b-Lactam antibiotic resistance: a current structural perspective
Mark S Wilke1, Andrew L Lovering1 and Natalie CJ Strynadka
Bacterial resistance to b-lactam antibiotics can be achieved by
any of three strategies: the production of b-lactam-hydrolyzing
b-lactamase enzymes, the utilization of b-lactam-insensitive
cell wall transpeptidases, and the active expulsion of b-lactam
molecules from Gram-negative cells by way of efflux pumps. In
recent years, structural biology has contributed significantly to
the understanding of these processes and should prove
invaluable in the design of drugs to combat b-lactam resistance
in the future.
Addresses
Department of Biochemistry and Molecular Biology, and the Center
for Blood Research, University of British Columbia, 2146 Health
Sciences Mall, Vancouver, BC, Canada V6T 1Z3
Corresponding author: Strynadka, Natalie CJ
(natalie@byron.biochem.ubc.ca)
1
Mark S Wilke and Andrew L Lovering contributed equally to this review.
Current Opinion in Microbiology 2005, 8:525–533
This review comes from a themed issue on
Antimicrobials
Edited by Christopher Walsh and Malcolm GP Page
Available online 29th August 2005
1369-5274/$ – see front matter
# 2005 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.mib.2005.08.016
Introduction
The introduction of b-lactam antibiotics into the health
care system in the latter stages of World War II represents
one of the most important contributions to medical
science in recent history. Today, b-lactams remain the
most widely utilized antibiotics owing to their compara-
tively high effectiveness, low cost, ease of delivery and
minimal side effects. b-lactams target transpeptidase
enzymes that synthesize the bacterial cell wall. The
desirable attributes of this class of antibiotic arise from
the facts that these enzymes are localized to the outer
leaflet of the bacterial cytoplasmic membrane (i.e. are
relatively accessible) and that they are specific to bacteria
(with no functional or structural counterpart in the human
host). In a practical sense, the low cost of production of
b-lactam antibiotics allows for a wide availability; thus, it
is imperative that we preserve the power of this valuable
clinical resource.
How do b-lactam antibiotics work? Bacteria of all species
rely on a heavily cross-linked peptidoglycan layer (cell
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wall) for the preservation of cell shape and rigidity. This
cell wall is comprised of a basic repeating unit of an
alternating disaccharide — N-acetyl glucosamine and
N-acetyl muramic acid. The latter sugar in this disacchar-
ide is modified by a characteristic pentapeptide. This
varies amongst the Gram-negative and Gram-positive
species, but always terminates in two D-alanine residues.
The individual peptidoglycan units are produced inside
the cell, but their final cross-linking is catalyzed outside
the cytoplasmic membrane by a group of membrane-
anchored bacterial enzymes known as the cell-wall trans-
peptidases. In this cross-linking reaction, a peptide bond
is formed between the penultimate D-alanine on one
chain and the free amino end of a diamino pimelic acid
(Gram-negative) or an L-lysine (Gram-positive) residue
on the other chain. The linkage is formed with the
penultimate D-alanine, causing the terminal D-alanine
to be cleaved in the process.
Transpeptidase enzymes utilize an active site serine and
perform their catalytic cycle by way of an acylation/
deacylation pathway. b-lactam antibiotics efficiently inhi-
bit the bacterial transpeptidases, therefore these enzymes
are often termed penicillin binding proteins or PBPs.
They are able to do this owing to the stereochemical
similarity of the b-lactam moiety with the D-alanine–D-
alanine substrate. In the presence of the antibiotic, the
transpeptidases form a lethal covalent penicilloyl–
enzyme complex that serves to block the normal trans-
peptidation reaction. This results in weakly cross-linked
peptidoglycan, which makes the growing bacteria highly
susceptible to cell lysis and death.
As with most antimicrobial agents, b-lactams are rendered
inactive against bacteria by way of three primary mechan-
isms of resistance (Figure 1). The most common mechan-
ism is the production of enzymes that degrade or modify
the antibiotic before it can reach the appropriate target
site. In this case, the b-lactamase family of enzymes
degrade b-lactam antibiotics and are found widely dis-
seminated amongst Gram-positive and Gram-negative
bacteria. The second mechanism is alteration of the
antibiotic target site. In this case, the b-lactam-resistant
cell-wall transpeptidases perform this role; this is now a
major cause of resistance in several pathogens including
the problematic Gram-positive Staphylococcal and Strepto-
coccal species. The final mechanism is prevention of
access of the antibiotic to the target by way of altered
permeability or forced efflux. For example, this can be
performed by the MexA,B–OprM antibiotic efflux pump,
which is a major cause of resistance in Pseudomonas and in
other pathogenic Gram-negative species.
Current Opinion in Microbiology 2005, 8:525–533
526 Antimicrobials

Figure 1

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 b-Lactam antibiotic resistance Wilke, Lovering and Strynadka 527

In this review we describe seminal new discoveries in
which structural biology has provided new insights into
these b-lactam antibiotic resistance phenomena as well as
new strategies for drug development.
Antibiotic-modifying enzymes:
the b-lactamases
The b-lactamases confer significant antibiotic resistance
to their bacterial hosts by hydrolysis of the amide bond of
the four-membered b-lactam ring. These enzymes are
especially important in Gram-negative bacteria as they
constitute the major defense mechanism against b-lac-
tam-based drugs. The spread of b-lactamase genes has
been greatly exacerbated by their integration within
mobile genetic elements, such as plasmids or transposons,
which facilitate the rapid transfer of genetic material
between microbes. Even more ominous is the organiza-
tion of b-lactamase genes within integrons as part of
multi-drug resistance cassettes that bestow mechanisms
for resistance not only to b-lactams but also to other
antibiotic classes such as aminoglycosides, macrolides,
sulphonamides and chloramphenicol (for a recent review,
see [1]). Once expressed, b-lactamases are secreted into
the periplasmic space (in Gram-negative bacteria), bound
to the cytoplasmic membrane, or excreted (in Gram-
positive bacteria).
Structure and mechanism
The >470 b-lactamases known to date [2] are typically
organized into four classes (A to D) on the basis of
sequence similarity. Crystal structures are currently avail-
able for representatives of each class (for a recent review,
see [2]). Classes A, C and D share a similar fold and all
have a mechanism that involves creation of a serine
nucleophile by deprotonation of an active site serine with
a general base, nucleophilic attack of the b-lactam ring to
form an acyl-enzyme intermediate, and hydrolysis of the
intermediate using a general base activated water mole-
cule. The differences between the catalytic mechanisms
of the serine b-lactamase classes center around the type of
general base residues used in acylation and deacylation.
The class B b-lactamases are zinc metalloenzymes and
are completely distinct from the serine b-lactamases in
terms of sequence, fold and mechanism. There are three
subclasses of class B metallo-b-lactamases (B1 to B3).
Classes B1 and B3 are able to bind one or two zinc ions [3],
whereas the class B2 enzymes appear to be mononuclear
[4]. In the binuclear metallo-b-lactamases, the zinc ions
are proximal to each other and are separated by a bridging
hydroxide that has been proposed to be the attacking
nucleophile in b-lactam hydrolysis. The class B1 and B3
metallo-b-lactamases can also function as mononuclear
enzymes, in which a single zinc ion (that occupies the Zn1
site) coordinates the nucleophilic hydroxide; this
mechanism has been proposed to predominate in the
presence of substrate under physiological conditions
[5]. The crystal structure of a class B2 metallo-b-lacta-
mase (CphA from Aeromonas hydrophilia) has only been
published recently [4]. Its proposed catalytic mechan-
ism differs from the class B1 and B3 mononuclear
mechanisms in that the zinc ion occupies the Zn2 site,
a general base activates the nucleophilic water, and the
zinc ion forms a bond with the amine nitrogen of the
hydrolyzed b-lactam amide (Figure 2).
A new generation of b-lactamases
The b-lactamases are ancient enzymes that were rela-
tively rare until b-lactam antibiotics were introduced into
medicine and agriculture half a century ago [6]. The
widespread use of carbapenems, the monobactam aztreo-
nam, cephamycins and oxyimino-cephalosporins in the
past few decades has led to the evolution of a new
generation of b-lactamases, which have an extended sub-
strate spectrum (i.e. extended-spectrum b-lactamases or
ESBLs), as well as the development of novel carbapene-
mases and plasmid-mediated AmpC b-lactamases (for
recent reviews, see [2,7,8]). Common ESBLs include
varieties from the class A b-lactamases TEM, SHV and
CTX-M and the class D b-lactamase OXA. These
enzymes are typified by a broad substrate spectrum that
includes oxyimino-cephalosporins, aztreonam and, in the
case of some OXA and CTX-M enzymes, cefepime.
Recently, several CTX-M structures have been made
available including inhibitor-bound structures, which pro-
vide snapshots of two reaction cycle transition states, and
the acyl-enzyme intermediate, which can aid in the
design of inhibitors [9] (Figure 3). In addition, several
atomic resolution CTX-M structures demonstrate that
the enhanced ceftazidimase activity of these enzymes is a
result of the increased active site flexibility; however, this

(Figure 1 Legend) Structural depiction of proteins involved in b-lactam resistance. The proteins responsible for resistance are sub-divided
into the b-lactamase, PBP and efflux pump groups. Also shown are two of the repressors for resistance operons — BlaI and MexR (genes are
shown as solid arrows). The structures of the glycosyltransferase domain of the class A PBPs and the cytoplasmic protease domain of BlaR that
are not yet determined are replaced by blue and green solid shapes, respectively. The diagram also shows the interaction of b-lactams with
these subgroups — the b-lactam-dependent signaling of BlaR, ring hydrolysis by the b-lactamases, acylation of the PBPs (poor in PBP2x and 2a),
and proton antiport of the antibiotics by the efflux pump systems. The recently solved structure of S. pneumoniae PBP1b is used in place of the
closely-related resistance determinant PBP1a, and the structures of MexB and OprM are used here as models derived from the co-ordinates
of their respective homologues, AcrB and TolC (docked together manually). Details of the precise interaction of MexA with MexB–OprM remain
unknown, as does the nature of the effector responsible for terminating MexR repression. Abbreviations: gm +/ , Gram-positive/negative; IM,
inner membrane; OM, outer membrane; PG, peptidoglycan. CTXM9 and CphA are shown as representatives of the >40 available serine and
metallo-b-lactamase structures.

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528 Antimicrobials
Figure 2
The fold and active site of CphA bound by the substrate biapenem
(PDB code 1X8). (a) Ribbon representation of the CphA fold. (b) Stick
representation of several active site residues, colored by atom type
(C atoms, yellow; O, red; N, blue; and S, orange). Biapenem is
displayed with purple carbon atoms to distinguish it from the protein
carbons shown in yellow. A single catalytic zinc and a water molecule
are shown as dark grey and cyan spheres, respectively. Hydrogen
bonds are represented by green dashes.
increase in flexibility is at the cost of protein stability [10].
Carbapenemases are derived from classes A, B and D and
they provide resistance to carbapenems as well as to
oxyimino-cephalosporins and cephamycins. The class B
metallo-b-lactamase CphA (mentioned above) is a carba-
penamase. Its crystal structure in complex with the car-
bapenem substrate biapenem (Figure 2) has been
determined; this might prove useful in the design of
inhibitors or of non-hydrolyzable antibiotics [4].
Historically, combination therapies in which the action of
b-lactams is supplemented with b-lactamase inhibitors
Current Opinion in Microbiology 2005, 8:525–533
Figure 3
The fold and active site of cefoxitin-acylated CTXM9 (PDB code 1YMX).
(a) Ribbon representation of the CTXM9 fold. (b) Stick representation of
several active site residues, colored by atom type (C atoms, yellow; O,
red; N, blue; and S, orange). The covalently-bound cefoxitin is shown
with purple carbon atoms to distinguish it from the protein carbons
displayed in yellow. An active site water molecule is indicated with a
cyan sphere.
have successfully restored the antibiotic activity of
b-lactam drugs against resistant pathogens. Although
outside the scope of this review, several novel compounds
have been reported in the past two years that have
inhibitory activity against class A, B and C b-lactamases;
these molecules include peptides [11], succinic acid
derivatives [12], substituted penam sulfones [13], mer-
captomethyl-penicillinates [14], bridged bicyclic octa-
nones [15], thiophene-carboxy derivatives [16], and
tricylclic 6-methylidene penems [17].
Regulation of resistance
b-lactamase expression is often induced by b-lactams
through a novel regulation system that consists of the
repressor BlaI and the receptor BlaR (Figure 1). A com-
plete description of the induction process is currently
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 b-Lactam antibiotic resistance Wilke, Lovering and Strynadka 529
unavailable, but recent determination of the structures of
BlaI [18,19] and the extracellular PBP-like sensor
domain of BlaR [20–22] have characterized important
features of the b-lactamase regulation machinery.
BlaR is a transmembrane protein composed of an extra-
cellular sensor domain that is acylated by b-lactam anti-
biotics, a transmembrane domain (consisting of four
membrane-spanning helices) that transduces the b-lac-
tam-binding signal across the membrane, and an intra-
cellular zymogenic zinc metalloprotease domain that is
activated by autoproteolysis upon acylation of the sensor
domain. Interestingly, the b-lactam sensor domain of
BlaR resembles the b-lactamases in terms of its fold
(especially the class D b-lactamases) as well as its use
of a serine nucleophile to form an acyl-enzyme inter-
mediate. However, unlike the b-lactamases and akin to
the PBPs, the covalent b-lactam adduct is stabilized as it
appears to lack a functional general base residue capable
of deacylation. No significant conformational changes
accompany acylation of the sensor domain [21] and it
has been proposed that the large extracellular BlaR loop
designated L2 that is positioned adjacent to the sensor
domain in the BlaR receptor might mediate the b-lactam-
binding signal to the transmembrane domain through an
interaction with the BlaR sensor [23].
The cytosolic repressor BlaI forms a dimer and binds an
operator in the bla divergon that encodes BlaI, BlaR and the
b-lactamase structural gene. Once activated, the BlaR
metalloprotease domain elicits cleavage of BlaI within
its dimerization domain, which prevents operator binding
and permits b-lactamase expression. The BlaI monomer
consists of an amino-terminal winged helix DNA-binding
domain and a carboxy-terminal dimerization domain,
which are separated by a flexible hinge region. This flex-
ibility allows the BlaI dimer to not only bind the bla
operator but bind the operator sequence for the mec diver-
gon as well, allowing BlaR and BlaI to additionally mediate
the expression of PBP2a (the protein encoded by mecA)
[19]. PBP2a expression in methicillin-resistant Staphylo-
coccus aureus (MRSA) is controlled by a highly similar
regulation system that consists of the repressor MecI and
receptor MecR. Although many MRSA strains have MecI
deletions that inactivate PBP2a repression, these strains
appear to be co-regulated by BlaR–BlaI [24]. Indeed, the
bla or mec regulatory genes appear to be required for the
maintenance and expression of mecA, which suggests that
PBP2a expression incurs a fitness cost on the host [25].
Therefore, the bla and mec regulation machinery might
prove useful as targets for the design of inhibitors to counter
b-lactamase and PBP2a-mediated resistance, particularly if
further structural details are forthcoming. Alternatively,
the creation of non-b-lactam lead compounds that circum-
vent the activation of such regulatory mechanisms are a
logical step in inhibitor design, as highlighted recently by
Tondi et al. [16] and Zervosen et al. [26].
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Altered antibiotic targets: the cell wall
transpeptidases (PBPs)
PBPs are divided into two subgroups: low molecular
mass (LMM) and high molecular mass (HMM) enzymes.
The HMM enzymes are further subdivided into the
bifunctional class A enzymes (which possess a b-lac-
tam-insensitive transglycosylase domain and the tradi-
tional b-lactam-sensitive transpeptidase domain) and the
monofunctional transpeptidase class B enzymes. The
soluble LMM PBPs have no identified role in b-lactam
resistance and are primarily involved in carboxypeptidase
reactions and peptidoglycan trimming. Despite their low
sequence identity with the more essential HMM counter-
parts, the LMM PBPs have been a common vehicle for
inhibitor studies owing to their soluble nature. Inhibitors
that have been most recently used include peptidoglycan-
mimetic b-lactams and b-sultams [27,28]. By contrast,
inhibitor studies of the membrane-anchored HMM PBPs
have been hindered by their under-representation in the
Protein Data Bank (PDB) structural database, primarily
owing to the difficulty of working with membrane pro-
teins and the scarce lipid II substrate. However, struc-
tures of the major determinants in class B PBP-associated
b-lactam resistance have recently become available to
increase understanding of these phenomena, for example,
PBP2x from the penicillin-resistant Streptococcus pneumo-
niae (PRSP) [29–31], PBP2a from MRSA [32] and
PBP5fm from the naturally resistant Enterococcus faecium
[33]. The transpeptidase domain of the class A S. pneu-
moniae PBP1b has also been solved [34] and might
provide a basis on which to model mutations in the
resistant PBP1a enzyme.
There are several PBP-mediated mechanisms of b-lactam
resistance, including: acquisition of a ‘new’ less-sensitive
enzyme, mutation of an endogenous PBP to lessen the
reaction with b-lactams (while maintaining some trans-
peptidase activity), or upregulation of PBP expression.
The latter option appears to be the least effective,
although the converse (lowering expression of some
resistant PBPs) might prove to be useful in the control
of b-lactam-resistant bacteria [35].
PBP2x structures
The mutation of PBP2x in PRSP has been studied
extensively [29–31] (Figure 4). Resistance occurs in a
mosaic pattern, with many mutations occurring in differ-
ent clinical isolates. This can cause problems in isolation
of the main determinants of resistance and also presents
the requirement for screening crystallization conditions
anew. The structures of PBP2x from several resistant
strains have been determined [29–31].
Strain sp328 harbors the most clinically important muta-
tion, T338A, which was found to result in the loss of an
important active site water molecule, thus weakening the
hydrogen bonding network that stabilizes the acyl-
Current Opinion in Microbiology 2005, 8:525–533
530 Antimicrobials
Figure 4
The fold and active site of S. pneumoniae PBP2x. (a) Surface
representation overlaid with protein fold. The transpeptidase domain is
colored red and the other domains blue. (b) Detail of active site topology.
This shows the S337 nucleophile and residues responsible for the
development of resistance to b-lactams. Residues are represented in
stick format and are colored according to atom type (C atoms, yellow;
O, red; N, blue; and S, orange). The protein co-ordinates used are
from PDB 1RP5, with a molecule of cefuroxime (C atoms, yellow in
[a], magenta in [b]) placed in an equivalent position to that observed in
the complex from PDB 1QMF. Note that because 1RP5 is used, the
native amino acid Q552 is mutated to a glutamate residue.
Current Opinion in Microbiology 2005, 8:525–533
enzyme complex [31]. The S389L and N514H mutations
that are also present in this strain were found to sterically
hinder favorable interactions with the b-lactam, reducing
the acylation rate. An additional mutation, M339F, con-
fers higher-level resistance to strains that possess the
T338A mutation. The structure of this variant was found
to re-orientate the S337 nucleophile and lower the reac-
tion rate by 4–10-fold [29].
PBP2x from the PRSP strain sp5259 has also been struc-
turally characterized [30] and appears to offer an alter-
native mechanism of resistance that shares features with
other class B enzymes. The mutation of Q552 to gluta-
mate introduces a negative charge near the edge of the
active site; this might act like a similarly positioned
residue in PBP5fm, disfavoring interaction with nega-
tively charged b-lactams. Other mutations in this strain
act in a similar way to residues from PBP2a, altering the
conformation of b-strand 3 so that an energetically costly
rearrangement is required for acylation. However, the
observation that resistant class B PBPs share these fea-
tures might not assist in the development of new broad-
spectrum antibiotics. Molecular dynamics simulations on
a group of enzymes that share the transpeptidase fold
raised the possibility that there are different mechanisms
of acylation dependent on the particular combination of
enzyme and antibiotic [36]; this leaves bacteria many
possibilities to develop resistance against new b-lactams.
Other PBP structures
It is also prudent to note that PBP1a and PBP2b are
involved in PRSP resistance. Sanbongi et al. [37]
sequenced six S. pneumoniae PBPs, all of which are pre-
sent in 40 clinical isolates, and thereby confirmed the
correlation of mutations in these proteins to b-lactam
minimum inhibitory concentrations (MICs). Mutations
in PBP2b resulted in higher MICs for penicillins and
carbapenems, whereas MIC increases for cephalosporins
were associated with PBP2x. Alteration of the class A
PBP1a conferred additional resistance to strains already
bearing mutations in PBPs 2b and 2x.
PBP2a of MRSA is encoded by the mecA gene, which is
believed to have arisen as a result of horizontal transfer
from an undisclosed species. When challenged with
b-lactams, MRSA will utilize the transglycosylase activity
of PBP2 (the only class A enzyme of S. aureus) and the
transpeptidase functionality of PBP2a to synthesize the
cell wall. It has recently been shown that PBP2 is able to
mutate to a resistant form in the laboratory [38], but
thankfully it is not responsible for the emergence of a
second alternate form of MRSA in the environment. The
structure of PBP2a [32] suggested that the poor acylation
by b-lactams was caused by a b-strand 3 alteration, and a
route to more effective antibiotics could be to increase the
length of the b-lactam compound to improve non-cova-
lent interactions. Several such new compounds have been
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 b-Lactam antibiotic resistance Wilke, Lovering and Strynadka 531
described at length by Bush et al. [39], including
BAL9141, which is active against both PRSP and MRSA.
The use of specific chemical groups to mimic the differ-
ences in peptidoglycan sub-structure between various
bacterial pathogens, or indeed to facilitate the conforma-
tional changes required for acylation, might also prove
fruitful, as suggested recently by Fuda et al. [40].
In contrast to S. aureus and S. pneumoniae, the bacterium
E. faecium is naturally resistant to b-lactam antibiotics.
The PBP responsible for this resistance, PBP5fm, has
been structurally characterized [33], although no co-ordi-
nates for this enzyme are available in the PDB at present.
The reason for the endogenous low-level affinity for
b-lactams in E. faecium isn’t immediately apparent, but
could be owing to the reduced active site accessibility and
also to charge repulsion with the b-lactam carboxylate
group. E. faecium can demonstrate higher-level resistance
by mutation of PBP5fm [41], including an insertion after
S466 that is present in a loop that shifts upon acylation.
Bifunctional PBPs
The first crystal structure of a transpeptidase domain from
a class A enzyme — PBP1b from S. pneumoniae — has
recently been reported [34]. The role of PBP1b in
b-lactam resistance is minor at best, but its structure
can be used as a model for mutational effects in the
resistant PBP1a enzyme (45% sequence identity between
the transpeptidase region of PBP1a and PBP1b). It is also
noted that crystallization conditions for PBP1a have been
reported [42]. Like PBPs 2x, 2a and 5fm, the enzyme
possesses a classical transpeptidase domain, which is
flanked by regions of unknown function that might play
a role in association with other cell wall modifying pro-
teins. The postulation of multi-enzyme complexes for
HMM PBPs is particularly interesting when applied to
this structure as the active site appears ‘closed’ and
activity might be regulated by interaction with other
proteins. One consequence of open and closed active
sites in these enzymes is that resistant PBPs are expected
to favor open conformations, allowing transpeptidation of
the bulkier substrates that result from inhibition of the
LMM PBP carboxypeptidases by b-lactams. The resis-
tance-conferring mutation of T371S/A in PBP1a is ana-
logous to that of T338A described for PBP2x. Other
mutations of b-lactam-insensitive PBP1a can be mapped
to a conserved proline near the classical SXN motif as well
as to a range of mutations along b-strands 4 and 5 [34].
Given the structural data thus far and the requirement of
conformational changes in catalysis of the HMM but not
of the LMM PBPs, there is some concern about using the
latter as model systems for inhibitor design. It is therefore
important to obtain structures of other HMM PBPs to
better understand the commonality of this active site
plasticity. In addition, structures of the full-length class
A enzymes might enable the generation of new antibio-
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tics that target the essential transglycosylase domain.
Such inhibitors would undoubtedly work well in combi-
nation therapy with b-lactams, as shown by the essential
nature of PBP2 and PBP2a co-operation in MRSA strains.
Altered antibiotic permeability and efflux:
the Gram-negative efflux pumps
With the exception of some strains of the Streptococci,
Enterococci and Staphylococci ‘superbugs’, Gram-negative
bacteria are generally more resistant to a large variety of
antibiotics and chemotherapeutic agents than are Gram-
positive bacteria. It is now recognized that a major con-
tribution to antibiotic resistance in Gram-negative species
is the presence of broad-specificity drug-efflux pumps.
One of the best-characterized of these is the drug efflux
system MexAB–OprM (recently reviewed in [43]) of the
opportunistic pathogen, Pseudomonas aeruginosa. This tri-
partite pump (composed of the inner membrane RND
transporter ‘pump’ MexB, the outer membrane porin
OprM, and the soluble periplasmic MexA) acts on a wide
range of antibiotics, including tetracycline, chloramphe-
nicol, quinilones, novobiocin, macrolides and trimetho-
prim, as well as b-lactams and b-lactamase inhibitors such
as clavulanic acid. The past four years have seen a
tremendous increase in our understanding of the struc-
tural features of the individual components of the tripar-
tite efflux pumps (for a recent review see [44]); the
orthologous outermembrane porin and innermembrane
pump components TolC and AcrB from E. coli have been
determined to 2.1 and 3.5 Å resolution, respectively, and
the periplasmic component MexA from Pseudomonas aer-
uginosa to 3.5 Å resolution. A structure of the inner
membrane pump AcrB in the presence of several hydro-
phobic small molecule compounds has also been deter-
mined [45], which implies a diverse binding mode for
individual ligands, at least in this component of the efflux
pump. Although these structures have provided a tre-
mendous new level of understanding of the distinct
architecture of the three proteins that make up these
pumps, there are still many unanswered questions with
regard to the way in which these components interact to
form a single path for extruded antibiotic ligands. These
systems represent logical targets for novel antibiotic
design, and development of lead compounds in this area
is evolving rapidly (as reviewed recently by Kaatz [46]).
Conclusions
Recent years have witnessed a substantial increase in
our understanding of the mechanisms responsible for
b-lactam resistance. The structures of the molecular
determinants of resistance — particularly in complex
with antibiotics or inhibitors — are poised not only to
explain resistance, but also to inspire novel methods of
combating it. The b-lactam class of antibiotics has proven
itself to be invaluable in the treatment of bacterial infec-
tions, and structural biology will undoubtedly play a
Current Opinion in Microbiology 2005, 8:525–533
532 Antimicrobials
central role in ensuring that b-lactams remain therapeu-
tically effective.
Acknowledgements
We are grateful for support from the Howard Hughes Medical Institute
(to NS), the Canadian Institute of Health Research (to NS and MW), and
the Michael Smith Foundation for Health Research (to MW and AL).
References and recommended reading
Papers of particular interest, published within the annual period of
review, have been highlighted as:
 of special interest
 of outstanding interest
1. Weldhagen GF: Integrons and beta-lactamases — a novel
perspective on resistance. Int J Antimicrob Agents 2004,
23:556-562.
2. Fisher JF, Meroueh SO, Mobashery S: Bacterial resistance to
 beta-lactam antibiotics: compelling opportunism, compelling
opportunity. Chem Rev 2005, 105:395-424.
An excellent and comprehensive review about the mechanisms of b-
lactam resistance.
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lactam-tolerant enzymes, including the re-arrangement of strain b3 (akin
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The first structural information obtained for a class A PBP, both in the
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representative of the classical PBP fold, but also provides information on
the interaction with the transglycosylase domain and the regulation of
enzymatic activity by way of active site opening. This work also discusses
the structural effects of mutations in the related enzyme PBP1a, which is
implicated in b-lactam-resistant forms of S. pneumoniae.
www.sciencedirect.com
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Current Opinion in Microbiology 2005, 8:525–533