Chapter 8

Biochemical Logic of Antibiotic Inactivation

and Modification

Vanessa D’Costa and Gerard D. Wright

1 Introduction ability. In Table 1 we itemize representative enzymes and

mechanisms, differentiating between mechanisms that mod-
ify the antibiotic (e.g., acylation, phosphorylation), and those
Bacterial resistance to antibiotics manifests itself in both
that essentially cause irreversible destruction (e.g., hydroly-
general and specific protection mechanisms. Consequently,
sis). A general observation evident from Table 1 is that most
the characteristics of resistance can be paralleled to those of
antibiotics that are either natural products or are based on
the mammalian immune response. Antibiotic resistance can
natural product chemical scaffolds are more susceptible to
be differentiated into: (1) nonspecific mechanisms that con-
some form of enzyme-based inactivation, while antibiotics
fer general innate immunity to a class of antibiotics (e.g.,
of synthetic origin (e.g., fluoroquinolones) are not (however,
broad spectrum efflux mechanisms, target modification), and
enzyme-based inactivation of certain fluoroquinolones has
(2) highly precise responses that include selective enzyme-
been reported (2) ). These relatively enzyme-impervious
based mechanisms that mirror the acquired immune response
antibiotics are nonetheless still susceptible to resistance
with respect to target specificity and potency. Bacteria deploy
mechanisms, often substrates for efflux pumps.
both types of mechanisms in response to the presence of
Walsh described the cellular impact and rationale of bio-
cytotoxic antibiotics.
chemical reactions as “molecular logic” (3) and this termi-
Although antibiotic resistance via target modification or
nology works very well in dissecting mechanisms of
efflux mechanisms results in the survival of the resistant
antibiotic resistance. Thus enzyme-catalyzed antibiotic resis-
organism, the concentration of antibiotic that the bacte-
tance is functionally and structurally linked to the mode of
rium is exposed to remains unaffected. Thus, other proximal
action of these agents. For example, modification of key
susceptible organisms can still be targeted by the antimicro-
functional groups on an antibiotic can sterically or electroni-
bial agent. In contrast, enzyme-catalyzed detoxification of
cally block interaction with target (see Sect. 3 below for
antibiotics effectively (and often irreversibly) lowers the con-
examples). This review describes mechanisms of antibiotic
centration of the drug and as a result has the potential for a
destruction and modification resulting in resistance in the
much broader impact on microbial growth. The presence of
context of the mode of action of the antibiotic. Our aim is not
an antibiotic-resistant microbe can, at least in theory, promote
to provide a comprehensive examination of the details of all
the growth of adjacent bacteria that otherwise would be sus-
known resistance mechanisms but rather to focus on selected
ceptible to the antibiotic by inactivating the drug in the local
examples to decode the molecular basis and biological
environment. This can occur even if it is the susceptible organ-
impact of these inactivation strategies.
ism, not the resistant strain, which is the cause of infection.
As a result, enzyme-catalyzed antibiotic inactivation can have
a significant and broad impact on antimicrobial therapy.
Since the first reports of penicillin inactivating strains of 2 Destruction of Antibiotics
bacteria in the early 1940s (1), virtually all antibiotics have
been shown to be modified or destroyed by a cadre of
We classify antibiotic destruction as a mechanism that results
enzymes with hydrolytic, chemical group transfer or redox
in either ablation of a key reactive centre or massive structural
rearrangement that is not readily reversed under normal
G.D. Wright ( ) physiological conditions. Hydrolysis of the reactive β-lactam
M.G. DeGroote Institute for Infectious Disease Research,
Antimicrobial Research Centre, Department of Biochemistry and
ring of penicillin and cephalosporin antibiotics by
Biomedical Sciences, McMaster University, Hamilton, ON, Canada β-lactamases is an example of the first class, and linearization
wrightge@mcmaster.ca of the cyclic depsipeptide of Type B streptogramins by Vgb

D.L. Mayers (ed.), Antimicrobial Drug Resistance, 81

DOI 10.1007/978-1-59745-180-2_8, © Humana Press, a part of Springer Science+Business Media, LLC 2009
82 V. D’Costa and G.D. Wright

Table 1 Survey of enzymatic mechanisms of antibiotic resistance

Antibiotic destruction
Antibiotic Mechanisms Enzyme(s)
β-Lactams Hydrolysis β-Lactamase
Macrolides Hydrolysis Macrolide esterase
Type B streptogramins C–O-bond cleavage Vgb lyase
Tetracyclines Mono-oxidation TetX
Fosfomycin Hydrolysis Epoxidase
Thiol transfer Thiol transferase
Antibiotic modification
Antibiotic Mechanisms Enzyme
Aminoglycosides Acylation Acetyltransferase
Phosphorylation Kinase
Adenylylation AMP-transferase
Macrolides Phosphorylation Kinase
Glycosylation UDP-glucosyl
transferase
Lincosamides Adenylylation AMP-transferase
Rifamycin Glycosylation TDP-glucosyl
transferase
Phosphorylation Kinase
Chloramphenicol Acylation Acetyltransferase
Type A streptogramins Acylation Acetyltransferase

lyase is an example of the second. In all classes, the action of
resistance enzymes tactically impacts the mode of action of
the affected antibiotics to disrupt their biological activity.
Examples of each class are discussed below.
Fig. 1 Structure of the bacterial peptidoglycan unit. Peptidoglycan
consists of repeating disaccharyl units (N-acetylglucosamine-N-
acetylmuramic acid), to which a pentapeptide is linked each
N-acetylmuramic acid. Crosslinking between adjacent pentapeptides
provides rigidity to the bacterial cell

2.1 b-Lactam Antibiotics

The β-lactams remain some of the most successful and

widely used antibiotics in modern chemotherapy. These
natural products and their semi-synthetic derivatives act by
covalently modifying so-called penicillin binding proteins
(PBPs) (4). PBPs include membrane-associated enzymes
important in bacterial peptidoglycan assembly and mainte-
nance. Covalent modification of this subclass of PBPs by
β-lactams blocks their enzymatic activity thereby inhibiting
cell wall metabolism, which results in impaired wall integ-
rity and cell death. PBPs include the transpeptidases and
dd-carboxypeptidases that act on the pentapeptide portion
of the peptidoglycan repeating unit that consists of the
disaccharyl unit, N-acetylglucosamine-N-acetylmuramic
acid, to which a d-Ala-d-Ala terminating pentapeptide is
linked through the lactyl group of N-acetylmuramic acid
(Fig. 1). Transpeptidases and dd-carboxypeptidases use
canonical Ser hydrolase chemistry to either rigidify the cell
wall by synthesizing interstrand peptidoglycan crosslinks
between the d-Ala-d-Ala termini of adjacent peptidoglycan
strands (transpeptidases) or control cell wall strength and
flexibility by cleaving the terminal d-Ala-d-Ala peptide
Fig. 2 Comparison of the β-lactam penicillin and the d-Ala-d-Ala
peptidoglycan terminus
bond of the pentapeptide (Fig. 1). Strominger noted
40 years ago that the β-lactam antibiotics sterically and
electronically mimic the acyl-d-Ala-d-Ala terminus of the
pentapeptide (Fig. 2) (5). This model overlaps the highly
strained (and thus chemically reactive) β-lactam ring over
the scissile d-Ala-d-Ala peptide bond. Attack of the nucleo-
philic Ser hydroxyl onto the β-lactam ring carbonyl opens
the cyclic structure and generates a covalent intermediate
that is resistant to hydrolysis (Fig. 3), thereby chemically
titrating PBPs into inactive complexes and shutting down
cell wall synthesis.
8 Biochemical Logic of Antibiotic Inactivation and Modification
In order to overcome the action of cytotoxic β-lactams,
bacteria have evolved secreted enzymes that hydrolytically
cleave the β-lactam ring of penicillins and cephalosporins (6)
(Fig. 4). The molecular logic of this resistance mechanism
therefore involves the destruction of the reactive “warhead”
of the β-lactam antibiotics, thereby eliminating the essential
chemical structure necessary for PBP inactivation.
These hydrolytic enzymes, appropriately named
β-lactamases, fall into two general structural classes: Ser
β-lactamases and metallo-β-lactamases (Fig. 4). The former
group share structural homology with the dd-carboxypeptidases
and operate by similar Ser hydrolase chemistry. However the
hydrolytic step, which is slow in PBPs, is fast in β-lactamases,
resulting in highly efficient detoxification of the antibiotics.
Metallo-β-lactamases adopt analogous hydrolytic chemistry
Fig. 3 Mechanism of action of β-lactam antibiotics on bacterial trans-
peptidases and dd-carboxypeptidases. Nucleophilic attack of the PBP
Ser hydroxyl on the β-lactam ring carbonyl results in an opening of the
Fig. 4 Mechanisms of enzymatic inactivation of β-lactam antibiotics.
β-lactamases catalyze the hydrolytic cleavage of β-lactam rings. (a)
Serine-β-lactamases form a transient enzyme-antibiotic intermediate,
83
employed by metallo-proteases to cleave the reactive
β-lactam ring.
2.2 Fosfomycin
Destruction of a reactive chemical warhead is also employed
by enzymes that inactivate fosfomycin. The key structural
element of this antibiotic is a reactive epoxide that is attacked
by its intracellular target, the cell wall biosynthetic enzyme
MurA (Figs. 5 and 6a). This enzyme is essential for synthe-
sis of N-acetylmuramic acid and covalent modification of a
key Cys residue by fosfomycin efficiently inactivates the
enzyme.
β-lactam ring. The active site machinery of transpeptidases or dd-
carboxypeptidases is effectively captured as the subsequent covalent
intermediate cannot be hydrolyzed
which is quickly hydrolyzed. (b) Metallo-β-lactamases utilize a bound
Zn2+ to activate water for hydrolytic attack of the β-lactam ring
84
The bacterial countermeasure to inactivate this antibi-
otic is an epoxide ring opening reaction using one of two
distinct chemical tactics. The first, catalyzed by FosX, is a
metal-dependent hydrolytic process that generates the
Fig. 5 Interactions of fosfomycin with its bacterial target, MurA.
Fosfomycin forms a covalent bond with MurA’s active site Cys.
Additional interactions with MurA are designated as arrows. MurA
residues are labeled in grey. Adapted from (7)
Fig. 6 Mechanism of fosfomycin action and
inactivation. (a) Fosfomycin targets the active-site
Cys residue of MurA, forming a covalent
intermediate. (b) FosX-mediated inactivation of
fosfomycin results in the formation of a diol.
(c) The product of FosA-mediated fosfomycin
inactivation is a glutathione-fosfomycin adduct.
(d) FosB-mediated resistance to fosfomycin
results in a ring-opened inactivated product with
free Cys
V. D’Costa and G.D. Wright
vicinal diol (8) (Fig. 6b). The second is via a thiol-
dependent ring opening by enzymes that use abundant
intracellular thiols such as glutathione (FosA) (9) (Fig. 6c)
and cysteine (FosB) (10) (Fig. 6d). Either strategy results
in efficient destruction of the antibiotic’s reactive centre,
thereby blocking its action on the target MurA.
2.3 Macrolide Antibiotics
The macrolide antibiotics include natural products such as
erythromycin and semi-synthetic derivatives (e.g., clarithro-
mycin). These antibiotics are assembled via a polyketide
assembly line, cyclized to form a macrolactone ring struc-
ture, and subsequently modified by glycosylation to generate
a mature antibiotic (11). Macrolides inhibit bacterial transla-
tion by binding to the large ribosomal subunit in the vicin-
ity of the peptide exit tunnel (12). This interaction requires
an intact cyclic macrolide ring and in most cases the amino
sugar desosamine (Fig. 7).
Enzymatic resistance to macrolide antibiotics occurs
either by modification of the desosamine sugar (see Sect. 3
below) or by linearization of the macrolactone ring (Fig. 8).
The latter mechanism is catalyzed by esterases that hydro-
lytically cleave the lactone resulting in ring opening and
consequently the inability to effectively bind to the peptide
exit tunnel. The erythromycin esterases EreA and EreB
have been identified in E. coli integrons and R-plasmids
(14–17).
8 Biochemical Logic of Antibiotic Inactivation and Modification 85

Fig. 7 Interactions of the macrolide

erythromycin with the bacterial ribosomal
RNA. Key 23S rRNA residues are shown
in grey and the interactions are designated
as arrows. The hydroxyl group that serves
as a site of inactivation interacts with
A2058. Adapted from (13)

Fig. 8 Inactivation of the macrolide

erythromycin by hydrolysis.
Macrolides can be inactivated by
hydrolysis of the macrolactone ring.
This reaction is mediated by the
esterase Ere

2.4 Type B Streptogramins occurs through a ring-opening reaction catalyzed by the

The streptogramins are natural product inhibitors of bacte-
rial translation that consist of two structurally distinct
classes, denoted as Type A and Type B (18). Type B strepto-
gramins are cyclic depsipeptides that, like macrolides, bind
to a region of the bacterial ribosome’s peptide exit tunnel
(19, 20) (Fig. 9). Type A streptogramins are mixed
peptide-polyketide antibiotics that bind to the peptidyl-
transferase centre of the ribosome. Enzymatic resistance to
Type A streptogramins occurs via an acetyltransfer mecha-
nism, while enzymatic resistance to Type B streptogramins
enzyme Vgb. Vgb, originally identified in streptogramin
resistant Staphylococcus aureus, cleaves the cyclic peptide
(21), resulting in depsipeptide linearization. The resulting
structure no longer exhibits affinity for the bacterial ribo-
some, mirroring the biochemical logic of macrolide
esterases. However, the mechanism of ring opening is quite
distinct. Rather than causing a hydrolytic reaction at the
thermodynamically vulnerable ester bond of Type B strep-
togramins, Vgb catalyzes a lyase reaction that results in a
ring opening of the peptide by a C–O cleavage strategy
(22) (Fig. 10).
86
2.5 Tetracycline
The tetracycline antibiotics have found extensive clinical
use for almost half a century. This class of antibiotics binds
divalent metals and acts by blocking bacterial translation by
binding to the small ribosomal subunit (23) (Fig. 11). The
principal mechanisms of clinical tetracycline resistance are
Fig. 9 Streptogramin B interactions with bacterial ribosome.
Quinupristin, a Type B Streptogramin, binds to the bacterial ribosome’s
polypeptide exit tunnel. Key interactions with the 23S rRNA are desig-
nated as arrows. Adapted from (19, 20)
Fig. 10 Vgb-catalyzed inactivation of the type B streptogramin quinupristin. Quinupristin undergoes a ring-opening elimination reaction, result-
ing in an inactive derivative
V. D’Costa and G.D. Wright
efflux and ribosomal protection (23, 25). However, an enzy-
matic mechanism of tetracycline resistance, originally dis-
covered in Bacteroides (26), has been identified that
inactivates the antibiotic via an oxygen-dependent process.
Purification of the enzyme that catalyzes this reaction, TetX,
followed by careful analysis of the products of the reaction
showed that the enzyme first facilitates mono-hydroxylation
of the antibiotic at position 11a, effectively disrupting the
essential metal-binding site on the molecule (27) (Fig. 12).
Furthermore, this step triggers a nonenzymatic decomposi-
tion of the antibiotic to a form of unknown structure that
turned the growth media black. This enzyme is also capable
of mono-hydroxylation of the latest generation of tetracy-
cline antibiotics, the glycylcyclines, resulting in resistance,
but not the subsequent nonenzymatic decomposition of the
antibiotic (28).
3 Antibiotic Modification
The most diverse class of resistance enzymes catalyzes the
covalent modification of antibiotics. This strategy confers
resistance by means of group transfer and includes both O-
and N-acetylation, O-phosphorylation, O-nucleotidylylation,
O-ribosylation, and O-glycosylation. Covalent modification
of antibiotics by this class of enzymes does not destroy the
essential active warheads of the compounds, as described in
the previous section, but rather obstructs interaction of the
antimicrobial with its target. This is accomplished by func-
tionally derivatizing the antibiotic at structural location(s)
that play an essential role in binding with the target. By doing
so, key interactions (e.g., hydrogen bonding, ionic inter-
actions, steric complementarity, etc.) are disrupted by the
introduction of the modifying group, resulting in an overall
8 Biochemical Logic of Antibiotic Inactivation and Modification 87

Fig. 11 Interactions of tetracycline with the

bacterial 16S rRNA. Interactions are
designated by grey arrows and key ribosomal
RNA residues are indicated in grey. Adapted
from (24)

Fig. 12 TetX-mediated
inactivation of tetracycline.
TetX catalyzes the
hydroxylation of the
antibiotic, which interferes
with the metal-binding site
required for activity.
Adapted from (27)

decrease in the affinity of the antibiotic derivative for its tar-
get in comparison to the unmodified counterpart.
This antibiotic inactivation tactic requires the presence of
a co-substrate for enzyme activity, such as acetyl-CoA, ATP
or UDP-glucose. Consequently, enzyme activity is localized
to the bacterial cytosol. The inactivation products are com-
monly stable in the cellular environment, thus the reactions
are considered to be irreversible in the absence of an enzyme
that counteracts the reaction. However it is conceivable that
the presence of such reversing enzymes (e.g., phosphatases,
acylases) can undo resistance in vivo.
3.1 Aminoglycosides
The aminoglycoside class of antibiotics is a diverse group of
hydrophilic aminocyclitols modified by amino and neutral
sugars that consist of both natural products and their
semi-synthetic derivatives. Polycationic aminoglycoside
antibiotics, as previously mentioned, act by interacting with
the 16S rRNA region of the bacterial ribosome’s A-site,
impairing its decoding mechanism and consequently result-
ing in a misreading of the mRNA (29–32). X-Ray crystallo-
graphic studies of aminoglycoside antibiotics and the small
88
ribosomal subunit or fragments of the 16S rRNA reveal that
interactions between aminoglycosides and the ribosome span
the entire length of the antibiotic (24, 33–36). The primary
mode of interaction is through predicted hydrogen bonding
and ionic contacts between the antibiotic amino and hydroxyl
groups and the 16S rRNA (Fig. 13).
The most prevalent mode of clinically relevant aminogly-
coside resistance is via enzymatic modification (38). Three
classes of enzymes, whose reactions differ with respect to
the functional group transferred and the acceptor site, are res-
ponsible for aminoglycoside modification. Aminoglycoside
acetyltransferases (AACs) modify amino groups, aminogly-
coside phosphotransferases (APHs) target hydroxyl groups,
and aminoglycoside nucleotidyltransferases (ANTs) modify
hydroxyl groups (Fig. 14). There are numerous examples of
each group and the genes encoding aminoglycoside-modify-
ing enzymes are commonly located on mobile genetic
elements such as plasmids or transposons, although some
have been identified within chromosomal DNA (39–41).
The action of all three classes of modifying enzyme changes
the electronic properties of the antibiotic, in addition to its
size and structure. These alterations result in steric and
electronic clashes between the modified antibiotic and the
16S rRNA, impairing efficient binding and resulting in
resistance.
Fig. 13 Interactions of the aminoglycoside
gentamicin C1a with the bacterial 16S rRNA. Key
16S rRNA residues are shown in grey and the
interactions are designated as arrows. Adapted
from (37)
V. D’Costa and G.D. Wright
3.1.1 Aminoglycoside Acetyltransferases
(AAC Family)
Aminoglycoside acetyltransferases (AACs) utilize intracellu-
lar acetyl-CoA as a co-substrate, catalyzing the formation of
a biologically stable amide with the aminoglycoside. Although
AACs primarily modify amino groups (N-acetylation),
O-acetylation has been documented with the acetyltransferase
domain of the bifunctional enzyme AAC(6′)-APH(2′′) (42)
and the mycobacterial enzyme AAC(2′)-Ic (43).
AACs are members of the GCN5 superfamily of proteins
(44, 45). Although all enzymes of this class do not exhibit
significant primary sequence homology or conserved cata-
lytic residues, analysis of available X-ray crystal structures
of four enzymes (AAC(6′Ii), AAC(3)-Ia, AAC(2′)-Ic, and
AAC(6′)-Iy) indicates that the aminoglycoside binding
pocket commonly contains a highly negatively charged sur-
face to accommodate the polycationic antibiotic (45–48).
AACs are further classified based on the site of acetyla-
tion along the aminoglycoside structure. By convention, the
position along the amino sugar/aminocyclitol targeted is
indicated in brackets, and the amino sugar/aminocyclitol
modified is designated in brackets after the position of attack.
For example, in Fig. 14, AAC (3) indicates acetylation of the
3-position of the central aminocyclitol moiety, the term (2′)
8 Biochemical Logic of Antibiotic Inactivation and Modification 89

Fig. 14 Inactivation of the amino-

glycoside gentamicin C1a by
aminoglycoside-modifying enzymes.
Aminoglycosides can be modified by
the addition of acetyl groups,
phosphate groups or AMP moieties.
These enzymatic reactions are
catalyzed by aminoglycoside
acetyltransferases (AACs), phospho-
transferases (APHs), and nucleotidyl-
transferases (ANTs) respectively.
(a) Sites of aminoglycoside
inactivation. Groups targeted are
labeled by the corresponding
resistance enzymes. (b) The products
of aminoglycoside acetyltransferases,
phosphotransferases, and
nucleotidyltransferases

suggests modification of the 2-position of the 4-substituent 3.1.3 Aminoglycoside Nucleotidyltransferases

diaminohexose, and (2′′) indicates modification of the
2-position of the 6-substituent aminohexose.
Modification of aminoglycosides by AACs results in neu-
tralization of the positive charge on the target amino group,
eliminating key ionic interactions and sterically blocking
interaction with the 16S rRNA.
3.1.2 Aminoglycoside Phosphotransferases
(APH Family)
Aminoglycoside phosphotransferases catalyze the phosphory-
lation of specific aminoglycoside hydroxyl residues (Fig. 14),
using intracellular ATP as a phosphate donor. Classification of
phosphotransferases is based on the site of action, analogous
to the system described above for acetyltransferases. The APH
enzymes are subdivided into seven classes, based on their site
of action on the aminoglycoside: APH(2′′), APH(3′), APH(3′′),
APH(4), APH(6), APH(7′′), and APH(9). There exists very
little primary sequence homology among the subclasses of
APHs; however common signature sequences and residues
essential for catalysis are evident (49).
The largest subclass of APHs modifies the 3′-hydroxyl of
the aminoglycoside and is consequently called APH(3′) (49).
Crystal structure analysis of the enzyme APH(3′)-IIIa bound
to ADP has established a remarkable similarity to known
protein kinases, despite the low primary sequence similarity
(50). This may be evidence that APH(3′) and protein kinases
evolved from a common ancestor.
Modification of aminoglycosides by APH-catalyzed
phosphorylation results in changes in overall charge and size
of the antibiotic. This results in electronic and steric clashes
with the 16S rRNA and a 103-fold impairment of binding to
the target 16S rRNA (51).
(ANT Family)
Aminoglycoside nucleotidyltransferases utilize the co-substrate
ATP to transfer an AMP moiety to selected aminoglycoside
hydroxyl groups. This class of inactivating enzymes has been
identified in some Gram-positive bacterial isolates, as well as
a broad range of Gram-negatives (40).
ANTs display very little primary sequence homology,
however they exhibit a common core signature region (49).
The enzyme ANT(4′)-Ia has been crystallized and its atomic
structure determined alone and in complex with the sub-
strates kanamycin and a nonhydrolyzable ATP analogue (52,
53). Although the primary sequence homology is only 10%,
the putative active site was determined to be structurally
equivalent to that of rat DNA-polymerase β, one of the small-
est and simplest of the polymerases (54), and catalyzes a
similar chemical reaction.
Paralleling the strategies of the other classes of aminogly-
coside modifying enzymes, the action of ANTs causes a
change in antibiotic structure that results in both a steric and
electronic clash between the antibiotic and its target. This
theme and molecular logic finds other examples in antibiotic
resistance as outlined below.
3.2 Macrolides
Macrolide antibiotics are a large class of antibiotics that
include both natural products and semi-synthetic derivatives.
Most macrolides are derived from bacterial fermentation
products, particularly from species of the actinomycete genus
Streptomyces. Erythromycin was the first member of this
class to be identified (1952), a natural product of Streptomyces
90
erythrae (now known as Saccharopolyspora erythrae). The
name macrolide is derived from the macrolactone ring that
characterizes the class, which can consist of 14–16 members
and is commonly attached to one or two sugar moieties.
Macrolides have found an important role in the treatment of
clinical pathogens. Since their introduction in the 1950s, efforts
to expand the spectrum of activity and deal with the inevitable
resistance that followed have resulted in a number of different
classes of derivatives. Azalides incorporate an endocyclic
nitrogen into the macrolactone ring. Azithromycin, the first
azalide approved for clinical use, exhibits increased potency
against a number of Gram-negative organisms, as well as a
longer apparent half-life. Ketolides, which have a keto group
in place of the l-cladinose in the 3-position, exhibit increased
activity against a number of macrolide resistant strains.
Macrolides, as described previously, act by binding with
the 23S rRNA of the bacterial 50S ribosomal subunit adja-
cent to the peptide exit tunnel, blocking polymerization at
the peptidyltransferase centre and inducing premature pep-
tide dissociation (13, 20). Interactions with the ribosomal
RNA occur primarily through hydrogen bonding, as shown
with erythromycin in Fig. 7. Much of the hydrogen bonding
ability of macrolides can be attributed to their hydroxyl and
amino groups, which interact with the nitrogenous bases or
backbone phosphate groups of the rRNA. As shown, the
Fig. 15 Inactivation of the
macrolide erythromycin by
Mph and Mgt. Macrolides
can be modified by the
addition of phosphate and
glucose moieties. The
hydroxyl group targeted and
the subsequent modifica-
tions are labeled in grey
V. D’Costa and G.D. Wright
hydroxyl residue of the desoamine sugar plays a key role in
the interaction of the macrolide with its target rRNA.
The second mode of enzymatic macrolide inactivation
occurs by modification of this essential desosamine sugar
(Fig. 15). Modification of the 2′ hydroxyl residue can occur
by either phosphorylation or glycosylation. This hydroxyl
group, as mentioned, plays an important role in macrolide-
target binding, serving as a multiple contact site of hydrogen
bonding with the 23S rRNA (Fig. 7). Modification of the
antibiotic at this site therefore results in loss of vital struc-
tural connections with the target and also results in steric
impairment of complex formation.
3.2.1 Macrolide Kinases (Mph Family)
Clinical resistance to macrolides has been documented by
means of phosphate transfer from ATP by a family of
macrolide-inactivating phosphotransferases, encoded by the
mph genes (Fig. 15). These enzymes have been identified in
both Gram-positive and Gram-negative pathogens (55–57).
Members of the Mph class of resistance enzymes appear to
be extremely diverse with respect to the nucleotide sequences
that encode the enzymes. The gene mphA exhibits a 66% G + C
content, uncharacteristically high for the organism it was
8 Biochemical Logic of Antibiotic Inactivation and Modification 91

originally identified in (E. coli, G + C content approximately

50%) (58). The sequences of mphB and mphC, conversely,
display a G + C content of only 38%. The structure of these
enzymes has yet to be elucidated, however they share canoni-
cal phosphate transfer residues with APHs and probably
resemble these aminoglycoside resistance enzymes.

3.2.2 Macrolide Glycosyltransferases (Mgt Family)

Resistance to macrolides in antibiotic-producing strains of

bacteria as well as other soil-dwelling organisms is com-
monly accomplished by intracellular glycosylation of the
antibiotic prior to export. This is catalyzed by a class of
enzymes called macrolide glycosyltransferases (Fig. 15).
Members of this class include Mgt, from the nonmacrolide-
producing Streptomyces lividans (59, 60), as well as OleD
(61) and GimA (62) from the macrolide-producing S. antibi-
oticus and S. ambofaciens respectively.
Members of the Mgt family are extremely similar with
respect to both DNA and primary amino acid sequences,
however each enzyme appears to display a unique substrate
specificity in vitro (63).
Fig. 16 Interactions of the rifampicin with the bacterial β-subunit of
RNA polymerase. Key amino acid residues are shown in grey and the
interactions are designated as arrows. Adapted from (64)
3.3 Rifamycins

The rifamycin family of antibiotics includes semisynthetic

derivatives of a natural product synthesized by the actinomy-
cete Amycolatopsis mediterranei. Rifampicin was first intro-
duced in 1968, but the most widely used member of the group
is rifamycin, which has become an integral component of the
multiantibiotic gold-standard treatment for Mycobacterium
tuberculosis infections.
Rifamycins target the bacterial β-subunit of RNA poly-
merase. The crystal structure of rifampicin bound to the RNA
polymerase of Thermus aquaticus has been determined to
3.3Å (64). Twelve amino acid residues were shown to asso-
ciate closely with rifampicin, six of which participate in
hydrogen bonding, as shown in Fig. 16. The majority of these
interactions occur at four crucial hydroxyl residues on the
rifampicin molecule including a key interaction between the
hydroxyl group at position 23 and the amide of Phe394.
Resistance to rifampicin commonly occurs through
amino acid mutations in the RNA polymerase β-subunit.
However, inactivating enzymes have also evolved to modify
the antibiotic (Fig. 17). Group transfer can result in ADP-
ribosylation, phosphate addition, and glycosylation of the
rifampicin’s 23-hydroxyl. Through the addition of a bulky
functional group, rifampicin’s tight binding to its target is
impaired.
3.3.1 ADP-Ribosyltransferases (ARR Family)
Although both eukaryotic and prokaryotic proteins are com-
monly modified by means of ADP-ribosyl transfer, this
mechanism of antibiotic resistance has so far only been doc-
umented for the rifamycin class. Resistance to rifampicin by
means of ADP-ribosylation has been documented in numer-
ous nontuberculosis Mycobacterium strains, such as M. smeg-
matis. Modification is due to a unique ADP-ribosyltransferase
known as ARR (65). Another inactivating ribosyltransferase
(ARR-2) with 55% identity to ARR has been identified in a
multidrug resistant integron in a Gram-negative Acinetobacter
strain (66).
The resistance enzymes from the ARR family are
characteristically small (approximately 200 amino acids) and
do not display sequence similarity to protein ADP-ribosyl
transferases. They target the hydroxyl residue at position 23
of rifampicin (Fig. 17), and utilize nicotinamide adenine
dinucleotide (NAD+) as a donor for the ADP-ribosyl moiety.
It has also been shown that this ADP-ribosylated antibiotic
can undergo subsequent decomposition to release the ADP
moiety (65, 67, 68).
92
Fig. 17 Inactivation of rifampicin. Rifampicin can be enzymatically modified by the addition of ADP-ribose, glucose, and phosphate moieties.
The hydroxyl group targeted and the subsequent modifications are labeled in grey
3.3.2 Rifampicin Kinases
Inactivation of rifampicin by phosphorylation (Fig. 17) has
been documented by species of Nocardia (69, 70), Rhodo-
coccus (71), as well as Bacillus (72). The kinases responsible
for this inactivation have yet to be identified or studied.
Phosphorylation of rifampicin’s hydroxyl at position 23 logi-
cally impedes interaction with the RNA polymerase target,
although little has been done to elucidate the details of this
mechanism.
3.3.3 Rifampicin Glycosyltransferases
Glycosylation of the 23-position of rifampicin has also been
reported in Nocardia species (69, 73) (Fig. 17). Glycosyla-
tion at this position prevents hydrogen bonding with the
23-hydroxyl, hindering effective target binding to RNA
polymerase β. The genes encoding the enzymes that catalyze
these reactions have yet to be elucidated.
4 Summary and Conclusions
Bacteria use enzymes to strategically incapacitate and neu-
tralize antibiotics. Tactically this includes deployment of
mechanisms that either destroy the essential chemical
V. D’Costa and G.D. Wright
“warhead” or “active site” of the antibiotic (e.g., cleavage of
the β-lactam ring by β-lactamases), or mechanisms that
modify key structural elements that are essential for binding
of the antibiotic to target (e.g., phosphorylation of aminogly-
cosides). The molecular logic of these approaches is revealed
with knowledge of the interaction of the active antibiotic
with its cellular target. Study of enzymatic resistance there-
fore not only can inform on molecular aspects of antibiotic-
target interactions, but can serve to guide target identification
where this is not yet known.
Another spin-off of the study of these mechanisms is the
opportunity to develop strategies to overcome the resistance
activity. For example, the observation that aminoglycosides
were inactivated by phosphorylation of the hydroxyl group at
position 3′ of the 6′-aminohexose ring guided the development
of antibiotics such as tobramycin, which lack this hydroxyl and
which were consequently resistant to this mechanism. A second
approach is to develop inhibitors of resistance enzymes. This
strategy has been very successful in the β-lactam arena where
combinations of an antibiotic and a resistance enzyme inhibi-
tor, such as amoxicillin and clavulanic acid respectively
(Augmentin), have emerged as billion-dollar drugs.
Finally, antibiotic-modifying enzymes also have the
opportunity to be exploited as novel reagents in antibiotic
semi-synthesis as protecting agents. In some cases, antibiotic-
modifying proteins are employed by antibiotic-producing
bacteria as a means of self-protection. For example, during
streptomycin biosynthesis in Streptomyces griseus, the
8 Biochemical Logic of Antibiotic Inactivation and Modification
enzyme StrA modifies mature antibiotic to the inactive
6-phosphoderivative. Export of this “pro-drug” is followed
by unmasking of the cytotoxic agent by an extracellular
phosphatase (74). These enzymes could serve as reagents to
chemically protect and deprotect sensitive structural elements
in the synthesis of libraries of semi-synthetic antibiotics.
Enzymatic resistance therefore provides both challenges
and opportunities in new drug development. Through a com-
bination of rigorous biochemical analysis and parallel efforts
in the determination of enzyme structure and target identifi-
cation, new approaches that circumvent these selective and
potent agents can be developed to extend antibiotic lifetime
and efficacy.
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