Beta Lactamase

b-Lactam antibiotic resistance: a current structural perspectiveMark 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

([email protected])1Mark 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

IntroductionThe 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

www.sciencedirect.com

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 withmost antimicrobial agents, b-lactams are rendered

inactive against bacteria by way of three primary mechan-

isms of resistance (Figure 1). The most commonmechan-

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.


526 Antimicrobials

Figure 1

Current Opinion in Microbiology 2005, 8:525–533 www.sciencedirect.com

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-lactamasesThe 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).

(Figure 1 Legend) Structural depiction of proteins involved in b-lactam resi

into the b-lactamase, PBP and efflux pump groups. Also shown are two of

shown as solid arrows). The structures of the glycosyltransferase domain of

are not yet determined are replaced by blue and green solid shapes, respec

these subgroups — the b-lactam-dependent signaling of BlaR, ring hydroly

and proton antiport of the antibiotics by the efflux pump systems. The rece

closely-related resistance determinant PBP1a, and the structures of MexB a

of their respective homologues, AcrB and TolC (docked together manually).

unknown, as does the nature of the effector responsible for terminating Me

inner membrane; OM, outer membrane; PG, peptidoglycan. CTXM9 and Cp

metallo-b-lactamase structures.


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

stance. The proteins responsible for resistance are sub-divided

the repressors for resistance operons — BlaI and MexR (genes are

the class A PBPs and the cytoplasmic protease domain of BlaR that

tively. The diagram also shows the interaction of b-lactams with

sis by the b-lactamases, acylation of the PBPs (poor in PBP2x and 2a),

ntly solved structure of S. pneumoniae PBP1b is used in place of the

nd OprM are used here as models derived from the co-ordinates

Details of the precise interaction of MexA with MexB–OprM remain

xR repression. Abbreviations: gm +/�, Gram-positive/negative; IM,

hA are shown as representatives of the >40 available serine and


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.

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.

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


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



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 bladivergon that encodesBlaI,BlaRand 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 blaoperator but bind the operator sequence for the mec diver-gon as well, allowingBlaR andBlaI to additionallymediate

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

proveuseful as targets for thedesignof inhibitors to counter

b-lactamaseandPBP2a-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].


Altered antibiotic targets: the cell walltranspeptidases (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 themore essential HMM counter-

parts, the LMM PBPs have been a common vehicle for

inhibitor studies owing to their soluble nature. Inhibitors

that have beenmost 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-mediatedmechanisms 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-


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.


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 tomore 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



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 SXNmotif 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-


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 pumpsWith 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 inGram-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 beendetermined to 2.1 and 3.5 A resolution, respectively, and

the periplasmic component MexA from Pseudomonas aer-uginosa to 3.5 A 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]).

ConclusionsRecent 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


532 Antimicrobials

central role in ensuring that b-lactams remain therapeu-

tically effective.

AcknowledgementsWe are grateful for support from the Howard Hughes Medical Institute(to NS), the Canadian Institute of Health Research (to NS and MW), andthe Michael Smith Foundation for Health Research (to MW and AL).

References and recommended readingPapers of particular interest, published within the annual period ofreview, have been highlighted as:

� of special interest

�� of outstanding interest

1. Weldhagen GF: Integrons and beta-lactamases — a novelperspective on resistance. Int J Antimicrob Agents 2004,23:556-562.

2.�

Fisher JF, Meroueh SO, Mobashery S: Bacterial resistance tobeta-lactam antibiotics: compelling opportunism, compellingopportunity. Chem Rev 2005, 105:395-424.

An excellent and comprehensive review about the mechanisms of b-lactam resistance.

3. Heinz U, Adolph HW: Metallo-beta-lactamases: two bindingsites for one catalytic metal ion? Cell Mol Life Sci 2004,61:2827-2839.

4.��

Garau G, Bebrone C, Anne C, Galleni M, Frere JM, Dideberg O: Ametallo-beta-lactamase enzyme in action: crystal structuresof the monozinc carbapenemase CphA and its complex withbiapenem. J Mol Biol 2005, 345:785-795.

This article reports the crystal structures of wild-type CphA, its N220Gmutant, and a complex of N220G CphA with the carbapenem substratebiapenem. As well as providing the first structures of a class B2 metallo-b-lactamase, the authors propose a novel monozinc carbapenemasemechanism based on the details of the CphA active site in complex withsubstrate.

5. Wommer S, Rival S, Heinz U, Galleni M, Frere JM, Franceschini N,Amicosante G, Rasmussen B, Bauer R, Adolph HW: Substrate-activated zinc binding of metallo-beta-lactamases:physiological importance of mononuclear enzymes. J BiolChem 2002, 277:24142-24147.

6. Palumbi SR: Humans as the world’s greatest evolutionaryforce. Science 2001, 293:1786-1790.

7. Poole K:Resistance to beta-lactam antibiotics. Cell Mol Life Sci2004, 61:2200-2223.

8. Jacoby GA, Munoz-Price LS: The new beta-lactamases.NEngl JMed 2005, 352:380-391.

9.�

Chen Y, Shoichet B, Bonnet R: Structure, function, andinhibition along the reaction coordinate of CTX-M beta-lactamases. J Am Chem Soc 2005, 127:5423-5434.

The crystal structures of CTX-M-9 and/or CTX-M-14 are reported incomplex with glycylboronic acid transition state analogues and withthe inhibitor cefoxitin. These structures represent snapshots of CTX-Mprogressing through b-lactam hydrolysis, (i.e. in an acylation transitionstate, as an acyl-enzyme intermediate, and in a deacylation transitionstate). To explain the inhibitory activity of cefoxitin, steric hindrance owingto the 7a-group of the cefoxitin was implicated in blocking formation ofthe deacylation transition state.

10. Chen Y, Delmas J, Sirot J, Shoichet B, Bonnet R: Atomicresolution structures of CTX-M beta-lactamases: extendedspectrum activities from increased mobility and decreasedstability. J Mol Biol 2005, 348:349-362.

11. Sanschagrin F, Levesque RC: A specific peptide inhibitor of theclass B metallo-beta-lactamase L-1 from Stenotrophomonasmaltophilia identified using phage display. J AntimicrobChemother 2005, 55:252-255.

12. Moloughney JG, Thomas JD, Toney JH: Novel IMP-1 metallo-beta-lactamase inhibitors can reverse meropenem resistancein Escherichia coli expressing IMP-1. FEMSMicrobiol Lett 2005,243:65-71.


13. Phillips OA, Reddy AV, Setti EL, Spevak P, Czajkowski DP,Atwal H, Salama S, Micetich RG, Maiti SN: Synthesis andbiological evaluation of penam sulfones as inhibitors ofbeta-lactamases. Bioorg Med Chem 2005, 13:2847-2858.

14. Buynak JD, Chen H, Vogeti L, Gadhachanda VR, Buchanan CA,Palzkill T, Shaw RW, Spencer J, Walsh TR: Penicillin-derivedinhibitors that simultaneously target bothmetallo- and serine-beta-lactamases. Bioorg Med Chem Lett 2004, 14:1299-1304.

15. Bonnefoy A, Dupuis-Hamelin C, Steier V, Delachaume C, Seys C,Stachyra T, Fairley M, Guitton M, Lampilas M: In vitro activity ofAVE1330A, an innovative broad-spectrum non-beta-lactambeta-lactamase inhibitor. J Antimicrob Chemother 2004,54:410-417.

16. Tondi D, Morandi F, Bonnet R, Costi MP, Shoichet BK: Structure-based optimization of a non-beta-lactam lead results ininhibitors that do not up-regulate beta-lactamase expressionin cell culture. J Am Chem Soc 2005, 127:4632-4639.

17. Venkatesan AM, Gu Y, Dos Santos O, Abe T, Agarwal A,Yang Y, Petersen PJ, Weiss WJ, Mansour TS, Nukaga M et al.:Structure-activity relationship of 6-methylidene penemsbearing tricyclic heterocycles as broad-spectrum beta-lactamase inhibitors: crystallographic structures showunexpected binding of 1,4-thiazepine intermediates.J Med Chem 2004, 47:6556-6568.

18. Melckebeke HV, Vreuls C, Gans P, Filee P, Llabres G, Joris B,Simorre JP: Solution structural study of BlaI: implications forthe repression of genes involved in beta-lactam antibioticresistance. J Mol Biol 2003, 333:711-720.

19.�

Safo MK, Zhao Q, Ko TP, Musayev FN, Robinson H, Scarsdale N,Wang AH, Archer GL: Crystal structures of the BlaI repressorfrom Staphylococcus aureus and its complex with DNA:insights into transcriptional regulation of the bla and mecoperons. J Bacteriol 2005, 187:1833-1844.

This report describes the first crystal structures of BlaI in a free form and incomplex with the mec operator. Although the BlaI structures closelyresemble the published structures of MecI, the authors describe an up-and-down model of repressor-binding to the mec operon (versus theside-by-side model for the bla operon) and propose that proteolyticcleavage of BlaI or MecI preferentially occurs in the DNA-bound form.

20. Kerff F, Charlier P, Colombo ML, Sauvage E, Brans A, Frere JM,Joris B, Fonze E: Crystal structure of the sensor domain of theBlaR penicillin receptor from Bacillus licheniformis.Biochemistry 2003, 42:12835-12843.

21. Wilke MS, Hills TL, Zhang HZ, Chambers HF, Strynadka NC:Crystal structures of the Apo and penicillin-acylated forms oftheBlaR1 beta-lactam sensor ofStaphylococcus aureus. J BiolChem 2004, 279:47278-47287.

22. Birck C, Cha JY, Cross J, Schulze-Briese C, Meroueh SO,Schlegel HB, Mobashery S, Samama JP: X-ray crystal structureof the acylated beta-lactam sensor domain of BlaR1 fromStaphylococcus aureus and the mechanism of receptoractivation for signal transduction. J Am Chem Soc 2004,126:13945-13947.

23. Hanique S, ColomboML, Goormaghtigh E, Soumillion P, Frere JM,Joris B: Evidence of an intramolecular interaction between thetwo domains of the BlaR1 penicillin receptor during the signaltransduction. J Biol Chem 2004, 279:14264-14272.

24. Rosato AE, Kreiswirth BN, Craig WA, Eisner W, Climo MW,Archer GL:mecA-blaZ corepressors in clinical Staphylococcusaureus isolates. Antimicrob Agents Chemother 2003,47:1460-1463.

25. Katayama Y, Zhang HZ, Hong D, Chambers HF: Jumping thebarrier to beta-lactam resistance in Staphylococcus aureus.J Bacteriol 2003, 185:5465-5472.

26. Zervosen A, Lu WP, Chen Z, White RE, Demuth TP Jr, Frere JM:Interactions between penicillin-binding proteins (PBPs) andtwo novel classes of PBP inhibitors, arylalkylidene rhodaninesand arylalkylidene iminothiazolidin-4-ones. Antimicrob AgentsChemother 2004, 48:961-969.

27. Llinas A, Ahmed N, CordaroM, Laws AP, Frere JM, Delmarcelle M,Silvaggi NR, Kelly JA, Page MI: Inactivation of bacterial DD-peptidase by beta-sultams. Biochemistry 2005, 44:7738-7746.



28. Silvaggi NR, Josephine HR, Kuzin AP, Nagarajan R, Pratt RF,Kelly JA: Crystal structures of complexes between the R61DD-peptidase and peptidoglycan-mimetic beta-lactams: anon-covalent complex with a ‘‘perfect penicillin’’. J Mol Biol2005, 345:521-533.

29. Chesnel L, Pernot L, Lemaire D, Champelovier D, Croize J,Dideberg O, Vernet T, Zapun A: The structural modificationsinduced by the M339F substitution in PBP2x fromStreptococcus pneumoniae further decreases thesusceptibility to beta-lactams of resistant strains. J Biol Chem2003, 278:44448-44456.

30.�

Pernot L, Chesnel L, Le Gouellec A, Croize J, Vernet T, Dideberg O,Dessen A: A PBP2x from a clinical isolate of Streptococcuspneumoniae exhibits an alternative mechanism for reductionof susceptibility to beta-lactam antibiotics. J Biol Chem 2004,279:16463-16470.

Following on from the work done on PRSP strain sp328 [31], the structureof PBP2x from strain sp5259 was obtained. In this case, the mutation ofresidues serves more to mimic resistance effects observed in otherlactam-tolerant enzymes, including the re-arrangement of strain b3 (akinto PBP2a from S. aureus) and the introduction of negative charge near theactive site (akin to PBP5fm from E. faecium). In tandem with the mutantsfrom sp328 that act to modify the environment around the active sitenucleophile, these results highlight the different techniques used to conferresistance in PBP2x.

31. Dessen A, Mouz N, Gordon E, Hopkins J, Dideberg O: Crystalstructure of PBP2x from a highly penicillin-resistantStreptococcus pneumoniae clinical isolate: a mosaicframework containing 83 mutations. J Biol Chem 2001,276:45106-45112.

32. Lim D, Strynadka NC: Structural basis for the beta lactamresistance of PBP2a from methicillin-resistantStaphylococcus aureus. Nat Struct Biol 2002, 9:870-876.

33. Sauvage E, Kerff F, Fonze E, Herman R, Schoot B, Marquette JP,Taburet Y, Prevost D, Dumas J, LeonardG et al.: The 2.4-A crystalstructure of the penicillin-resistant penicillin-binding proteinPBP5fm from Enterococcus faecium in complex withbenzylpenicillin. Cell Mol Life Sci 2002, 59:1223-1232.

34.�

Macheboeuf P, Di Guilmi AM, Job V, Vernet T, Dideberg O,Dessen A: Active site restructuring regulates ligandrecognition in class A penicillin-binding proteins. Proc NatlAcad Sci USA 2005, 102:577-582.

The first structural information obtained for a class A PBP, both in theunliganded form and when complexed with b-lactams. The structure isrepresentative of the classical PBP fold, but also provides information onthe interaction with the transglycosylase domain and the regulation ofenzymatic activity by way of active site opening. This work also discussesthe structural effects of mutations in the related enzyme PBP1a, which isimplicated in b-lactam-resistant forms of S. pneumoniae.


35. Tajima Y: Polyoxotungstates reduce the beta-lactamresistance of methicillin-resistant Staphylococcus aureus.Mini Rev Med Chem 2005, 5:255-268.

36. Oliva M, Dideberg O, Field MJ: Understanding the acylationmechanisms of active-site serine penicillin-recognizingproteins: a molecular dynamics simulation study. Proteins2003, 53:88-100.

37. Sanbongi Y, Ida T, Ishikawa M, Osaki Y, Kataoka H, Suzuki T,Kondo K, Ohsawa F, Yonezawa M: Complete sequences ofsix penicillin-binding protein genes from 40 Streptococcuspneumoniae clinical isolates collected in Japan. AntimicrobAgents Chemother 2004, 48:2244-2250.

38. Leski TA, Tomasz A: Role of penicillin-binding protein 2 (PBP2)in the antibiotic susceptibility and cell wall cross-linking ofStaphylococcus aureus: evidence for the cooperativefunctioning of PBP2, PBP4, and PBP2A. J Bacteriol 2005,187:1815-1824.

39. Bush K, Macielag M, Weidner-Wells M: Taking inventory:antibacterial agents currently at or beyond phase 1. Curr OpinMicrobiol 2004, 7:466-476.

40. Fuda C, Hesek D, Lee M, Morio K, Nowak T, Mobashery S:Activation for catalysis of penicillin-binding protein 2a frommethicillin-resistant Staphylococcus aureus by bacterial cellwall. J Am Chem Soc 2005, 127:2056-2057.

41. Rice LB, Bellais S, Carias LL, Hutton-Thomas R, Bonomo RA,Caspers P, Page MG, Gutmann L: Impact of specific pbp5mutations on expression of beta-lactam resistance inEnterococcus faecium. Antimicrob Agents Chemother 2004,48:3028-3032.

42. Job V, Di Guilmi AM, Martin L, Vernet T, Dideberg O, Dessen A:Structural studies of the transpeptidase domain of PBP1afrom Streptococcus pneumoniae. Acta Crystallogr D BiolCrystallogr 2003, 59:1067-1069.

43. Poole K:Efflux-mediated antimicrobial resistance. J AntimicrobChemother 2005, 56:20-51.

44. Eswaran J, Koronakis E, Higgins MK, Hughes C, Koronakis V:Three’s company: component structures bring a closer viewof tripartite drug efflux pumps. Curr Opin Struct Biol 2004,14:741-747.

45. Yu EW, McDermott G, Zgurskaya HI, Nikaido H, Koshland DE Jr:Structural basis of multiple drug-binding capacity of the AcrBmultidrug efflux pump. Science 2003, 300:976-980.

46. Kaatz GW: Bacterial efflux pump inhibition. Curr Opin InvestigDrugs 2005, 6:191-198.


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