Evolution of enzyme superfamilies
Margaret E Glasner1, John A Gerlt2 and Patricia C Babbitt1
Enzyme evolution is often constrained by aspects of catalysis.
Sets of homologous proteins that catalyze different overall
reactions but share an aspect of catalysis, such as a common
partial reaction, are called mechanistically diverse
superfamilies. The common mechanistic steps and structural
characteristics of several of these superfamilies, including the
enolase, Nudix, amidohydrolase, and haloacid dehalogenase
superfamilies have been characterized. In addition, studies of
mechanistically diverse superfamilies are helping to elucidate
mechanisms of functional diversification, such as catalytic
promiscuity. Understanding how enzyme superfamilies evolve
is vital for accurate genome annotation, predicting protein
functions, and protein engineering.
Addresses
1
Department of Biopharmaceutical Sciences, University of California,
San Francisco, California 94143, USA
2
Departments of Biochemistry and Chemistry, University of Illinois,
Urbana, Illinois 61801, USA
Corresponding author: Babbitt, Patricia C (babbitt@cgl.ucsf.edu)
Current Opinion in Chemical Biology 2006, 10:492–497
This review comes from a themed issue on
Mechanisms
Edited by Carol A Fierke and Dan Herschlag
Available online 28th August 2006
1367-5931/$ – see front matter
# 2006 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.cbpa.2006.08.012
Introduction
How has evolution produced an incredible variety of
enzymatic activities from a limited number of protein
folds? Both large-scale genomic surveys and studies of
individual protein superfamilies have demonstrated that
aspects of catalysis are often conserved between evolutio-
narily related proteins that catalyze different overall reac-
tions [1
,2,3]. This observation has led to a definition of
protein superfamilies based not only on structural con-
servation, but also on functional conservation. Mechan-
istically diverse superfamilies are sets of evolutionarily
related proteins that utilize a common mechanistic attri-
bute, such as a specific partial reaction, intermediate, or
transition state to catalyze different overall reactions with
different substrate specificities. Of superfamilies that are
classified only on the basis of structure, about one-third are
predicted to be mechanistically diverse [3,4]. Because
these studies were restricted to structurally characterized
enzymes, they may underestimate the true number of
Current Opinion in Chemical Biology 2006, 10:492–497
mechanistically diverse superfamilies in the whole protein
universe [5]. Understanding the evolution of these super-
families has implications for many areas of biology, includ-
ing genome annotation, function prediction and protein
engineering. In this review, we discuss evolutionary
themes in mechanistically diverse superfamilies and
some mechanisms that might drive their functional
diversification.
Evolutionary themes of mechanistically
diverse superfamilies
A small number of mechanistically diverse superfamilies
have been systematically investigated to characterize
common sequence, structural and mechanistic elements,
and to determine the range of catalyzed reactions. Some
representative superfamilies are shown in Table 1, and
others are described elsewhere [2,3]. Here, we focus on
four that have been reviewed recently (Figure 1).
The enolase superfamily is one of the best-characterized
mechanistically diverse superfamilies and is known to
catalyze at least 14 different reactions, including cycloi-
somerization, racemization and b-elimination of water or
ammonia [6]. All enolase superfamily proteins share a
common partial reaction in which a base abstracts a proton
a to a carboxylate to form an enolate anion intermediate
that is stabilized by a divalent metal ion. The conserved
catalytic residues are arranged around the inside of a
modified (b/a)8-barrel, and a second domain caps the
active site. The metal-coordinating residues are highly
conserved, but the identity and position of the general
base that abstracts the proton is variable. The capping
domain provides substrate specificity for some enolase
superfamily enzymes, but its lack of conservation among
the o-succinylbenzoate synthases suggests that its pri-
mary role might be solvent exclusion in some cases [7].
Several reviews have discussed the mechanism and pre-
valence of the Nudix (nucleotide diphosphates linked
to other moieties, X) superfamily [8
,9,10]. Members of
this superfamily hydrolyze nucleoside diphosphates
linked to phosphate, other nucleosides or sugars by
nucleophilic substitution at phosphorus or carbon. The
Nudix superfamily is characterized by a highly conserved
loop–helix–loop motif that appears to be optimized
for polyphosphate and metal binding. Although some
of the metal-binding residues are conserved, slight varia-
tions in the positions of additional metal-coordinating
residues, the number of required magnesium ions, and
the position and identity of the general base determine
the precise position of nucleophilic attack on the
substrate.
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 Evolution of enzyme superfamilies Glasner, Gerlt and Babbitt 493

Table 1

Examples of mechanistically diverse superfamilies.

Superfamily Fundamental common chemistry Representative superfamily members Refs

a
Enolase Proton abstraction a to a carboxylate to form Enolase [6]
metal-ion stabilized enolate intermediates Mandelate racemase
o-Succinylbenzoate synthase
Haloacid dehalogenase a Formation of covalent enzyme-substrate b-Phosphoglucomutase [12
]
intermediates via a conserved aspartate Phosphonoacetaldehyde hydrolase
Cu2+/H+-ATPase
Crotonase a Utilization of an oxyanion hole to stabilize 4-Chlorobenzoyl-CoA dehalogenase [2,35]
enolate-anion intermediates generally derived Methylmalonyl-CoA decarboxylase
from thioesters ClpP Protease
Amidohydrolase a Metal-assisted hydrolysis Urease [11
,36]
Phosphotriesterase
Adenosine deaminase
Vicinal oxygen chelate a Stabilization of oxyanion intermediates formed Dioxygenase [2,37]
by metal-dependent catalysis Glyoxylase I
Methylmalonyl CoA epimerase
N-acetylneuraminate lyase a Utilization of a protonated Schiff base as an N-acetylneuraminate lyase [38–40]
electron sink Dihydrodipicolinate synthase
2-keto-3-deoxygluconate aldolase
Terpene cyclase a Carbocation formation by elimination of Lanosterol synthase [28,41]
pyrophosphate or protonation of an Taxadiene synthase
epoxide or olefin Squalene-hopene cyclase
b
tDBDF Hydride transfer between NAD(P) and FAD Glutathione reductase
NADH ferredoxin reductase
Flavin-containing monooxygenase
Nudix Metal-assisted hydrolysis 8-oxo-dGTP pyrophosphohydrolase (MutT) [8
,9,10]
mRNA-50 -decapping enzyme
GDP-mannose-mannosyl hydrolase
a
Included in the Structure–Function Linkage Database [13

,42].
b
S Ojha, E Meng, PC Babbitt, unpublished data.

Seibert and Raushel recently reviewed structural and
catalytic diversity of the (b/a)8-barrel amidohydrolase
superfamily [11
]. Most members of this superfamily
catalyze amide or ester hydrolysis, but one catalyzes an
isomerization reaction. One or two Zn2+, Ni2+ or Fe2+ ions
are used to activate a water molecule for nucleophilic
attack, and most metal-coordinating residues are con-
served. However, seven different variations in other
metal-coordinating residues have been observed in 16
different crystal structures, resulting in variations in the
identity, number and position of the metal ions. These
catalytic residues are at the ends of the b strands, and
substrate diversity appears to be determined by variations
in the lengths and conformations of loops linking the
strands and helices of the barrel.
The haloacid dehalogenase (HAD) superfamily mainly
catalyzes phosphotransferase reactions resulting in clea-
vage of C–Cl, C–P, CO–P or PO–P bonds [12
]. A
magnesium bound to a conserved metal-binding motif
provides orientation and charge shielding for a two-step
reaction in which a conserved aspartate acts as a nucleo-
phile, then as a leaving group. The catalytic core includes
other catalytic residues, some of which are specific to
particular enzymes in the superfamily. Because the cat-
alytic core is essentially nonspecific and open to solvent,
several strategies have evolved to confer specificity. Two
subgroups of the HAD superfamily have different a-
helical domains that cap the catalytic core to exclude
solvent and provide additional catalytic residues and
substrate specificity. Lacking an accessory domain, mem-
bers of a third subgroup function as nonspecific phospha-
tases, bind large substrates such as tRNA and proteins
that effectively close the active site, or form oligomeric
proteins in which active sites are at the interface and are
shielded by adjacent subunits.
Although relatively few mechanistically diverse super-
families have been thoroughly characterized, these stu-
dies are proving useful for detecting misannotation in
public databases and predicting protein functions
[5,13

]. Several specialized databases will be useful for
expanding these studies to other superfamilies (Box 1).
By relating function to structure, SCOPEC and the
Catalytic Site Atlas are excellent starting points for iden-
tifying mechanistically diverse superfamilies, while
EzCatDB and MACiE offer a wealth of information about
enzyme reaction mechanisms [4,14
,15,16]. The Struc-
ture–Function Linkage Database combines this informa-
tion with sequences of uncharacterized proteins to derive
comprehensive analyses of several well-characterized
mechanistically diverse superfamilies [13

].

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494 Mechanisms

Figure 1

Representative reactions and conserved active site residues of several mechanistically diverse superfamilies. Metal ions are shown as spheres.
(a) Enolase superfamily proteins enolase (blue; 7ENL), mandelate racemase (green; 2MNR), and muconate lactonizing enzyme (purple; 1MUC).

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 Evolution of enzyme superfamilies Glasner, Gerlt and Babbitt 495

Box 1 Resources for studying mechanistically diverse superfamilies

1. Structure–Function Linkage Database (SFLD) [13

,42]

Description: The SFLD is explicitly designed to describe mechanistically diverse superfamilies. Assigns sequences from the NCBI
nonredundant database using Hidden Markov Models and literature references.
Features: Provides sequence alignments, catalytic residues, structures and step-by-step chemical reactions; can be searched by sequence or
partial chemical reaction.
Website: http://sfld.rbvi.ucsf.edu/

2. Catalytic Site Atlas (CSA) [14
]

Description: The CSA documents catalytic residues in structurally characterized proteins. Lists catalytic residues directly involved in catalysis
but excludes those that bind metal ions or cofactors.
Features: Provides sequence, structure and catalytic residues; also provides structural templates, which are geometric descriptions of active
site residues that can be used to predict functions of remote homologs and assess convergent evolution [43].
Website: http://www.ebi.ac.uk/thornton-srv/databases/CSA/

3. SCOPEC [4]
Description: SCOPEC maps Enzyme Classification (EC) numbers to domain structures.
Features: Lists EC numbers for structurally characterized proteins.
Website: http://www.enzome.com/databases/scopec.php

4. EzCatDB [15]
Description: EzCatDB classifies enzymes by catalytic mechanism and structure.
Features: Provides a unique hierarchy for classifying reactions based on reaction type, ligand group involved in catalysis, catalytic mechanism,
and catalytic residues/cofactors; includes some 3D models of reaction mechanisms.
Website: http://mbs.cbrc.jp/EzCatDB/ezcat.html

5. MACiE [16]
Description: MACiE is a database of reaction mechanisms, focusing on structurally characterized enzymes with well understood mechanisms.
Features: Shows step-by-step reaction mechanisms, including catalytic residues; mechanisms also shown as animations.
Website: http://www-mitchell.ch.cam.ac.uk/macie/

Mechanisms of functional diversification duplication before functional divergence, duplication

While chemical aspects of catalysis constrain the evolu-
tion of mechanistically diverse superfamilies, less is
known about mechanisms of functional diversification.
Generally, only a subset of the catalytic residues are
conserved. Mechanistic diversity arises from differential
placement of other catalytic residues, and substrate diver-
sity often arises from variation in loops and accessory
binding domains [6,8
,11
,12
]. Another mode of func-
tional diversification has been observed in the two dinu-
cleotide-binding domains flavoprotein (tDBDF)
superfamily, which uses NAD(P) and FAD to catalyze
oxidation/reduction reactions (S Ojha, E Meng, PC Bab-
bitt, unpublished data). All known substrates of this
superfamily are proteins or small molecules bound to
proteins. Reminiscent of a subset of the HAD super-
family discussed above, functional diversity in the
tDBDF superfamily evolved through diversification of
protein–protein interactions at this interface.
One hypothesis that explains functional diversification is
that new protein functions evolve through promiscuous
intermediates [1
,2,17–22]. Rather than requiring gene
may primarily allow optimization of different functions
of promiscuous evolutionary intermediates [23,24,25

].
Several lines of evidence support this hypothesis, includ-
ing the observation that a protein’s promiscuous activities
are often the biological activities of evolutionarily related
proteins and that engineered proteins are almost always
promiscuous for both their original and selected reactions
[1
,19–22,25

,26–28].
Several recent studies also support this hypothesis. Bridg-
ham et al. demonstrated that the ancestral sequence of
two hormone receptors was responsive to aldosterone
eons before aldosterone synthesis evolved, suggesting
that ancestral promiscuity for aldosterone binding was
exploited millions of years later [29]. In a study of the
enolase superfamily protein o-succinylbenzoate synthase
(OSBS), we determined that a subset of the OSBSs are
promiscuous for N-acylamino acid racemization.
Although some of these proteins are from species that
require OSBS activity, many are from species that lack
the pathway in which OSBS is an intermediate [7]. All
OSBSs that were predicted to have racemization activity

(Figure 1 Legend Continued) (b) Nudix superfamily proteins MutT (blue; 1PUN), diadenosine tetraphosphate hydrolase (green; 1KTG), and
GDP-mannose-mannosyl hydrolase (purple; 1RYA). A, adenosine; dG, 8-oxo-deoxyguanosine; G, guanosine. (c) Amidohydrolase superfamily
proteins phosphotriesterase (blue; 1EZ2), N-acetylglucosamine-6-phosphate deacetylase (green; 1O12), and adenosine deaminase (purple; 1add).
(d) Haloacid dehalogenase superfamily proteins haloacid dehalogenase (blue; 1QQ5), b-phosphoglucomutase (green; 1O03), and
phosphonoacetaldehyde hydrolase (purple; 1FEZ).

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496 Mechanisms
and have been experimentally characterized are promis-
cuous, and some have been shown to be part of a pathway
for amino acid racemization [30].
Other recent studies have added to the conceptual frame-
work of the catalytic promiscuity hypothesis. Tawfik and
colleagues examined promiscuity among products of pro-
tein engineering experiments and observed that, while
promiscuous activities improved more than 1000-fold,
parental activities decreased by threefold on average
[25

]. They suggest that native protein activities have
evolved to be robust to mutation, in part because muta-
tions that generate promiscuous activities often occur in
conformationally flexible loops [31]. This hypothesis is
intriguing, but some experiments fail to support it [32].
For example, a single point mutant in the enolase super-
family protein muconate lactonizing enzyme II endows it
with OSBS activity while slightly decreasing its native
activity, but the same mutation in another enolase super-
family protein, a dipeptide epimerase, makes it a poor
catalyst for both OSBS and its native activity [21].
Conclusions
The importance of chemical aspects of catalysis in
enzyme evolution is well established, and expanding
these studies to additional superfamilies will make sev-
eral contributions to biological understanding. First, ana-
lyzing mechanistically diverse superfamilies will lead to
better methods of genome annotation. For instance,
assigning proteins to a hierarchy of superfamilies (denot-
ing evolutionary relatedness) and families (denoting spe-
cific functions) will help avoid over-annotation problems
by only assigning specific functions to proteins that meet
stringent evidence criteria [13

]. Second, annotating
proteins at different levels in the hierarchy aids in iden-
tifying proteins of unknown function, and assigning these
proteins to mechanistically diverse superfamilies narrows
the potential range of reactions they are likely to catalyze.
This provides an avenue for predicting protein functions
through analysis of phylogeny, operon context, computa-
tional substrate docking, and experimental characteriza-
tion [7,30,33,34]. Finally, characterizing additional
mechanistically diverse superfamilies is likely to reveal
other mechanisms for functional diversification which will
be valuable for developing protein design methodologies
that more closely imitate natural protein evolution [1
].
Update
In SCOP, the N-acetylneuraminate lyase superfamily is
included in the structurally defined Class I aldolase family,
but it has not been known whether the entire Class I
aldolase family constitutes a mechanistically diverse
superfamily. Recently, Choi et al. established that several
other members of the Class I aldolase family belong in the
N-acetylneuraminate lyase superfamily because they
share very low but significant sequence similarity as well
as a common catalytic dyad consisting of the Schiff-base
Current Opinion in Chemical Biology 2006, 10:492–497
lysine and a general acid whose position, but not identity, is
conserved in the superfamily [44
].
Acknowledgements
This work was supported by National Institutes of Health Grant
GM60595 to PCB and Grants GM52594 and GM65155 to JAG. MEG is
supported by a Postdoctoral Fellowship in Informatics from the
Pharmaceutical Researchers and Manufacturers of America.
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