230
Definitions of enzyme function for the structural genomics era
Patricia C Babbitt
Questions are being asked about how enzyme function is
described at the molecular level and the strengths and
weaknesses of the EC system for this purpose. A new approach
to describing enzyme function has been proposed that might
improve our capabilities for functional inference for members of
enzyme superfamilies.
Addresses
Departments of Biopharmaceutical Sciences and Pharmaceutical
Chemistry, University of California, 513 Parnassus Street, San Francisco,
CA, 94143-0446, USA
e-mail: babbitt@cgl.ucsf.edu
Current Opinion in Chemical Biology 2003, 7:230–237
This review comes from a themed section on
Biocatalysis and biotransformation
Edited by Tadhg Begley and Ming-Daw Tsai
1367-5931/03/$ – see front matter
ß 2003 Elsevier Science Ltd. All rights reserved.
DOI 10.1016/S1367-5931(03)00028-0
Abbreviations
CMLE carboxymuconate lactonizing enzyme
EC Enzyme Commission
MLE muconate lactonizing enzyme
PDB Protein Databank
Introduction
The ability to predict functions for the protein products of
sequenced genomes is required for understanding the
relationships between structure and function, applying
that knowledge to important problems in protein engi-
neering and drug design, and for understanding the
molecular basis of disease. Yet, although large numbers
of genomes have been solved and annotated, many pre-
dicted protein products of unknown function remain.
Complicating the annotation problem, a still unknown
but probably significant level of misannotation exists
across the sequence databases [1,2,3
,4,5], further com-
promising our ability to understand protein function at
the molecular level. As the structural genomics projects
move into high gear [6–9], we can expect increasing
numbers of three-dimensional protein structures to
become available whose functions are either uncertain
or even entirely unknown. The promise of structural
genomics will be blunted without more effective
approaches for predicting protein function from sequence
and structural information. For protein products with
insufficient similarity to proteins of characterized func-
tion, functional inference from sequence and structure
remains a difficult problem.
Current Opinion in Chemical Biology 2003, 7:230–237
This review focuses on some of the problems associated
with describing the molecular functions of enzymes and
relating those descriptions to sequence and structural
information in a way that is useful for functional infer-
ence. We describe recent large-scale attempts to correlate
sequence and structural information with enzyme func-
tion and cite a few examples of individual enzyme super-
families whose study has provided special insight into the
problem. In the context of these observations, the current
system for describing enzyme function, the Enzyme
Commission (EC) system, is evaluated. Finally, a new
approach to describing enzyme function is proposed that
may be useful for improving our capabilities for functional
inference at the molecular level.
Evaluation of the EC system for describing
enzyme function
In the EC system, enzymes are named according to the
overall transformations they perform. Each enzyme name
is associated with a four digit numerical code describing
each distinct transformation [10]. The Nomenclature
Committee of the International Union of Biochemistry
and Molecular Biology (IUBMB) updates the system
regularly using specific rules that have been developed
for naming enzymes and assigning them EC numbers (see
http://www.chem.qmul.ac.uk/iubmb/enzyme/). The EC
system now describes most of the enzymes known,
although as increasingly large numbers of new enzymes
are discovered in the genome projects, the number of
enzymes without an EC designation continues to rise.
The EC system represents a hierarchy of functional
classification in which each digit of the four-digit system
represents a different level of granularity in describing
chemical function. The first digit describes an overall
class of enzyme reaction (e.g. transferases, hydrolases,
isomerases), whereas the second and subsequent digits
indicate the subclass, sub-subclass, and specific serial
number assigned to each individual enzyme. For each
of the six major EC classes (first digit), subclasses and sub-
subclasses may have somewhat different meanings. For
example, in class 1 (oxidoreductases), the second (sub-
class) digit describes the substrate type upon which the
EC 1. enzymes act; for class 5 enzymes (isomerases), the
subclass second digit describes different general types of
isomerases (e.g. EC 5.1, isomerases; EC 5.2, racemases
and epimerases; EC 5.3, intramolecular isomerases).
Because of its long-term use as a gold standard and its
ease of use in computational efforts, the EC system is
used almost universally to describe enzyme function in
databases of sequence, structural and metabolic pathway
information.
www.current-opinion.com
 Definitions of enzyme function for the structural genomics era Babbitt 231

Despite these significant advantages, the EC system also Figure 1

has several disadvantages for use in genome-era analyses
[3
,11]. For example, EC designations presume a 1:1:1 NH2 OH
relationship between gene, protein, and reaction, which Melamine
N N N N
does not take into account the fact that enzymes can have
deaminase
more than one function or that oligomeric proteins gen- H2N N NH2 H 2N N NH2
erated from the protein products of different genes may
perform a single function. Perhaps the most important
disadvantage of the EC system for describing function in Cl OH
the era of structural genomics is that it does not provide N N
Atrazine
N N
any information about the mechanism, especially in a way chlorohydrolase
that can be effectively associated with sequence or struc- N N N N N N
tural patterns of conservation. Although it has been noted Current Opinion in Chemical Biology
that similarity among sequences is strongly correlated
with similarities in mechanism, this is most frequently Chemical reactions of melamine deaminase and atrazine
so at the level of a common partial reaction, rather than at chlorohydrolase.
the level of an overall transformation such as described by
the EC system [2,11–14,15

]. In effect, because the
underlying conceptual framework of the EC system catalyzing the o-succinylbenzoate synthase reaction across
was developed before sequence or structural information several bacterial species is generally well below the 40%
was available, it is not well suited to mapping mechan- identity cutoff [20].
istically important functional characteristics to enzyme
structure, particularly in terms of families and superfa- The correlation between EC number and sequence/
milies of homologous proteins. structure conservation below the 30–40% sequence iden-
tity cutoff is much more problematic. This represents a
The consequences of this deficit can be evaluated by level generally associated by structural biologists with
examining the results of several recent attempts to cor- superfamily classification [21]. At the superfamily level,
relate enzyme functions to families and superfamilies of where the member proteins have often diverged to per-
protein sequences using the EC system. At high levels of form different overall functions, EC families often do not
sequence similarity, EC numbers correlate well, as might correlate well with structural families, suffering from two
be expected. Here, descriptions of overall chemical reac- general problems: first, the EC system may cluster struc-
tions as reflected in enzyme names and their associated turally dissimilar proteins as functionally similar; and
EC numbers can usually be used for transfer of functional second, the EC system may cluster structurally similar
annotation in highly similar proteins. In a recent assess- proteins as functionally dissimilar. Both pose significant
ment of functional variation across the members of homo- problems for transfer of functional annotation.
logous enzyme superfamilies represented in the Protein
Data Bank (PDB), Todd et al. [16

] found, in general An early example of the first problem is provided by the
agreement with others [3
], that even variation in the enzymes muconate lactonizing enzyme (MLE) and car-
fourth digit of the EC number is rare above a sequence boxymuconate lactonizing enzyme (CMLE), whose reac-
identity threshold of 40%. However, exceptions to this tions are illustrated in Figure 2. These two enzymes,
rule are prevalent, with one recent report describing the
sequences of two enzymes that perform different overall
Figure 2
transformations, melamine deaminase and atrazine chloro-
hydrolase (Figure 1), to be 98% identical [17]. Consistent
−
with this example, other groups have found the correla- O2C
CO2−
CO2−
tion between the level of sequence similarity and the CO2−
CO2−
level of EC classification similarity to be somewhat pro-
blematic, particularly with respect to the unevenness of MLE EC 5.5.1.1 EC 5.5.1.2 CMLE
the correlations [18,19]. According to these studies, reli-
able annotations are difficult to achieve using simple
CO2− CO2−
sequence identity cutoffs. Numerous other examples O O
O O
can be cited from the general literature in which sequence −
H O2C
identities >40% represent proteins with different EC
numbers and thus, different overall functions. Conver- Current Opinion in Chemical Biology
sely, homologs with much lower sequence identities
sometimes have the same or very similar EC codes. Chemical reactions of muconate lactonizing enzyme (MLE) and
For example, the overall sequence identity of the enzyme carboxymuconate lactonizing enzyme (CMLE).

www.current-opinion.com Current Opinion in Chemical Biology 2003, 7:230–237

232 Biocatalysis and biotransformation
members of the catachuate and protocatechuate bran-
ches, respectively, of the b-ketoadipate pathway, have
nearly identical EC numbers, differing only in the last
digit, which in this case denotes a difference only in
substrate specificity. The similarities in their overall
reactions as well as their analogous roles in parallel
branches of the same metabolic pathway presumably
led to their receiving nearly identical EC designations.
Because these numbers were assigned before the deter-
mination of their sequences, it was not possible to
ascertain whether or not they were structurally related.
Once their sequences were determined, however, it was
trivial to deduce that they evolved not only from dif-
ferent superfamilies but from different fold classes as
well [22]. For MLE and CMLE, it is clearly incorrect to
infer structural similarity from similarities in EC num-
ber. Thus, this example provides a caution for the
general inference of structural similarity on the basis
of common function. In MLE and CMLE, nature devel-
oped different structural (and mechanistic) strategies to
perform essentially the same overall reaction. While it is
difficult to assess how widespread this problem may be,
several large-scale studies have recently provided addi-
tional examples of this type of disconnection between
EC designations and protein superfamilies [23,24,25

].
In one of these studies, Galperin and co-workers [23]
found many enzymes without detectable sequence simi-
larities to one another for 105 EC numbers. In 34 of the
cases examined, these enzymes could be shown to
belong to different folds, similar to the MLE/CMLE
example. As the sequence and structure databases con-
tinue to grow, we expect that many additional examples
of such ‘analogous’ (but not homologous) enzymes will
be described.
Perhaps more problematic for transferring functional
information from sequence or structure relationships is
the second problem described above, that EC designa-
tions often differ for structurally similar proteins. In two
large-scale studies focusing on all of the protein super-
families in the PDB [16

] and, in greater detail, on the
superfamilies on the (a/b)8 (TIM barrel) fold [25

],
Thornton and colleagues found many superfamilies con-
taining more than one EC designation, even at the level
of the first digit. Of the 167 protein superfamilies studied
from the PDB, nearly half showed variation in their EC
classification; for 22 of these superfamilies, the EC des-
ignation was not conserved at any level. Extensive details
of this study are available at http://www.biochem.ucl.ac.
uk/bsm/FAM-EC/. In the study on (a/b)8 barrels, Nagano
et al. [25

] found that 61 EC numbers are represented in
the 21 superfamilies described. One of these superfami-
lies, the FMN-oxidoreductase/PP-binding proteins, con-
tains enzymes representing four of the six primary EC
classes! Moreover, when homologous sequences were
added into their analyses, the number of functions asso-
ciated with a given (a/b)8 superfamily often doubled.
Current Opinion in Chemical Biology 2003, 7:230–237
Additional evidence for disparities between EC func-
tional class and structural similarity has been recorded
by other investigators [24,26
].
How should we define enzyme function at
the superfamily level?
If the EC system does not map well to the structural
similarities seen at the superfamily level, what alterna-
tives can be suggested? It would seem that development
of more structurally contextual mappings between struc-
ture and function requires a better conceptual under-
standing of how conserved elements of function explicitly
map to conserved elements of structure. Several recent
studies have looked in detail at this problem, and from
these investigations, several theoretic models of protein
evolution have emerged [15

]. Because these models
have been described in some detail elsewhere, including
in the companion paper in this issue by Gerlt and Raushel
[27], only two are described briefly here.
The first model, based originally on the work of Horowitz
[28,29], can be termed ‘substrate-constrained’ and
describes the case in which substrate specificity is con-
served in divergent evolution. This model predicts that
the conserved elements of structure that can be mapped
to conserved elements of function are associated with the
ability to bind common ligands. A recent study of
enzymes in Escherichia coli suggests that the occurrence
of this model may be relatively uncommon [30]. The
second model, termed ‘chemistry-constrained evolution’,
builds on the earlier observations of Petsko et al. [31], and
predicts that conserved elements of structure that can be
mapped to conserved elements of function are chemical
capabilities (e.g. a specific partial reaction or chemical
capability) [2,12–14,15

,27]. Other investigators have
described related concepts for the association of function
and structure through evolutionary divergence and
detailed the prevalence of the ‘chemistry-constrained’
model in genomes that have been examined [11,16

,
25

,30,32,33
].
We and our collaborators have examined the chemistry-
constrained model using specific superfamilies, including
the enolase [34,35], crotonase [36], haloacid dehalogenase
[37], vicinal oxygen chelate [38], and amidohydrolase
[27] superfamilies, and discussed the importance of this
model for developing new ways of describing enzyme
function likely to be useful for functional inference. Many
other superfamilies that appear to follow one (or some-
times more) of these evolutionary models have been
described.
A lesson that has emerged from such studies, particularly
with respect to the chemistry-constrained model of super-
family evolution, is that describing enzyme function in
terms of the overall transformation, such as with the EC
designations, is not very useful for prediction of function
www.current-opinion.com
 Definitions of enzyme function for the structural genomics era Babbitt 233

Table 1 Figure 3

EC numbers and chemical capabilities associated with some

members of the enolase, crotonase and haloacid dehalogenase (a) 10
superfamilies. Cu++ATPase.Ec LDTVVF D KTGTLTEG
Cu++ATPase.Hs VKVVVF D KTGTITHG
Superfamily Fundamental partial reaction/chemical capability
Ca++ATPase.At ATTICS D KTGTLTTN
Enolase Metal-dependent abstraction of a-protons of Urf.Mj KVAIVF D SAGTLVKI
carboxylic acids to form stabilized enolate PhosSerPhos.Hs ADAVCF D VDSTVIRE
intermediates 2-DO-6-PPhos.Sc VDLCLF D LDGTIVST
EC number Overall reaction DL-Gly-3-Phos.Sc INAALF D VDGTIIIS
4.2.1.6 Galactonate dehydatase Phosphon.Pa LQAAIL D WAGTVVDF
4.2.1.11 Enolase Phosphon.St IHAVIL D WAGTTVDF
4.2.1.40 Glucarate dehydratase Phosphon.Bc IEAVIF D WAGTTVDY
4.3.1.2 Methylaspartate ammonia-;yase PhosGlycolPhos.Rs MPGVVF D LDGTLVHS
5.1.2.3 Mandelate racemase NtermDom.IGPD.Pp VQALLL D MDGVMAEV
5.5.1.1 Muconate lactonizing enzyme B-PhosGlucoMut.Ll FKAVLF D LDGVITDT
6.2.1.26 o-Succinylbenzoate-CoA synthase HaloAcidDehal.PspYL IKGIAF D LYGTLFDV
NtermDomEpoxHyd.Hs LRAAVF D LDGVLALP
Crotonase Stabilization of oxyanion intermediates derived EnolasePhos.Ko IRAIVT D IEGTTSDI
from thioesters
EC number Overall reaction
3.1.2.4 3-Hydroxyisobuyryl-CoA hydrolase (b)
3.4.21.92 Atp-dependent Clp protease
3.8.1.6 4-Chlorobenzoyl-CoA dehalogenase
4.1.1.41 Methymalonyl-CoA decarboxylase
4.1.3.36 Naphthoate synthase
4.2.1.17 Enoyl-CoA hydratase (crotonase)
5.3.3.- D3,5,D2,4-dienoyl-CoA isomerase D10

Haloacid Hydrolysis, phosphoryl group transfer via

dehalogenase hydrolytic nucleophilic substitution
3.1.3.3 Phosphoserine phosphatase
3.1.3.15 Histidinol phosphatase
3.11.1.1 Phosphonatase
3.1.3.18 Phosphoglycolate phosphatase
3.8.1.2 Haloacid dehalogenase
5.4.2.6 b-Phosphoglucomutase

of unknown reading frames with no close homologs in the

sequence databases. Thus, if the closest homolog for a
newly sequenced protein has diverged to perform a
different overall function than that of the unknown
sequence in question, annotation transfer on the basis
of this overall reaction will be wrong. Examples of super-
families that illustrate this problem are provided in
Table 1, which shows that several different overall reac-
tions (as exemplified by their EC numbers) can be
associated with a given superfamily. Yet for each super-
family set, all of the homologous member structures can
be explicitly associated with a single fundamental che-
mical capability. For one of these superfamilies, the
haloacid dehalogenase superfamily, the structural corre-
lates to the fundamental chemical capability are given in
Figure 3. Figure 3a shows the conserved sequence motif
that contains the aspartic acid (labeled as D10 in Figures
3a and 3b) that functions as an active site nucleophile in
the C–X, P–C and P–O bond cleavage reactions catalyzed
by members of the superfamily [37]. Figure 3b shows a
superposition of several conserved active site residues,
including the critical aspartic acid nucleophile, for some
structurally characterized members of the superfamily.
Members of this superfamily are so divergent that their
Conserved motifs important for function in the haloacid dehalogenase
superfamily. (a) Multiple sequence alignment showing the motif
containing the important active site nucleophile (labeled as D10 in the
alignment) common to the fundamental chemistry of members of the
haloacid dehalogenase superfamily. Modified from [37] with permission
(copyright, Biochemistry 1998, from which sequence accession
numbers can be obtained. (b) Structural superposition showing several
members of the haloacid dehalogenase superfamily. The active site
nucleophile designated in Figure 3a is also labeled as D10 in the
superposition. Structures shown are haloacid dehalogenase from
Xanthobacter autrophicus (white), PDB 1qq5 [47];
phosphonoacetaldehyde hydrolase from Bacillus cereus (cyan), PDB
1fez [39]; phosphoserine phosphatase from Methanococcus jannaschii
(green), PDB 117min [48]; probable phosphatase from Haemophilus
influenzae (magenta), PDB 1kle [49]; b-phosphoglucomutase from
Lactococcus lactis (yellow), PDB 1lvh; and polynucleotide kinase from
T4 phage (orange), 1ltq [50]. Structural superposition and visualization
for Figures 3 and 4 were generated using Chimera [51], available from
http://www.cgl.ucsf.edu/chimera.
sequences cannot easily be aligned, except around the
conserved active site residues whose sidechains are
shown in Figure 3b (see [39], for a sequence alignment
showing all of these active site motifs).

www.current-opinion.com Current Opinion in Chemical Biology 2003, 7:230–237

234 Biocatalysis and biotransformation
The value of structure–function mapping for
developing new definitions of enzyme
function
From the ideas described here emerges a general
approach for functional annotation of new superfamily
members. First, sequence and structural relationships at
the superfamily level should be determined and con-
served structural characteristics identified from multiple
alignments of sequence and structure. Next, the partial
reactions/catalytic capabilities of functionally character-
ized members of the superfamily should be deduced and
those that are common to all members of the superfamily
identified. Explicit mappings can then be made between
conserved elements of structure and conserved elements
of function. From this information, the model or models
of protein evolution that apply to the superfamily can be
identified. If the superfamily follows the ‘substrate-con-
strained’ model, new superfamily sequences are likely to
bind the specific ligand (or moiety of a ligand) bound by
other members of the superfamily. If the superfamily
follows the ‘chemistry-constrained’ model, a new super-
family sequence can be expected to perform the partial
reaction/chemical capability associated with the other
members of the superfamily, even though it may perform
a very different overall reaction and bind ligands different
from those bound by any other member of the super-
family. For one such superfamily, the enolase superfam-
ily, the usefulness of this approach has recently been
expanded for in vitro protein engineering (described in
the companion paper in this volume by Gerlt and Raushel
[27]) and for finding structurally distant superfamily
members using only the information from the conserved
active site architecture.
Figure 4
Structural superposition showing conserved active site residues of several members of the enolase superfamily. Structures shown are mandelate
racemase from Pseudomonas putida (white), PDB 1mdr [52]; muconate lactonizing enzyme from P. putida (cyan), PDB 1muc [53]; galactonate
dehydratase from E. coli (green) [54]; o-succinylbenzoate synthase from E. coli (magenta), PDB 1fhv [55]; enolase from Saccharomyces cerevisae
(yellow), PDB 1ebh [56]; and b-methylaspartate ammonia-lyase from Clostridium tetanomorphum (orange), PDB 1kcz [57].
Current Opinion in Chemical Biology 2003, 7:230–237
In this latter experiment, we asked whether the active
site residues associated with the fundamental partial
reactions common to all enolase superfamily members,
abstraction of a proton a to a carboxylate (see Babbitt et al.
[34] for details), could provide sufficient information to
find homologous structures in available databases. Simi-
lar in concept to several other published approaches
using active site templates to find structural homologs
[40–43], this experiment used only the information from
the positions of the a carbons and side-chain centroids for
five active site residues conserved across all of the
divergent superfamily members (Figure 4). Using the
SPASM algorithm [44], several enolase superfamily
member templates were generated and used to search
the PDB for active site homologs. The results showed
that the best of these templates could find all of the other
divergent superfamily members with both high specifi-
city and sensitivity (EC Meng and PC Babbitt, unpub-
lished results), providing additional support for the
notion that the tight coupling between conserved func-
tional characteristics and conserved structural character-
istics in enzyme superfamilies represents a fundamental
property of those enzymes. Definition of such structure–
function paradigms provides a powerful basis for infer-
ence of function, especially for ‘chemistry-constrained’
superfamilies.
Looking forward: problems and
unanswered questions
Although the concepts associated with the structure–
function paradigm described here have been shown to
be useful for functional inference and analysis for the
small number of enzyme superfamilies upon which they
www.current-opinion.com
 Definitions of enzyme function for the structural genomics era Babbitt 235
have been tested thus far, it will be important to evaluate
this approach for functional inference on many more
examples. To this end, we have begun a collaboration
with the UCSF Resource for Biocomputing, Informatics,
and Visualization to develop a Structure–Function Link-
age Database to capture superfamily information in terms
of the sequences, structures and overall and partial reac-
tions for a large number of enzyme superfamilies. This
effort is also intended to facilitate the development of
new enzyme function descriptors that are appropriate for
mapping structure to function and that can be handled
computationally. However, for such definitions to be
useful beyond the limits of the investigation of detailed
molecular function, it will be important that they be
integrated with computationally tractable descriptions
of higher levels of organization (e.g. overall chemical
function, metabolic paths/networks, cells and organisms).
At the next highest level of organization for enzyme
function, metabolic pathways, the Gene Ontology system
[45] and other computational resources for functional
analysis all use the EC system effectively. Consistent
with this observation, in a recent attempt at exploring the
evolution of enzymes in metabolism using a network
approach, Alves et al. [46] found the EC system to be
useful for connecting sequence information to substrates
and products. Thus, although the EC system can be
inappropriate for mapping conserved functional charac-
teristics to conserved elements of structure in divergent
enzyme superfamilies, it remains essential for describing
overall chemical functions and relating those overall
functions to higher-order biological organization. Devel-
opment of a new system based on structure–function
mappings at the finer level suggested here can best be
envisaged as an addition to, not a replacement for, the EC
system. Ultimately, such a system would be useful for
structure–function mapping at the individual enzyme,
family and superfamily levels.
Conclusions
Although the EC system is useful for describing
enzyme function at the level of overall reactions, these
definitions are problematic for correlating structure and
function at a finer level. Recent research has shown that
more refined definitions of enzyme function described
at the level of the partial chemical reactions that make
up the overall reactions described by the EC provide
more useful mappings between structural and func-
tional elements conserved across superfamilies. Devel-
opment of a new system for describing enzyme function
at this level of granularity is needed for the inference
of functional properties from sequence and structural
similarities.
Acknowledgements
We thank Elaine C Meng, PhD, for making Figures 3b and 4 and for helpful
discussions. The research in the author’s laboratory is supported by NIH
GM60595 and NC RR01081.
www.current-opinion.com
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