the genomic enzymology of antibiotic resistance

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GE44CH02-Wright ARI 17 July 2010 13:29

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The Genomic Enzymology of Antibiotic Resistance

Mariya Morar and Gerard D. Wright

M.G. DeGroote Institute for Infectious Disease Research and the Department of Biochemistry and Biomedical Sciences, McMaster University, Hamilton, Ontario,L8N 3Z5, Canada; email: [email protected]

Annu. Rev. Genet. 2010. 44:25–51

The Annual Review of Genetics is online atgenet.annualreviews.org

This article’s doi:10.1146/annurev-genet-102209-163517

Copyright c 2010 by Annual Reviews.All rights reserved

0066-4197/10/1201-0025$20.00

Key Words

antibiotic resistome, proto-resistance gene, antibiotic resistance

mechanism, enzyme structure-function.

Abstract

The need for new antibiotic therapies is acute and growing in large partbecause of the emergence of drug-resistant pathogens. A vast number

of resistance determinants are, however, found in nonpathogenic mi-

croorganisms. The resistance totality in the global microbiota is the

antibiotic resistome and includes not only established resistance genes

but alsogenes that have the potential to evolve into resistance elements.

We term these proto-resistance genes and hypothesize that they share

common ancestry with other functional units known as housekeeping

genes. Genomic enzymology is the study of protein structure–function

in light of genetic context and evolution of protein superfamilies. This

concept is highly applicable to study of antibiotic resistance evolution

from proto-resistance elements. In this review, we summarize some of

the genomic enzymology evidence for resistance enzymes pointing to

common ancestry with genes of other metabolic functions. Genomic

enzymology plays a key role in understanding the origins of antibiotic

resistance and aids in designing strategies for diagnosis and prevention

thereof.

25




Antibiotic resistome:

the totality of antibiotic resistanceelements in microbialpopulations across theglobe

INTRODUCTION

The Problem of Antibiotic Resistanceand the Concept of the Resistome

Antibiotics are the cornerstone drugs of mod-

ern medicine. Ever since their discovery in

the first half of the twentieth century, clini-

cians have exploited them not only to treat in-

fections, but also to allow numerous medical

procedures by prophylactically preventing in-

fectious disease. Many modern therapies from

cancer chemotherapy to organ transplantation

would simply not be conceivable without an-

tibiotics.They truly are wonderdrugs that have

changed the practice of medicine.

There are several classes of antibiotics avail-

able to clinicians (Table 1). These compoundstarget vital microbial biochemistry at vulner-

able metabolic and physiologic hubs such as

translation, DNA replication, and cell wall

biosynthesis. Antibiotics can arrest cell growth

(bacteriostatic) or kill the cells outright (bac-

tericidal) depending on their mode of action

and the biochemical and genetic idiosyncrasies

of individual species of pathogens. With a few

exceptions, all the members of our current ar-

senal of antibiotic drugs were discovered dur-

ing a brief 20-year period of tremendous pro-

ductivity in drug discovery following the first

clinical use of penicillin in the mid-1940s. Themajority of these antibiotics were discovered

by screening soil bacteria, mostly of the acti-

nomycete family, for the production of bioac-

tive molecules with microbial growth inhibition

activity. It is this small set of chemical scaf-

folds that has formed the basis of success of

antibiotic chemotherapy for over half a cen-

tury (45). At least for the foreseeable future,

these same scaffolds will be required to main-

tain our continued reliance on antibiotics in

medicine.

Soon after the first use of antibiotics in

medicine, resistant organisms were seen to arise

during therapy (1). The subsequent decades

have seen a cycle of emergence of resistant mi-

crobes, followed by the subsequent develop-

ment of new antibiotics. These have included

brand new classes of drugs as well as medicinal

chemical elaboration of established classes to

avoid resistance. This cyclical approach has

proven highly successful, and we are now em-ploying fourth, fifth, and sixth generation an-

tibiotics in many cases. The continued evolu-

tion andselection ofnew resistancegenes, along

with the emergence of multi-drug resistant or-

ganisms, are driving the need for new drugs

(16).

Paradoxically, despite a growing unmetclin-

ical need for new antibiotics, there has been a

general retreat over the past decades in new an-

tibiotic drug discovery by the pharmaceutical

industry. One reason is that it has proven to

be difficult to identify novel antibiotic chemi-

cal scaffolds using the screening methods thatwere so profitable in the past. At the same time,

newer target-based, high-throughput screen-

ing approaches that have been very successful

in other areas of drug discovery have so far

failed to produce in thearea of antibiotics (108).

There is therefore a chemical scaffold gap that

needs to be bridged to spur new directions in

antibiotic discovery. Compounding these chal-

lenges is the problem of resistance that contin-

ues to grow unabated.

Antibiotic resistance has evolved along with

the deployment of these cytotoxic chemicals.

The first resistance genes were no doubt asso-ciated with antibiotic-producing bacteria in the

environment and have evolved over the millen-

nia (6). Presently, the evolution and selection

of resistance is happening on a larger scale and

is being accelerated as we produce and use an-

tibiotics on the ton scale in human health and

agriculture (7, 61).

Not surprisingly, the focus of resistance re-

search has been principally on pathogenic mi-

crobes in healthcare settings. However, this is

an incomplete representation of the pool of re-

sistance elements that impact antibiotics on a

global scale. We recently proposed the con-cept of the antibiotic resistome to describe

the totality of resistance in microbial popu-

lations across the globe (36, 149). The resis-

tome includes not only those elements that

26 Morar ·Wright




Table 1 Antibiotics in clinical use and modes of resistance

Antibiotic class Examples Target Mode of resistance

β-lactams Penicillins (ampicillin) Peptidoglycan biosynthesis Hydrolysis

Cephalosporins (cephamycin) EffluxPenems (meropenem) Altered target

Monobactams (aztreonam)

Aminoglycosides Gentamicin Translation Phosphorylation

Streptomycin Acetylation

Spectinomycin Nucleotidylation

Efflux

Altered target

Glycopeptides Vancomycin

Teicoplanin

Peptidoglycan biosynthesis Reprogramming of peptidoglycan

biosynthesis

Tetracyclines Minocycline Translation Monooxygenation

Tigecycline Efflux

Altered target

Macrolides Erythromycin Translation Hydrolysis

Azithromicin GlycosylationPhosphorylation

Efflux

Altered target

Lincosamides Clindamycin Translation Nucleotidylation

Efflux

Altered target

Streptogramins Synercid Translation C-O lyase (type B streptogramins)

Acetylation (type A streptogramins)

Efflux

Altered target

Oxazolidinones Linezolid Translation Efflux

Altered target

Phenicols Chloramphenicol Translation AcetylationEfflux

Altered target

Quinolones Ciprofoxacin DNA replication Acetylation

Efflux

Altered target

Pyrimidines Trimethoprim C1 metabolism Efflux

Altered target

Sulfonamides Sulfamethoxazole C1 metabolism Efflux

Altered target

Rifamycins Rifampin Transcription ADP-ribosylation

Efflux

Altered target

Lipopeptides Daptomycin Cell membrane Altered target

Cationic peptides Colistin Cell membrane Altered target

Efflux

www.annualreviews.org • Enzymology of Antibiotic Resistance 27




Genomic

enzymology: study of protein structure andfunction taking intoaccount geneticcontext and evolutionwithin a proteinsuperfamily

Proto-resistanceelement: a gene thatmay have any functionbut has potential toevolve into a resistanceelement

are acute problems in the clinic but also takes

into account the vast reservoir of resistance

genes in nonpathogenic organisms (23). This

idea is important to fully understand the evo-lution of resistance, the maintenance of resis-

tance genes within microbial populations, and

the spread of these genes between species and

genera.

Resistance can arise as the result of altered

molecular targets, efflux of antibiotics from

within the cell, blockade of antibiotic entry into

the cell, and chemical modification or destruc-

tion of the compounds. In many cases, a sin-

gle antibiotic or class is impacted by more than

one, or even all, of these mechanisms (e.g., see

Table 1). Furthermore, these mechanisms can

be collected within a single organism resultingin combinatorial resistance. The range of re-

sistance mechanisms is common to pathogenic

and nonpathogenic bacteria and suggests a

complex natural history of evolution and selec-

tion. The evolution of enzymes that modify an-

tibiotic targets and the antibiotics themselves

are of particular interest. Unlike many efflux

systems, which tend to have been selected to

purge a wide variety of toxic organic molecules

from the cell (91), or single site mutations in

targets, which may confer resistance with neu-

tral evolutionary impact, enzymes likely have

evolved in specific response to antibiotics (148).Theseare particularlynoteworthy for theirsub-

strate specificity (target or antibiotic).

The wellspring of the resistome is the col-

lectionof genes that have thepotential to evolve

into resistance elements. We have termed these

proto-resistance genes as they are analogous to

the proto-oncogenes of the cancer field (98).

Proto-resistance genes are found in numerous

bacterial chromosomes and are often annotated

as putative antibiotic resistance elements during

genome sequencing. We hypothesize that it is

these proto-resistance genes, which may have

other functions in the cell and have little or noantibiotic resistance capacity, that are the ulti-

mate source of the highly efficient and robust

resistance elements whose function is to detox-

ify antibiotics (Figure 1a).

Genomic Enzymology as an Approach to Understanding Resistance Evolution

Gerlt & Babbit(50) have suggested theterm ge-nomic enzymology as a strategy to understand

the evolution of protein function within en-

zyme superfamilies. This concept is highly ap-

plicable to the study of the evolution of antibi-

otic resistance from proto-resistance enzymes

of the resistome (Figure 1b). Protein structure

and function studies clearly show that many

antibiotic resistance enzymes are members of

several enzyme superfamilies, providing clues

for the nature of ancestral proto-resistance

elements. Furthermore, bacterial genome se-

quences reveal numerous chromosomal genes

annotated as possible resistance elements, evenin the absence of the need for self protection of

antibiotic producers. These genes may encode

bona fide resistance proteins or could be proto-

resistance elements. Expression and character-

ization of the structure and function of these

proteins provides vital information for the un-

derstanding of resistance evolution and insight

into leveraging this knowledge to overcome re-

sistance in the clinic. In this review, we describe

the genomic enzymology of several classes of

resistance enzymes with a goalof understanding

how resistance evolves and what can be learned

from these studies that can have applicability

in drug discovery and medicine. We are care-

ful to note that in most cases the generally low

level of sequence similarity between resistance

enzymes and structurally similar enzymes can

often only suggest at common ancestry, but

lacks the robustness to unequivocally demon-

strate evolutionary relatedness. We therefore

have generally resisted comment on phylogeny

except where the evidence is clear.

ANTIBIOTIC HYDROLASES

AND LYASES

β-Lactamases

β-Lactam antibiotics include the penicillins,

cephalosporins, penems, and monobactams

28 Morar ·Wright




Ancestralprotoresistance

protein

Housekeepingprotein

Resistanceprotein

a

b

Identify homologsand protoresistance

genes

Characterizeenzyme

Vmax

0.5Vmax

K m

V

[S]

Determine3D structure

Figure 1

Key concepts in antibiotic genomic enzymology. (a) Representation of the concept of proto-resistancedepicting the ability of the same ancestral core to acquire a different function, housekeeping versusresistance. (b) The genomic enzymology strategy integrates enzyme structure, function, and genetic context.

that are mainstay drugs for the treatment of

bacterial infection. These antibiotics act by co-

valentbinding through an active siteSer residue

to so-called penicillin binding proteins (PBPs),

which are required for assembly and tailoring

of the peptidoglycan component of the bacte-

rial cell wall (92).

Resistance to β-lactam antibiotics is princi-pally the result of the expression of hydrolytic

enzymes, β-lactamases, that inactivate the

antibiotics via lactam ring opening hydrolysis

(Figure 2a). Two general chemical mecha-

nisms are recognized that accomplish this reac-

tion. The first is via canonical Ser nucleophile

attack at the lactam carbonyl carbon, resulting

in a covalent enzyme intermediate that mimics

the inactivation step of PBPs. The complex

then undergoes hydrolysis comparable to Ser

protease/esterasechemistry.The secondmech-

anism is by Zn2+-dependent activation of a

hydrolytic water molecule, here in mechanisticanalogy to metallo-peptidases. β-Lactamases

have been categorized based on protein se-

quence analysis [Ambler classification (3)] and

by function [Bush-Jacoby notation (21)]. The

Ambler notation separates β-lactamases into





N

S

HN

COOH

R

O

O

HH

PBP

NHS

HN

COOH

R

O

OH

O

Ser

NHS

HN

COOH

R

O

OH

O

NHS

HN

COOH

R

O

OH

HOSer

OH

NHS

HN

COOH

R

O

OH

HO

O

HZn

Ser

OH

Ser

OH

Class A, C, D

Class B

a

b c

Fez-1

IMP-1

Ancestralcore

Ancestralcore

GLX2

SDSA1AmpC

OXA

R61DDpep

TEM-1

H2O

Zn

α-helix

β-strand

Loop

30 Morar ·Wright




4 groups: A, B, C, and D. Class B enzymes are

Zn2+-dependent metallo enzymes, and class A,

C, D are active-site Ser enzymes. Phylogenetic

analysis reveals that the class B metallo-enzymes are distinct from the class A, C, and D

Serproteins. Theformer consistof twodiscrete

structural groups (B1–2 and B3 subclasses)

(Figure 2b) (54, 55). On the other hand,

the class A and D enzymes are homologues

and have diversified from a common shared

ancestor with the class C enzymes (52, 53).

In 1986, comparison of the three-dimensional

(3D) structure of class A enzymes with

PBPs revealed conservation of 3D structure

(Figure 2c ) and of biochemical mechanism (74,

92). Thedetermination of several 3D structures

of PBPs and class A, C, and D β-lactamasesin the past 25 years has served to solidify the

relationship between cell wall–metabolizing

enzymes and drug-resistance β-lactamases.β-Lactamases therefore share structure and

function with enzymes associated with cell wall

metabolism and general peptidase activities.

Phylogenetic evidence suggests that the class

A Ser β-lactamases emerged 2.4 billion years

ago from a common ancestor of PBPs (53).

The genes encoding these enzymes and the

related class D lactamases are widely circulated

in bacteria through the aegis of mobile genetic

elements such as plasmids. Class C enzymes,however, are chromosomally encoded (al-

though in the past decades, they have begun

to be associated with mobile genetic elements,

no doubt a recent event linked to antibiotic

selection) and diverged from the class A and

D enzymes approximately 1.8 billion years ago

(based on their association with Gram negative

proteobacteria) (53). Consistent with the

ancient divergence of β-lactamases from PBPs,

the former have no peptidoglycan biosynthetic

activity. Recent evolution has contributed

to the broadening of the antibiotic substrate

specificity of β-lactamases, and the numberof distinct enzymes with unique function

continues to grow and is approaching 1,000

entries (www.lahey.org/studies).

Streptogramin B Lyase

Thepublicationof the3D structuresof thebac-

terial ribosomal subunits in complex with an-

tibiotic inhibitors of translation over the past

decade has transformed our understanding of

antibiotic action and resistance. The large ribo-

somal subunit includes twomajorsitesof antibi-

otic binding: the peptidyl transfer center (theactive site of the ribosome) and the peptide exit

tunnel where nascent proteins transit from the

peptidyl transfer center and emerge from the

ribosomal complex. Streptogramin antibiotics

are composed of two structurally distinct com-

ponents, type A peptide-polyketide hybrids that

bind to the peptidyl transfer center and type B

cyclic depsipeptides that block the peptide exit

tunnel. These antibiotic are produced simulta-

neously by the same organism resulting in syn-

ergistic antibiotic activity (100).

Type B streptogramins are inactivated by

cleavage of the lactone link by an enzyme, vir-giniamycin B lyase (Vgb) (2). The structure-

function of this enzymehas been thoroughlyin-

vestigated showing that the ring opening takes

place via an elimination reaction, assisted by a

hexa-coordinated Mg2+ ion and a catalytic His

base (8, 78, 87, 99). Vgb has a seven-blade β-

propeller fold, similar to that of a muconate-

lactonizing enzyme (MLE) (Figure 3a). MLE

catalyzes a similar reaction, that of cyclization

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−

Figure 2

Genomic enzymology of β-lactamases. (a) Enzymatic reaction of a β-lactam hydrolysis catalyzed by PBPs versus class A, C, D serineβ-lactamases versus class B metallo β-lactamases, summarizing the similarities and differences in this transformation. Spheres representthe remainder of the enzyme. (b) Conservation in structure between class B1–2 (IMP-1 complexed with two Zn2+ ions; pdb id: 1DDK),B3β-lactamases (Fez-1 complexed with two Zn2+ ions; pdb id: 1L9Y), glyoxalase II (complexed with Zn2+ and Fe3+ ions; pdb id:1XM8), and alkylsulfatase (complexed with two Zn2+ ions; pdb id: 2CFZ). Their emergence from a common ancestor is represented by the common structural core. Structures are shown in ribbon diagram and color coded by secondary structure. The structural core is inblack and white. (c ) Conservation in structure between class A (TEM1 complexed with transition state analog; pdb id: 1M40), C (AmpC;pdb id: 1IEL), D β-lactamases (OXA complexed with Meropenem; pdb id: 1H8Y), and PBP DD-peptidase R61 (pdb id: 3PTE).





Ancestralcore

MLE Vgb

a

b

N

NHO

OH

HN

O

NO

O

ONH

N

O

CH3

N(CH3)2

ON

O

O

N

NHO

OH

HN

O

NO

O–

ONH

N

O

CH3

N(CH3)2

ON

O

OVgb, Mg2+

OO

MLE

CO2–

–O2C

CO2–

β-strand

Loop

Figure 3

Genomic enzymology of streptogramin B lyase (Vgb). (a) Conservation instructure between Vgb (complexed with quinupristin; pdb id: 2Z2P) and amuconate-lactonizing enzyme (MLE; pdb id: 1JOF). Presentation and colorcoding as in Figure 2. (b) Reaction catalyzed by MLE versus Vgb, withsimilarities in the transformation highlighted in green for the housekeepingprotein and in purple for the resistance protein.

APH: aminoglycosidephosphotransferase

rather than ring opening, and also makes use of

a His base (Figure 3b) (70). Analogous to the

macrolide glycosyltransferases discussed below,

Vgb binds its antibiotic substrate through very

few hydrogen bonds, and substrate recognition

is dominated by van der Waal interactions.

ANTIBIOTIC KINASES

Aminoglycoside Phosphotransferases

Aminoglycoside antibiotics are cationic small

molecules composed of an aminocyclitol ring

linked to various amino sugars. They include

such clinically important antibiotics as to-

bramycin, gentamicin, streptomycin, and

spectinomycin. Aminoglycosides bind to the

16S rRNA of the small ribosomal subunitand interfere with proper codon-anticodon

pairing, which results in mistranslation of

proteins (the exception is spectinomycin,

which blocks protein synthesis but does not

cause mistranslation) (25, 35). These abnormal

proteins are cytotoxic and result in cell death.

Binding of the antibiotics to the 16S rRNA

requires both charge-charge interactions and

close steric alignment. It is therefore not

surprising that bacteria have evolved a series

of enzymes that alter aminoglycoside charge

and structure as a means of efficient resistance.

Interestingly, three unrelated mechanismsare known: O-phosphotransferases (APH), N -

acetyltransferases, and O-adenylyltransferases.

Structure and function studies on each of these

classes have revealed that they share traits with

well-known families of metabolic enzymes.

APHs and ePKs share a common ancestor.

Over two dozen APHs have been identified,

with protein sequence identity ranging from

20% to 40% (127, 151). These enzymes are

classified based on regiospecificity of phospho-

rylation of the same aminoglycoside substrate,as well as substrate specificity and promiscuity.

Current knowledge of the structure-function

for several APH family members and phy-

logenetic analyses place them in the atypical

kinase superfamily with eukaryotic protein ki-

nase (ePK) fold, with the closest relatives being

small molecule kinases: choline kinase(CK) and

5-methylthioribose kinase (MTRK) (71, 125)

(Figure 4a,b).

To date, four crystallographic structures of

APHs have been reported (48, 64, 106, 156).

The structure of APH(3)-IIIa was the first to

reveal conservation of the ePK fold for theseenzymes and suggest a common ancestor (64).

In fact, all four structures have the core ePK

fold, and moreover, the primary structure for

theATP-binding, HXDXXXXN,knownas the

Brenner motif, is conserved as well (18).

32 Morar ·Wright




APH(9)-Ia

APH(3’)-IIIa

CAPK

CK

APH(2”)-IIa

MTRK

a

b

CPT SK

AK

c

d

Ancestral

coreAncestral

core

O

O

O

O

NH2 NH2

NH2

NH2

NH2

NH2

NH2

NH2

HOHO

HO

HO OH

OH

ATP ADP

ATP ADP

O

O

O

O

HO

HO

HO OH

OH

N+

HO

N+

NH

CHCl2 CHCl2

NO2NO2

OH

O

OH

NH

OH

O

OP

O

O––O

HO

OH

OH

OHO

OH

OH

OHO

OP

O

O––O

OP

O

O––O

OP

O

O––O

APH(3'), Mg2+

CK, Mg2+

ATP ADP

SK, Mg2+

ATP ADP

CPT, Mg2+

α-helix

β-strand

Loop

H2N H2N

Figure 4

Antibiotic phosphotransferases. (a) Conservation in structure between aminoglycoside phosphotransferases APH(2 )-IIa (pdb id:3HAV), APH(3)-IIIa (pdb id: 2B0Q), APH(9)-Ia (pdb id: 3I0O), choline kinase (CK pdb id: 2CKP), 5-methylthioribose kinase(MTRK; pdb id: 2OLC), and cAMP-dependent protein kinase (CAPK; pdb id: 1ATP). Presentation and color coding as in Figure 2.

APH(2

)-IIa is shown in complex with AMPPCP, CAPK in complex with ATP, and the remaining four are with ADP. (b) Reactioncatalyzed by CK versus APH(3)-IIIa, with similarities in the transformation highlighted in green for the housekeeping protein and inpurple for the resistance protein. (c ) Conservation in structure between chloramphenicol phosphotransferase (CPT complexed withATP; pdb id: 1QHX), adenylate kinase (AK complexed with ADP; pdb id: 1M7G), and shikimate kinase (SK complexed withAMPPNP; pdb id: 3BAF). Presentation and color coding as in Figure 2. (d ) Reaction catalyzed by SK versus CPT, with similarities inthe transformation highlighted in green for the housekeeping protein and in purple for the resistance protein.





Of the four APHs with known structure,

APH(9)-Ia is thought to be the closest link to

ePKssofar.LikeePKs,thedomainsinAPH(9)-

Ia undergo conformational changes upon sub-strate binding, which are unseen in other

APHs (48). In ePKs conformational changes are

needed for activation, whereas APHs thus far

have been thought to be constitutively in the

“on” conformation. The conformational flex-

ibility of APH(9)-Ia could either be a vestige

of ePK relation or perhaps could serve some

as yet unknown regulatory function. The ge-

ometry of nucleotide binding in APH(9)-Ia is

also more ePK-like. Furthermore, like CK and

MTRK, the APH(9)-Ia substrate binding site is

quite rigid and small in contrast with APH(3)

isozymes and APH(2

)-IIa (80, 90, 110). Thelatter have a large flexible active site pocket

capable of accommodating multiple substrates

(46, 47).

The similarity of APHs with ePKs extends

beyond structural components; these enzymes

alsoshare mechanistic aspects. Enzymatic stud-

ies have shown that like ePKs, APHs operate

via a direct phosphate transfer mechanism from

ATP to antibiotic substrate, with the Asp from

the Brenner motif responsible for activation of

the attacking hydroxyl ontoγ-phosphate of the

ATP (96, 97, 132, 139). However, for the most

thoroughly studied antibiotic kinase, APH(3)-IIIa, the transfer takes place via a dissociative

transition state (138), and this transition state

was also suggested for MTRK (80), whereas

for a well-studied ePK:cAMP-dependent pro-

tein kinase the transition is associative (157).

Mechanistic connection between APHs and

ePK is further strengthened by studies showing

that APHs can phosphorylate ePK peptide and

protein substrates, and are inhibited by ePKs

inhibitors (14, 30, 31).

Macrolide PhosphotransferasesMacrolide antibiotics include the first gen-

eration natural product drug erythromycin

as well as the semisynthetic derivatives clar-

ithromycin and azithromycin.These antibiotics

block translation by binding to the peptide exit

tunnel of the large ribosomal subunit (140).

Macrolide resistance in the clinic is most often

associated with enzyme-catalyzed methylationof the23S rRNA by Ermmethyltransferases;an

action that simultaneously confers resistance to

the structurally unrelated lincosamide and type

B streptogramins. Recently, however,genesen-

coding macrolide kinases (MPHs) have been

found to be associated with emerging resistance

(112). These enzymes inactivate macrolides

by phosphorylation of the 2-OH in a GTP-

dependent manner (128). The MPHs charac-

terized so far come in two isoforms that are dif-

ferentiated based on substrate specificity. No

detailed structure-function analysis has been

published for these enzymes. However, Sawaiand coworkers (137) have noted a presence

of well-conserved C-terminal regions between

MPHs and APHs, one of them being the

Brenner motif. Mutagenesis of the Asp in this

motif confirmed it being essential for enzyme

activity, leading the authors to suggest that like

APHs and ePKs, this residue acts as a general

base activating the 2-OH. Further characteri-

zation of this enzyme will show whether MPHs

like APHs are a result of divergent evolution

from the vast ePK superfamily.

Chloramphenicol Phosphotransferase

Like macrolides, chloramphenicol also binds to

the largesubunit of the ribosome. In the crystal

structure of chloramphenicol, with a large sub-

unit of the bacterium Haloarcula marismortui ,

theantibiotic overlaps with the macrolidebind-

ing site (57), whereas in the Deinococcus radiodu-

rans structure, it binds at a different site adja-

cent to the peptidyltransfer center (126). These

distinct binding sites are nonetheless consis-

tent with biochemical and resistance mutation

data that suggest that both sites may be relevant

to chloramphenicol action. The antibiotic hasbeenassociated with significant toxicity, includ-

ing rare but irreversible aplastic anemia, but

it still sees limited clinical use throughout the

world.

34 Morar ·Wright




CPT:

chloramphenicolphosphotransferase

AAC: aminoglycosideacetyltransferase

CPT is a member of nucleoside monophos-

phate kinase family. The producer of chlo-

ramphenicol, Streptomyces venezuelae, uses a 3-

O-phosphotransferase (CPT) to modify theC3-hydroxyl of the antibiotic and render it in-

active. Although this microorganism utilizes

the same inactivation strategy for chloram-

phenicol as described for aminoglycosides and

macrolides, the similarity does not extend to

structure. X-ray crystallographic characteriza-

tion of CPT revealed that this enzyme belongs

to the monophosphate kinase family, which is

unrelated to ePKs. The closest structural rel-

atives of CPT are shikimate kinase (SK) and

adenylate kinase (AK) (Figure 4c ) (39, 65, 79).

Like SK and AK, CPT possesses a signature

Gly-rich P-loop, which forms an oxy-anionhole and accommodates the phosphate tail of

ATP. A second conserved motif, the B-motif,

contains an Asp to act as the catalytic base

for C3-OH activation. CPT is proposed to act

via direct phosphate transfer and an associative

transition state (65). The same type of mech-

anism was proposed for SK (Figure 4d ) (58).

Therefore, although thefold andpositioning of

the catalytic residues is different for CPT and

the monophosphate kinase family from APHs

and ePKs, the fundamental mechanistic fea-

tures are retained.

ANTIBIOTICACETYLTRANSFERASES

Aminoglycoside AcetyltransferasesAre Members of the GNAT Superfamily

Aminoglycoside inactivation via aminoglyco-

side acetyltransferase (AAC) enzymes was the

second bacterial resistance mechanism discov-

ered after penicillinases (107). Since then ex-

tensive structure-function characterization of

these enzymes has been ongoing. AACs belongto the GCN5 N-acetyl transferase (GNAT) su-

perfamily. This superfamily of enzymes utilizes

acetyl-CoA as a cofactor and contains an im-

pressive number of approximately 10,000mem-

bers (146). Of these, AACs were the first to

be identified (33, 130) and were subsequently

shown to have conserved sequence motifs with

eukaryotic transcription factor GCN5, giving

the superfamily its name (130).

Structure and function of AACs. AACs are

divided into four groups based on the re-

giospecificity of the aminoglycoside modifica-

tion and designated AAC(1), (3), (2), and (6).

Structures are available for plasmid-encoded

AAC(3)-Ia, chromosomally encoded AAC(2)-

Ic, and several AAC(6) isozymes (143, 147,

153). The structures show conservation of the

GNAT core, despite very little identity or sim-

ilarity in the primary sequence (Figure 5a)

(142). One sequence motif R/QxxGxG/A is in-

variant in these proteins and is responsible forrecognition of 3-P ADP moiety of the CoA

cofactor (105, 147). Structure of the AAC(6)-

Ii dimer is similar with AAC(3)-Ia and even

more so with eukaryoticHpa2-encoded histone

acetyltransferase (20, 153), while being distinct

from AAC(2)-Ic.

AACs have been extensively investigated

biochemically with most reports available for

AAC(6)-Ii and -Iy and AAC(2)-Ic groups.

The acetylation reaction follows a random [for

AAC(2)-Ic] or sequential [AAC(6)-I] mecha-

nism, with acetyl-CoA binding first followed by

aminoglycoside, andin eithercase, thepresenceof both substrate and cosubstrate is required

for acyl transfer, with rate-limiting product

release (37, 60, 89, 118). Further investigation

of molecular mechanism via mutagenesis of

potential catalytic residues revealed that muta-

tions ofmostactivesiteresidues donot yield in-

active enzymes. These observations are consis-

tent with the enzyme’s primary function being

binding and positioningof the substrates,rather

than specific catalytic activation via direct par-

ticipationof amino acidfunction groupsin roles

such as a general acid/base (38, 59). Substrate

specificity investigations have shown a remark-ably broad range of aminoglycosides acetylated

by both AAC(6) isozymes and AAC(2)-Ic (29,

60,117, 150). Moreover AAC(6) isozymes were

shown to acetylate eukaryotic histone proteins

and human histone H3 peptide (144, 153).





CrAT

E2pCD CAT

a

c d

b

VatDLpxA

XAT

Ancestralcore

Ancestralcore

Ancestralcore

O

O

O

O

NH2

HOHO

HO

HO OH

OH

O

O

O

O

NH

HOHO

HO

HO OH

OH

OHPA2

AAC(6')

SCoA

O

HSCoA

SCoA

O

HSCoA

NH

O

NH

CHCl2 CHCl2

NO2 NO2

OH

O

OH

CAT

SCoA

O

HSCoA

NH

OH

O

OO

O

O–OH

O

O–O

N+

N+ SCoA

O

HSCoA

CrAT

O

AAC(2’)-Ic

AAC(6’)-IiHpa2

NH2

Lys Lys

NH2

NH2

NH2

NH2

NH2NH2

H2N H2N

α-helix

β-strand

Loop

36 Morar ·Wright




CAT:

chloramphenicolacetyltransferase

Divergent evolution of GNAT superfamily.

Despite a low amino acid sequence iden-

tity, the GNAT superfamily members known

to date share the core tertiary structure andfollow similar mechanisms of acetyl transfer

(Figure 5c ). In catalysis, the positioning of the

substrates appears to be most important, rather

than the presence of specific functional moi-

eties. AACs do not share the most conserved

quaternary structure among themselves. Some

function less discriminatively and are capable

of acetylating substrates other than aminogly-

cosides. Owing to this loose conservation of

tertiary and quaternary structure, as well as a

very broad substrate specificity, the true physi-

ologic function of AACs, especially those chro-

mosomally encoded, remains to be confirmed,placing these enzymes at the interface of bona

fide and proto-resistance elements in the resis-

tome (143, 150). For these same reasons, AACs

are thought the oldest members of the GNAT

family from which other members, such as eu-

karyotic histone acetyltransferases and fluoro-

quinolone acetyltransferases (see below), can

evolve toward novel function (144).

Fluoroquinolone acetyltransferases. Flu-

oroquinolones such as ciprofloxacin are

synthetic antibiotics that target DNA gy-rase and topoisomerases necessary for DNA

synthesis. Resistance is usually the result of

point mutations in target genes (e.g., gyrA,

parC ) or via efflux. By virtue of their synthetic

origins, it was considered unlikely that there

would be enzyme-catalyzed inactivation modes

of resistance. However, an AAC(6) variant,

AAC(6)-Ib-cr, containing two amino acid mu-

tations, has evolved to confer resistance fluoro-quinolone antibiotics with an available nitrogen

on a piperazine heterocycle (122). Structure-

function characterization of this enzyme led

to the mechanistic proposal for the enzyme’s

ability to acetylate both aminoglycosides and

fluoroquinolones. However, the mechanistic

details remain to be worked out (94, 145).

These findings further underscore the sig-

nificance of broad specificity for AACs and

our lack of understanding of their physiologic

function. The adaptability of these enzymes is

remarkable as only small numbers of changes

arerequiredfor theevolution of a newfunction.

AACs, CATs, and XATs are a result of

convergent evolution. Two variants of chlo-

ramphenicol acetyltransferases (CATs) have

been reported to date. Classic CATIII activity

was first identified in 1967 encoded on a plas-

mid (136). Biochemical characterization of this

enzyme yielded a mechanism consistent with

randomorder of substratebinding andthe pres-

ence of both a chloramphenicol substrate and

the acetyl-CoA cofactor for direct acetylation

(76, 77). Although the mechanism is reminis-cent of that for AACs, site-directed mutagen-

esis points to an important difference. Unlike

AACs, whose primary purpose in catalysis is

proposed to be binding and orientation of sub-

strates, CATs have an essential histidineresidue

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−

Figure 5

Antibiotic acetyltransferases. (a) Conservation in structure between aminoglycoside acetyltransferases AAC(6)-Ii (pdb id: 1B87),AAC(2)-Ic (CK pdb id: 1M4D), and Hpa2 histone acetyltransferase (Hpa2; pdb id: 1QSO). Presentation and color coding as inFigure 2. AAC(6)-Ii and AAC(2)-Ic are both shown in complex with Coenzyme A (CoA). (b) Conservation in structure betweenaminoglycoside acetyltransferases:chloramphenicol acetyltransferase (CAT complexed with chloramphenicol; pdb id: 3CLA), carnitine

acetyltransferase (CrAT; pdb id: 2H3P), and dihydrolipoyl transacetylase (E2pCD; pdb id: 1EAB). Presentation and color coding as inFigure 2. CrAT and E2pCD are shown in complex with acetyl-CoA and CoA, respectively. (c ) Reactions catalyzed by Hpa2 and CrATversus AAC(6) and CAT, with similarities in the transformations highlighted in green for the housekeeping proteins and in purple forthe resistance proteins. Spheres represent the remainder of the enzyme. (d ) Conservation in structure between xenobioticacetyltransferases chloramphenicol acetyltransferase (XAT complexed with chloramphenicol and desulfo-CoA; pdb id: 2XAT),streptogramin group A acetyltransferase (VatD complexed with acetyl-CoA; pdb id: 1KK4), and UDP N -acetylglucosamineO-acyltransferase (LpxA; pdb id: 1LXA). Presentation and color coding as in Figure 2.





XAT: xenobiotic

acetyltransferaseANT: aminoglycosidenucleotidyltransferase

acting as a general base(85, 103). Furthermore,

CAT structure reveals a completely different

fold, quaternary organization, and substrate

binding mode from that of AACs (83, 84).CATs show high specificity for their substrate,

although one variant is able to bind and acety-

late the steroidal antibiotic, fusidic acid (102).

CATs share structure-function similarity

with carnitine acetyltransferase (68, 152) and

dihydrolipoyl transacetylase (93), the latter two

also being CoA-dependent acetyltransferases

(Figure 5b,c ). Remarkably, CAT and dihy-

drolipoyl transacetylase are both trimers with

the active site located at the interface, and their

monomer organization is similar with the two

domain organization of carnitine acetyltrans-

ferase. Furthermore, the catalytic histidine ispresent at the same position for all (67).

There exists a second variant of enzyme ca-

pable of chloramphenicol acetylation, termed

CATB, that belongs to xenobiotic acetyltrans-

ferase (XAT) class (104). XAT class includes an

enzyme, VatD, responsible for group A strep-

togramin acetylation (17, 121). Structures of

both CATB and VatD have been determined

(10, 73, 135) and show an unusual left-handed

parallel β-helix (LβH) fold (119). Other en-

zymes with LβH fold are also CoA-dependent

acetyltransferases, the exception beingcarbonic

anhydrase (Figure 5d ) (75, 119). Similaritiesbetween VatD and CATB extend to primary

structure, with the conservation of key catalytic

residues. Although theLβH fold is unrelated to

the classic CAT, XATs also form trimers with

the active site located at the monomer interface

and an active site histidine proposed to serve as

catalytic base (10).

In summary, nature has adapted a least

three different protein folds to catalyze CoA-

dependent acetyl transfers, and antibiotic

acetyltransferases span all three.

ANTIBIOTIC NUCLEOTIDYLYLTRANSFERASES

Divergent Evolution of Aminoglycoside and Lincosamide NTs

Aminoglycosides can be inactivated by an

ATP-dependent adenylylation of various hy-

droxyl moieties. Enzymes responsible for this

modification, aminoglycoside nucleotidyltrans-

ferase (ANTs), are classified based on their

substrate and regiospecificity (88, 148). Of these, ANT(2 )-Ia and ANT(4)-I have been

thoroughly investigated biochemically, and al-

though these enzymes show no relatedness in

primary sequence, they follow a similar mech-

anism. The adenylyl group is transferred di-

rectly from ATPto theantibiotic via an ordered

Bi-Bi mechanism and an associative transition

state (26, 49, 51, 141). The 3D structure of

ANT(4)-I further supports this mechanism and

points to a glutamate residue as a potential cat-

alytic base responsible for substrate activation

(109, 123). The ANT(4)-I structure also re-

veals thesimilarity of this enzyme fold with thatof DNA polymerase β (Figure 6a) (63, 124).

Another class of antibiotics inactivated by

adenylylation is the lincosamides. Clindamycin

is the lincosamide antibiotic that is most of-

ten used clinically. It binds to the peptide exit

tunnel of the bacterial ribosome in the same

region as the macrolide antibiotics, consistent

−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−→

Figure 6

Antibiotic adenylyltransferases and glycosyltransferases. (a) Conservation in structure between aminoglycoside adenylyltransferase[ANT(4)-I in complex with AMPCPP; pdb id: 1KNY], lincosamide adenylyltransferase (LinB in complex with AMPCPP; pdb id:

3JZ0), DNA polymeraseβ (PolB in complex with dUMPNPP; pdb id: 2FMS) and polyA polymerase (PAP in complex with 3

-dATP;pdb id: 1Q78). Presentation and color coding as in Figure 2. (b) Reactions catalyzed by PAP versus ANT(4)-I and LinB, withsimilarities in the transformations highlighted in green for the housekeeping protein and in purple for the resistance proteins.(c ) Conservation in structure between macrolide glycosyltransferases OleI (ternary complex with UDP and oleandomycin; pdb id:2IYA) and OleD (ternary complex with UDP and erythromycin; pdb id: 2IYF), triterpene UDP-glucosyltransferase (UGT71G1 incomplex with UDP; pdb id: 2ACV), and vancomycin aglycone UDP-glucosyltransferase (GtfB; pdb id: 1IIR). Presentation and colorcoding as in Figure 2. (d ) Reactions catalyzed by GtfB versus OleD, with similarities in the transformations highlighted in green forthe housekeeping protein and in purple for the resistance protein.

38 Morar ·Wright




ANT(4’)-I

LinB

PAP

PolB

a b

c d

Ancestralcore

Ancestralcore

HN Cl

ON

OO

OHHO

N

N N

N

NH2

O P

O

O–

LinB

ORO

OHHO

N

N N

N

NH2

O P

O

O–

ATP PPi

PAP

ORO

OHO

N

N N

N

NH2

O P

O

O–

O

OHHO

N

N N

N

NH2

O P

O

O–

ATP PPi

ATP PPi

ANT4'

O

O

O

O

NH2

NH2

NH2

NH2

HO

HO

HO

HO

OH

OH

O

O

O

O

NH2

NH2

NH2

NH2

OHO

H2N

HO

HO OH

OH

O

OHHO

N

N N

N

NH2

O P

O

–O

O

SOH

HO

HO

HN Cl

ON

O

SOH

HO

O

O

O

O

OHHO

O

OOH

O

OHO

NOH

O

O

O

O

OHHO

O

OOH

O

OO

NOHUDP-Glc

OleD

UDP O

OH

HO OH

HO

NHO

HN

O

NHO

HN

OHNH

O HO

O

OH

OH

HO

OCl

O

NH2

O

O

NH

Cl

HO

O

HN

HO

NH

O

HNO

NHO

HN

OHNHO HO

O

OHOH

HO

OCl

ONH2

O

O

NH

Cl

HO

O

HN

O

UDP-Glc

GtfB

UDP

O

HO

HOHO

HO

Utg71G1

OleI OleD

GtfB

H2N

α-helix

β-strand

Loop





MGT: macrolide

glycosyltransferase

with the cross resistance afforded by Erm 23S

RNA methyltransferases (140). The responsi-

ble clindamycin adenylating enzymes are en-

coded by lin genes (40, 82) and are dividedinto two major groups, LinA and LinB, based

on substrate and regiospecificity. Analogous to

ANT(2 )-Ia and ANT(4)-I, LinA and LinB

show no homology in sequence. Structure-

function characterization of LinB revealed that

similar to ANTs this enzymefollows an ordered

Bi-Bi mechanism with direct AMP transfer

(Figure 6b) (98). Moreover, it has the same

3D fold as ANT(4)-I, DNA polβ, and polyA

polymerase (PAP) (Figure 6a) (34, 123, 124).

Conservation is alsoobservedfor positioning of

ATP, as well as location and positioning of the

catalytic glutamate and Mg2+

ions responsibleforstabilizationof thetransition state. LinB and

ANT(4) are thereforea resultof divergentevo-

lution from family X polymerases (4), as these

polymerases not only share the structural simi-

larities with antibiotic NTsbut alsofunctionvia

a direct associative mechanism like antibiotic

NTs (11, 133). ANT(2 )-Ia and LinA variants

show low sequence homology with each other

and none with family X polymerases, ANT(4 )-

I, or LinB (111). Structural characterization of

these enzymes will aid in further understanding

the relation of these resistance genes with the

rest of the resistome.

OTHER ANTIBIOTIC GROUPTRANSFERASES

Macrolide GlycosyltransferasesAre Related to the GT-1 Family

Macrolide glycosyltransferase (MGT) encodes

an enzyme that inactivates macrolides such as

erythromycin using UDP-glucose as cofactor.

This activity has been detected in environmen-

tal organisms such as members of the genus

Streptomycetes , both in antibiotic producers andnonproducers (66, 81). MGT inactivates the

antibioticsby glycosylation of the 2-OHofa6-

deoxyhexose moiety; however, variants of this

enzyme have different substrate specificities

(113, 116). This is the same regiospecificity of

the MPH resistance kinases and reflects the

importance of this hydroxyl group in binding

of macrolide antibiotics to the large subunitof the ribosome as revealed by the X-ray

structures, where a hydrogen bond between

the 2-OH and A2058 (E. coli numbering) is

evident (140). The MGT catalyzed reaction

proceeds via an ordered mechanism, with

both substrate and cofactor required for direct

transfer, and antibiotic binding first followed

by UDP-glucose (114–116). Structures of two

MGTs, OleI and OleD, revealed that these

enzymes belong to glycosyltransferase-1 family

(GT-1) (15, 27) and share similarity with the

GtfB enzyme, which is responsible for gly-

cosylation of vancomycin aglycone (101) andflavonoid/triterpene GT (Figure 6c ) (129). As

proposed for GtfB, the OleI/D-substrate com-

plexes showed primarily hydrophobic interac-

tions between the enzymes and the substrate.

Another unifying factor for these enzymes is

the mechanistic postulate, which involves the

activation of hydroxyl moiety via a general

base, followed by direct transfer (Figure 6d ).

Fosfomycin Inactivation EnzymesBelong to Vicinal Oxygen

Chelate Superfamily One mechanism of fosfomycin inactivation is

via epoxide ring opening using one of three re-

sistancegenes: FosA, FosB, and FosX (5, 24,41,

42). These enzymes utilize different substrates:

FosA uses glutathione, FosB uses L-cysteine,

FosX uses water. Each enzyme shows a differ-

ent metal dependency: Mn2+/K +, Mg2+, and

Mn2+ for FosA, ForB, and FosX, respectively

(13, 24, 43, 134). Despite these differences the

three enzymes show 30% to 35% protein se-

quence identity andshare thesame fold belong-

ing to the vicinal oxygen chelate (VOC) family

of metallo-enzymes (43).FosA and FosX structure-functions have

been characterized more extensively, and these

not only share a common fold but also metal

and substrate binding sites (43, 44, 120). FosX

40 Morar ·Wright




catalyzes direct addition of water with Mn(II)

and a base involved in activation of the nucle-

ophile(43, 44), anddirect addition hasalso been

proposed for the glutathione transferase FosA (43, 44). Furthermore, the relationshipbetween

the two enzymes is strengthened by identifica-

tion ofa FosX homolog from Mesorhizobium loti

containing both activities (43). A triple mutant

of this enzyme improved FosA catalytic effi-

ciency and abolished that of FosX, demonstrat-

ing theease of manipulation of theproperties of

these enzymes (19). Besides Fos enzymes, VOC

family includes extradiol dioxygenase, glyox-

alase I, and methylmalonyl-CoA epimerase

(Figure 7a,c ) (22, 56, 95). All are metallo-

enzymes with a requirement for a catalytic base

and with a conserved metal binding site. Inter-estingly, Fos proteins show no structural simi-

laritywith an ancient superfamilyof glutathione

transferases, catalyzing analogous reactions of

glutathione-mediated small molecule detoxifi-

cation (131), and therefore represent an exam-

ple of convergent evolution.

ADP-Ribosylation of Rifamycin Antibiotics

Rifamycin antibiotics are inactivated by ADP-

ribosylation reaction catalyzed by NAD-

dependent Arr enzymes (28). This is theonly small molecule ADP-ribosylation enzyme

known to date. Structure-function analysis of

Arr from Mycobacterium smegmatis revealed that

despite nonexistent sequence homology, Arr

is a member of the ADP-ribosyltransferase

(ART) superfamily (9). All ART enzymes cat-

alyze transfer of ADP-ribose moiety with re-

lease of nicotinamide (62, 154). The conser-

vation of Arr structure is most significant for

the NAD+ binding core to that of RNA 2-

phosphotransferase (72) and bacterial toxins:

exotoxin A domain III (86) and cholera toxin

(69) (Figure 7b,d ). Of these, Arr representsthe minimal unit necessary for NAD-binding

and catalysis (9). Besides the tertiary structure,

Arr also contains three catalytically essential

residues characteristic of the ART family. Two,

His and Tyr, are part of a conserved H-Y-

E motif, proposed to be directly involved in

NAD binding. However, the catalytic Glu of

ART family is not conserved in Arr; instead Arrutilizes an Asp found elsewhere in the struc-

ture. This enzyme, capable of modifying both

natural-product and semisynthetic rifamycins,

is an excellent example of nature exploiting ex-

istingprotein scaffoldsin evolution of antibiotic

resistance.

OPPORTUNITIES ANDOUTCOMES OF ANTIBIOTICRESISTANCE GENOMICENZYMOLOGY

Antibiotic resistance is an integral componentof the natural history of antibiotics. The most

parsimonious hypothesis on the origins of re-

sistance is that it must have first coevolved with

biosynthesis as a means of auto-immunity to

the production of toxic secondary metabolites.

This chemical strategy then could have inde-

pendently evolved de novo in neighboring or-

ganisms as a means of protection or could have

been imported via horizontal gene transfer. As

long as there was selection, the genes for resis-

tance would have been maintained and even-

tually stably integrated into the genome. The

number of resistance elements that we nowsee scattered in virtually all bacterial genomes

may reflect prior exposure during the evolu-

tion of the species. This idea is not incompati-

ble with the new hypothesis that naturally pro-

duced antibiotics are not in fact antibiotics at

all in the concentrations produced in the envi-

ronment, but rather signaling molecules (155).

Resistance elements could have evolved as

receptors or mediators of such signaling

molecules. The fact that resistance is so

widespread in the environment and that we can

readily select for resistance even to completely

synthetic antibiotics, speaks to the depth andplasticity of the resistome.

The genomic enzymology strategy offers an

integrated approach to the study of the evolu-

tion of resistance and possible solutions to the





a

c

b

d

Ancestralcore

Ancestralcore

O

P–O

OHO

FosX, Mn2+

OH

HO

P

O–

HO

O

OH

OH O2

DHBD, Fe2+O

OH–O

O

OO

R1

OH

HO

NHO

OH

OOO

O

OO

O

OHHO

N

N N

N

NH2

O P

O

O

OP

O– O

OHHO

O–

OO

R1

OH

HO

NHO

OH

OOO

O

O

NAD+

ARR

R2R2

N

NH2

O

OHO

OHHO

N

N N

N

NH2

O PO

O

ORP

O

PARPelongationO

O

OHHO

N

N N

N

NH2

O P

O

OOP

O– O

OHHO

O–O

OHO

N

N N

N

NH2

O

NAD+

N

NH2

O

–OOHP

O

O

ORP

O–O

DHBDFosA

FosXGlxI

ARR

PARPpeIII

RNA2P

H2O

α-helix

β-strand

Loop

42 Morar ·Wright




challenge resistance poses for the therapeutic

use of antibiotics. Using structure and function

studies, it is now evident as discussed above that

either resistance has emerged directly from el-ements with other metabolic functions or that

they share common ancestors. This idea first

emerged with the comparison of the struc-

tures of β-lactamases and PBPs over 20 years

ago (74) and the observations of Benveniste &

Davies (12) on aminoglycoside resistance al-

most 40 years ago. The availability of micro-

bial genomes coupled with theability to express

enzymes in quantity and study both structure

and function have only strengthened the links

between the genes and proteins of antibiotic

resistance and cellular metabolism. Given the

vast numbers of microbes on the planet (>1030

)(146), the depth of metabolic enzymes that can

serve as proto-resistance elements, and the ra-

pidity of bacterial cell growth, antibiotic resis-

tance is inevitable.

While the inevitability of resistance is

reflected in the clinical experience of the

past 50 years, it is a reality that can serve

to strengthen antibiotic drug discovery and

stewardship. For example, the identification

of resistance and proto-resistance elements

in environmental organisms early in the drug

discovery process can be very valuable. First, it

can serve to tailor candidate molecules duringlead optimization to avoid or otherwise modify

particularly vulnerable features, e.g., hydroxyl

groups that can be modified by kinases.

Second, by identifying resistance elements

early, scans of bacterial genomes can inform

on the distribution and density of resistance

genes. Third, this information could be used

to prepare molecular diagnostics that can be

used to routinely survey clinically relevant

organisms for the emergence of resistanceduring therapy. Finally, by understanding the

molecular basis of resistance, strategies such as

the codevelopment of inhibitors of resistance

can be pursued. For example, the relationship

between aminoglycoside kinases and eukariotic

Ser-Thr-Tyr protein kinases led to a study

demonstrating that protein kinase inhibitors

block the action of resistance kinases (31).

Because many pharmaceutical companies have

extensive chemical libraries targeted towards

protein kinase inhibition, leads for antiresis-

tance molecules that could be coadministered

with antibiotics may be readily identified andrepurposed for infectious disease therapy.

Given the continuing emergence of multi-

drug resistant pathogens, the need for new an-

tibiotics is acute and growing. With the ad-

ventof nextgenerationgenome sequencingthat

promises to ever increase the rapidity and num-

ber of sequenced bacterial genomes along with

downward pressures on the per genome costs,

there will be a large influx of new genomic

data from pathogenic and nonpathogenic bac-

teria over the next decade. The application of

this information to infectious disease biology

and drug discovery requires robust platformsthat can link gene with function and struc-

ture. Genomic enzymology is a modern inte-

grated strategy that offers a pathway to study

antibiotic resistance and apply this knowledge

in the development and management of new

drugs.

←−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−−

Figure 7

Genomic enzymology of fosfomycin inactivation enzymes and rifamycin ADP-ribosyltransferase. (a) Conservation in structure betweenFosA (complexed with fosfomycin; pdb id: 1LQP), FosX (pdb id: 1R9C), glyoxalase I (GlxI complexed with methyl-gerfelin; pdb id:

2ZA0), and biphenyl-cleaving extradiol dioxygenase (DHBD; pdb id: 1HAN). Presentation and color coding as in Figure 2.(b) Conservation in structure between Arr (complexed with rifampicin; pdb id: 2HW2), PARP (complexed with NAD+ analogue; pdbid: 1A26), exotoxin A domain III (peIII complexed with NAD+ analogue; pdb id: 1AER), and RNA 2-phosphotransferase (RNA2P;pdb id: 1WFX). Presentation and color coding as in Figure 2. (c ) Reactions catalyzed by DHBD versus FosX, with similarities in thetransformations highlighted in green for the housekeeping protein and in purple for the resistance protein. (d ) Reactions catalyzed by poly (ADP-ribose) polymerase (PARP) versus rifamycin ADP-ribosylase (ARR), with similarities in the transformations highlighted ingreen for the housekeeping protein and in purple for the resistance protein.





SUMMARY POINTS

1. The antibiotic resistome accounts for all bone fide and potential antibiotic resistance

elements found not only in the clinic but also in any other environments (e.g., soil). Inorder to fully understand various aspects of antimicrobial drug resistance, all parts of the

resistome need to be investigated.

2. Proto-resistance elements are genes in the resistome that have potential to become re-

sistance determinants. Proto-resistance genes may share common ancestry with genes of

other cellular functions.

3. Genomic enzymology can be used to study the evolution of antibiotic resistance from

proto-resistance enzymes. Genomic enzymology of several classes of antibiotic resistance

enzymes revealed their evolutionary relation with other cellular functionalities.

4. Genomic enzymology of antibiotic resistance is a powerful approach for learning the

dynamics of antibiotic resistance. This knowledge can be applied in development and

management of antibiotic therapies.

FUTURE ISSUES

1. How can we track and differentiate resistance and proto-resistance elements in microbial

communities?

2. Can resistance genes in nonpathogenic microbes be directly linked to the emergence of

resistance in pathogens? What is the role of horizontal gene transfer in this process?

3. Can we identify the metabolic roles of proto-resistance elements in the resistome?

4. What will aid leveraging genomic enzymology strategies in drug discovery?

DISCLOSURE STATEMENT

The authors are not aware of any affiliations, memberships, funding, or financial holdings that

might be perceived as affecting the objectivity of this review.

ACKNOWLEDGMENTS

Research in the author’s lab on antibiotic resistance is supported by a Canada Research Chair (to

GDW)and the Canadian Institutes of Health Research and the Natural Sciences and Engineering

Research Council of Canada.

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