enzymatic activities of
several ()8-barrels further supports their common
evolutionary origin. Moreover, the high structural
similarity between the N- and C-terminal ()4 units of
two ()8-barrel enzymes from histidine biosynthesis
indicates that the contemporary proteins evolved by
tandem duplication and fusion of the gene of an ancestral ‘half-barrel’ precursor. In support of this hypothesis, recombinantly produced ‘half-barrels’ were
shown to be folded, dimeric proteins.
Key words: Directed evolution / Enzyme fold / Histidine
biosynthesis / Protein families / TIM-barrel / Tryptophan
biosynthesis.
lationship), superfamilies (probable common evolutionary origin) and folds (Murzin et al., 1995). Proteins are defined to have a common fold if they have the same major
secondary structures in the same spatial arrangement
and the same topological connections. SCOP currently
distinguishes between several hundred different folds.
The (βα)8- (or TIM-)barrel is a frequently encountered
fold, which comprises about 10% of all known protein
structures (Gerlt, 2000). (βα)8-barrel enzymes catalyse a
vast range of different reactions, functioning as either oxidoreductases, transferases, hydrolases, lyases or isomerases (Pujadas and Palau, 1999; Wierenga, 2001). The
(βα)8-barrel consists of a core of eight twisted parallel βstrands, the β-barrel, which are connected by eight α-helices which form the outer layer of the structure (Figure 1).
In all known (βα)8-barrel enzymes, the active site residues
are located at the C-terminal face of the β-barrel and
within the loops that connect the β-strands with the subsequent α-helices. Many (βα)8-barrel enzymes contain
extensions to this canonical topology, either at the N- or
C-termini of the sequence, or in loop segments (Pujadas
and Palau, 1999).
The evolution of the versatile (βα)8-barrel fold has been
Introduction
The rapidly growing number of determined amino acid
sequences and three-dimensional structures allows to
investigate how the efficient and specific enzymes of
modern metabolic pathways are evolutionarily related,
and how they developed from less efficient and specific
precursors (Gerlt and Babbitt, 2001). The Structural Classification of Proteins (SCOP) database divides proteins
according to their amino acid sequence, structural and
functional similarities into families (clear evolutionary re-
Fig. 1 The (βα)8-Barrel Fold.
(A) View onto the C-terminal ends of the eight β-strands, which
form a cylindrical parallel β-sheet. This β-barrel is surrounded by
the eight α-helices.
(B) Topologic diagram of the eight (βα) modules. The active site
residues of all known (βα)8-barrel enzymes are located at the
C-terminal ends of the β-strands and in the loops that connect
the β-strands with the subsequent α-helices.
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M. Henn-Sax et al.
discussed for many years, and arguments in favour of either convergent evolution to a stable fold or, alternatively,
divergent evolution from a common ancestral barrel have
been put forward (Lesk et al., 1989; Farber and Petsko,
1990; Raine et al., 1994; Reardon and Farber, 1995). Here
we summarize recent sequence and structure analyses,
as well as protein engineering studies, which provided
new insights into the evolution of (βα)8-barrels. The results suggest that the members of several important superfamilies and probably a large fraction of all known
(βα)8-barrels have a common evolutionary origin (Altamirano et al., 2000; Babbitt and Gerlt, 2000; Copley and
Bork, 2000; Jürgens et al., 2000). Moreover, evidence for
the evolution of the (βα)8-barrel fold from ancestral ‘halfbarrel’ precursors will be presented (Thoma et al., 1998;
Lang et al., 2000; Höcker et al., 2001).
The Phosphate-Binding Superfamiliy
The program PSI-Blast (Altschul et al., 1997) was used to
perform iterative rounds of sequence comparisons between (βα)8-barrels (Copley and Bork, 2000). The search
was started with N’-[(5’-phosphoribosyl)formimino]-5aminoimidazole-4-carboxamide-ribonucleotide (ProFAR)
isomerase of histidine biosynthesis (HisA), which contains two phosphate-binding sites. A number of other
(βα)8-barrels with significant sequence similarity were detected. All of them contain a characteristic glycine-rich
phosphate-binding motif at the C-terminal end of βstrand 7, which was identified earlier by analysing smaller sets of amino acid sequences (Bork et al., 1995) or
three-dimensional structures (Wilmanns et al., 1991). The
detected enzymes include (βα)8-barrels from tryptophan
biosynthesis, triosephosphate isomerase and fructose
bisphosphate aldolases. Since there are eight equivalent
β-strands in (βα)8-barrels, the identical location of the
phosphate-binding site of all of these enzymes at the Cterminal end of β-strand 7 suggests that this superfamiliy
evolved from a common ancestor.
Divergent evolution is further supported by the combined structural and functional analyses of phosphatebinding (βα)8-barrels from histidine and tryptophan
biosynthesis. HisA and imidazole glycerol phosphate
synthase (HisF) catalyse two successive reactions
in histidine biosynthesis. As a consequence, both enzymes bind the common ligand N’-[(5’-phosphoribulosyl)formimino]-5-aminoimidazole-4-carboxamide-ribonucleotide (PRFAR), which is the product of HisA and the
substrate of HisF (Figure 2A). The crystal structures of
HisA and HisF from Thermotoga maritima were determined at high resolution (Lang et al., 2000). The superposition of their backbone atoms yielded a root mean square
(rms) deviation of only 1.79 Å (Figure 2B). Furthermore,
Fig. 2 Two Sequential Reactions of Histidine and Tryptophan Biosynthesis with Similar Chemistries are Catalysed by Related (βα)8Barrels.
(A) HisA and TrpF catalyse Amadori rearrangements of aminoaldoses into aminoketoses. HisF and TrpC catalyse the closure of the imidazole and indole ring to yield ImGP and IGP, respectively. The second product of the HisF reaction, AICAR, is further used in de novo
purine biosynthesis. To form ImGP and AICAR, HisF uses nascent ammonia produced by the glutaminase HisH (Beismann-Driemeyer
and Sterner, 2001).
(B) Experimental evidence for evolutionary relatedness of HisF, HisA, TrpF and TrpC. The residual HisA activity of HisF, and the interconversions of HisA and TrpC into TrpF are indicated by broken arrows. The rms deviations resulting from pairwise structural superpositions using
all main chain, non-hydrogen atoms are shown. The percentages of identical residues in the corresponding structure-based alignments are
given in brackets. The calculations were carried out with the program ALIGN-PDB (Cohen, 1997). Abbreviations: AICAR: 5-aminoimidazole4-carboxamide ribotide; CdRP: 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate; HisA: ProFAR isomerase; HisF: synthase subunit
of ImGP synthase; HisH: glutaminase subunit of ImGP synthase; IGP: indole glycerol phosphate; ImGP: imidazole glycerol phosphate; PRA:
phosphoribosyl anthranilate; PRFAR: N’-[(5’-phosphoribulosyl)formimino]- 5-aminoimidazole-4-carboxamide-ribonucleotide; ProFAR: N’[(5’-phosphoribosyl)formimino] -5-aminoimidazole-4-carboxamide-ribonucleotide; TrpC: IGP synthase; TrpF: PRA isomerase.
Enzyme Evolution
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Fig. 3 Synopsis of a Four-Fold Structure-Based Sequence Alignment of the Amino-Terminal (HisA-N, HisF-N) and the Carboxy-Terminal Halves (HisA-C and HisF-C) of HisA and HisF.
The arrows represent β-strands, the cylinders α-helices. β-strands and α-helices 1 – 4 correspond to HisA-N and HisF-N, β-strands and
α-helices 5 – 8 correspond to HisA-C and HisF-C. β-strands 1’ and 5’, and α-helices 2’, 4’, 6’, 8’ are extensions to the limit (βα)8-barrel
fold (Lang et al., 2000). Invariant residues are shown in upper case letters and residues that are identical in three of the four sequences
in lower case letters. The invariant aspartate residues (D) that are essential for catalysis are located at the C-terminal ends of β-strands
1 and 5.
the two aspartate residues of HisA and HisF that are important for catalysis are located at equivalent positions at
the C-terminal ends of β-strands 1 and 5 (Figure 3). Therefore, both enzymes were tested for their mutual residual
activities. Whereas HisA does not show detectable HisF
activity, HisF catalyses the HisA reaction, albeit with low
efficiency (Lang et al., 2000).
In analogy to HisA and HisF, phosphoribosyl anthranilate (PRA) isomerase (TrpF) and indole glycerol phosphate synthase (TrpC) are (βα)8-barrel containing enzymes that catalyse two successive reactions in the
biosynthesis of tryptophan. They bind the common ligand 1-(o-carboxyphenylamino)-1-deoxyribulose 5-phosphate (CdRP), which is the product of TrpF and the substrate of TrpC (Figure 2A). The rms deviation of the
backbone atoms of TrpF and TrpC from Escherichia coli
is 2.03 Å (Figure 2B). In an attempt to establish TrpF activity on the scaffold of TrpC, Altamirano et al. (2000) used
a combination of rational design and directed evolution.
In a first step, the N-terminal extension to the limit (βα)8barrel of TrpC was removed and several loops at the Cterminal end of the β-barrel were designed to be similar to
the corresponding loops in TrpF. Subsequently, random
mutagenesis and selection in a trpF deficiency strain
were performed. With this approach a TrpC variant with
high TrpF activity but lacking TrpC activity was isolated.
The establishment of TrpF activity on the TrpC scaffold
and the residual HisA activity of HisF indicate a common
evolutionary origin of (βα)8-barrel enzymes within histidine and tryptophan biosynthesis.
Both HisA and TrpF catalyse mechanistically similar reactions, namely Amadori rearrangements of an aminoaldose into an aminoketose (Figure 2A), and the superposition of their backbone atoms yielded an rms deviation of
2.55 Å (Figure 2B). There is strong experimental evidence
for a close inter-pathway relationship between these enzymes. Using random mutagenesis and selection in a
trpF-deficiency strain, HisA variants were generated that
catalysed the TrpF reaction. Moreover, one of these variants retained significant HisA activity (Jürgens et al.,
2000). A closer analysis revealed that a single amino acid
exchange in the active site region was sufficient to interconvert the substrate specificity from HisA to TrpF, although the enzymes share a sequence identity of only
about 10%.
Taken together, these experiments suggest an evolu-
tionary network that links the (βα)8-barrels HisA, HisF,
TrpF and TrpC (Figure 2B).
Evolution of the Enolase Superfamily
The sequence similarities between the members of the
enolase superfamily are often low and their substrates
are chemically quite diverse. However, their similar
three-dimensional structures and catalytic mechanisms
indicate a common evolutionary origin (Babbitt and
Gerlt, 2000). Members of the enolase superfamiliy consist of two domains: a larger (βα)7β barrel domain, which
is a modified version of the (βα)8-barrel, and a mixed α/β
domain that is formed by the N- and C-terminal parts of
the sequence (Babbitt et al., 1996). The mixed α/β domain is an important determinant of the substrate specificity and caps the barrel domain at the C-terminal ends
of the β-strands, where the residues that are essential
for catalysis are located. The mechanistic similarity within the superfamiliy is the abstraction of the α-proton of a
carboxylate anion substrate, which is assisted by electrostatic stabilization of the resulting enolate intermediate by a metal ion. The enolate intermediate is then converted to different products via different mechanisms in
the various active sites. However, the chemical nature
and the location of the residues essential for catalysis
within the barrel are highly conserved. Within the enolase superfamily, therefore, new enzymatic activities obviously evolved by retaining a crucial step in the catalytic mechanism while changing substrate specificity. An
interesting member of the enolase superfamily is an enzyme from Amycolaptosis sp., which acts both as N-acyl
amino acid racemase and as o-succinylbenzoate synthase (Palmer et al., 1999). These two reactions are considerably different with regard to the substrate and the
overall chemical mechanism. The recently solved structure of o-succinylbenzoate synthase from Escherichia
coli shows that most interactions between the bound
product and the active site are either indirect via water
molecules or via hydrophobic interactions (Thompson et
al., 2000). It was speculated that this plasticity within the
active site contributes to the dual substrate specificity of
the homologous enzyme from Amycolaptosis sp., which
might examplify ‘evolution in action’ (Babbitt and Gerlt,
2000).
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M. Henn-Sax et al.
Evolutionary Links between Superfamilies
Enolase catalyses the formation of phosphoenolpyruvate
from 2-phosphoglycerate. However, it does not bind the
phosphate group of the substrate at the end of β-strand
7, in contrast to the phosphate-binding superfamily. Nevertheless, through a sequence family of unknown structure and function, significant sequence similarities
between the enolase and the phosphate-binding superfamilies were detected (Copley and Bork, 2000). Circular
permutation of the sequence might have occurred in the
course of divergence of these superfamilies, because βstrand 3 of the enolase superfamily aligns with β-strand 5
of the phosphate-binding (βα)8-barrels. This similarity is
equivalent to a conserved metal-binding residue in βstrand 7 of the enolase superfamily with the conserved
glycines of the phosphate-binding (βα)8-barrels. Probably, the conformations of these sites make them
favourable locations for ligand-binding residues, even if
the ligands are different. Further rounds of PSI-Blast
searches that were performed with relaxed inclusion
thresholds detected significant similarities of the phosphate-binding superfamily to members of the phosphoenolpyruvate/ pyruvate superfamily and to additional
(βα)8-barrels, which are involved in leucine and lysine
biosynthesis, and in gluconeogenesis (Copley and Bork,
2000).
Horowitz (1945) speculated that anabolic pathways
evolved by several duplication and diversification events,
starting with the gene encoding the last enzyme of the
contemporary pathway (‘retrograde evolution’). The appeal of this model is that no new ligand binding site has to
be invented upon evolving new enzymatic activities. Although the Horowitz model is probably not correct in a
strict sense (Roy, 1999), (βα)8-barrel enzymes that catalyse successive reactions within the same pathway almost certainly evolved upon retention of a common ligand binding site. However, there are also (βα)8-barrel
enzymes from different pathways that bind the same ligand. For example, transaldolase, the α-subunit of tryptophan synthase, triosephosphate isomerase and fructose-1,6-bisphosphate aldolase all catalyse reactions
with D-glyceraldehyde 3-phosphate as one of the products. This may indicate a common ancestor for these enzymes with a D-glyceraldehyde 3-phosphate binding
site. Similarly, both enolase and pyruvate kinase bind
phosphoenolpyruvate (PEP) and Mg2+, and neither
shows the standard phosphate-binding motif. Probably,
both enzymes may have arisen from a common PEP and
Mg2+-binding ancestor. Other (βα)8-barrel enzymes, for
example HisA and TrpF, and the members of the enolase
superfamily, use similar mechanisms to catalyse reactions of different substrates (Babbitt and Gerlt, 2000; Jürgens et al., 2000). This suggests that these enzymes are
derived from a common ancestor with a broader substrate specificity (Jensen, 1976). One has to conclude,
therefore, that (βα)8-barrel enzymes are derived from ancestors with similar functions, which can either be the
binding of the same ligand or a particular catalytic activity. In summary, numerous (βα)8-barrels within and between pathways appear to have a common evolutionary
origin.
Evolution of the ()8-Barrel Fold from
Ancestral ‘Half-Barrels’
Which of the contemporary (βα)8-barrels is most similar
to the putative common ancestor? The sequences and
structures of HisA and HisF, which have substrates with
two phosphate moieties (Figure 2A), show an internal
duplication that is not observed in other (βα)8-barrels
(Fani et al., 1994; Thoma et al., 1998; Lang et al., 2000).
It follows that HisA and HisF have retained ancestral features, which were lost during the evolution of other barrels. The superposition of the N- and C-terminal halves
HisA-N, HisF-N, HisA-C, and HisF-C from Thermotoga
Fig. 4 Model for the Evolution of the (βα)8-Barrel Fold.
The first tandem gene duplication and fusion generates two
identical, fused half-barrels that then adjust to form the ancestral
(βα)8-barrel. Further gene duplications and diversifiactions lead
to HisA, HisF, and to other contemporary (βα)8-barrels. Adapted
with permission from Lang et al., Science 289, 1546 – 1550.
© (2000) American Association for the Advancement of Science.
Enzyme Evolution
maritima yielded close overall similarities with rms deviations of only 1.4 to 2.1 Å (Lang et al., 2000). The resulting four-fold structure-based sequence alignment revealed a number of conserved residues that are located
at structurally identical positions in all four half-barrels
(Figure 3). Among them are the aspartate residues essential for catalysis, which are located at the ends of βstrands 1 (in HisA-N and HisF-N) and β-strands 5 (in
HisA-C and HisF-C). These structural data suggest that
HisA and HisF evolved by tandem duplication and fusion
of a gene encoding an ancestral half-barrel comprising
only four (βα) units. To test this hypothesis, HisF-N and
HisF-C were produced recombinantly in E. coli, purified
and characterized (Höcker et al., 2001). Separately,
HisF-N and HisF-C are proteins with well-defined secondary and tertiary structures, but are catalytically inactive. Upon co-expression in vivo or joint refolding in vitro, HisF-N and HisF-C assemble to the catalytically fully
active HisF-NC complex. These findings support the notion that HisA, HisF and probably a large fraction of the
known (βα)8-barrels evolved from an ancestral half-barrel (Figure 4). This half-barrel was probably not a
monomeric protein. Instead, in order to shield the hydrophobic face of the putative half-barrel from the solvent, it may have formed homo-dimers. Along these
lines, isolated HisF-N and HisF-C molecules form
oligomers, predominantly of the (HisF-N)2 and (HisF-C)2
type (Höcker et al., 2001).
Conclusions and Outlook
The present post-genomic era offers the unprecedented
opportunity for comprehensive comparisons of protein
folds and functions. Such comparisons help to understand the evolution of enzymes, which is important for
both basic and applied research. The presented comparisons of both sequences and structures clearly support a common evolutionary origin of a large fraction of
the known (βα)8-barrel enzymes, which are used by nature to catalyse numerous and quite diverse biochemical
reactions. Since few amino acid exchanges can be sufficient to interconvert the catalytic activities of (βα)8-barrels, this fold appears to be particularly suited for enzyme design. Finally, the surprising finding that
(βα)8-barrels were derived from ‘half-barrels’ will motivate the search for ancestral domains within other apparent single-domain protein folds (Petsko, 2000; Gerlt
and Babbitt, 2001).
Acknowledgements
We thank Professor Kasper Kirschner for discussions and comments on the manuscript. Work in the authors’ laboratories is
supported by the Deutsche Forschungsgemeinschaft (grants to
R.S. and M.W.) and the Deutsche Bundesstiftung Umwelt (a
grant to R.S.). M.H.-S. is a fellow of the Hans-Böckler-Stiftung.
1319
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