The enzymes of bacterial censusand censorshipWalter Fast1 and Peter A. Tipton2

1 Division of Medicinal Chemistry, College of Pharmacy, University of Texas, Austin, TX 78712, USA2 Department of Biochemistry, University of Missouri, Columbia, MO 65211, USA

Review

Glossary

Autoinducer: signaling molecule that is produced by a microorganism,

accumulates in the growth medium and leads to induction of a subset of

genes in the same organism. These signals are used in quorum sensing, i.e. to

sense the local cell density.

Holo-ACP synthase: acyl-carrier protein synthase bearing a serine residue that

has been post-translationally modified to carry a 40-phosphopantetheinyl

substituent. This modification introduces a thiol-containing flexible ‘‘arm’’ that

covalently binds the acyl chain (linked as a thioester) that is used as a substrate

for AHL synthesis.

Lactonization: formation of a cyclic ester. In the reaction catalyzed by AHL

synthases, this process results in the formation of an a-amino-g-butyrolactone

ring.

Metalloforms: variants of a metalloprotein in which the identity or stoichio-

metry of the metal content differs. These variants can be found endogenously

or can be reconstituted in an experimental system.

Nucleophilic attack: donation of an electron pair from a donor atom to an

acceptor electrophile to form a covalent chemical bond.

Ping-pong mechanism: double-displacement bireactant mechanism. Binding

of the first substrate is followed by release of a product and (usually) formation

of a covalent enzyme intermediate before the second substrate is bound and

processed to complete the reaction.

Sociomicrobiology: study of group behavior in microorganisms. Group

N-Acyl-L-homoserine lactones (AHLs) are a major classof quorum-sensing signals used by Gram-negativebacteria to regulate gene expression in a population-dependent manner, thereby enabling group behavior.Enzymes capable of generating and catabolizing AHLsignals are of significant interest for the study of micro-bial ecology and quorum-sensing pathways, for under-standing the systems that bacteria have evolved tointeract with small-molecule signals, and for their pos-sible use in therapeutic and industrial applications. Therecent structural and functional studies reviewed hereprovide a detailed insight into the chemistry and enzy-mology of bacterial communication.

The social lives of bacteriaHumans are certainly not the first to use informationwarfare. Nature is rife with examples of communicationfor cooperation and for subterfuge, with the growing field ofsociomicrobiology (see Glossary) representing possibly themost reductionistic extreme [1]. In one example, variousGram-negativebacteriaproduce cell-permeableN-acylated-L-homoserine lactones (AHLs) at a low basal rate. If thesesignals are allowed to accumulate, they bind cognatetranscriptional regulators and act as autoinducing signals.Because the concentration of autoinducers oftenmirrors thelocal population density, they act as a sort of census toregulate gene expression in a population-dependent man-ner. Several strategies to interfere with these signalingpathways have been discovered, including AHL-degradingenzymes with activities that work as a type of censorship toblock interbacterial communication. The study of theseprocesses enhances our understanding of microbial ecologyand suggests novel therapeutic strategies. Towards theseends, recent advances in the study of AHL generation anddecay have facilitated a detailed understanding of the en-zymology that underlies the social lives of bacteria.

Bacterial census: AHL synthesisAlthough bacteria have evolved many chemically diversecommunication systems (Box 1), in the present review weconsider only AHLs, which are produced by Proteobacteria.The two protein components of the AHL signaling systemwithin the producing organism are the inducer protein (I)and the receptor protein (R). Inducer proteins are thesynthases responsible for the formation of AHLs; theyare designated LuxI-type AHL synthases, after the LuxI

Corresponding authors: Fast, W. (waltfast@mail.utexas.edu); Tipton, P.A.(tiptonp@missouri.edu).

0968-0004/$ – see front matter � 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.tibs.2011.

protein fromVibrio fischeri. The number of AHLs that havebeen characterized as quorum-sensing molecules in bacte-ria greatly exceeds the number of corresponding synthasesthat have been studied in detail. Nevertheless, sequencehomology among AHL synthases suggests that they arestructurally similar and probably follow similar chemicalmechanisms. Early studies of the archetypal AHLsynthase LuxI from V. fischeri established that the enzymesubstrates are S-adenosylmethionine (SAM) and an acyl-ated acyl carrier protein (ACP) [2]. Two distinct chemicalreactions are required to form the AHL: acyl transfer fromthe ACP to the amino group of SAM, and lactonization ofSAM with concomitant expulsion of S-methylthioadeno-sine (MTA) (Figure 1). The usual metabolic function ofSAM is as a methyl group donor, so its role as the source ofthe amino acid in AHL synthesis is unusual.

The structures of three AHL synthases have been de-termined by X-ray crystallography: EsaI from Pantoeastewartii, which catalyzes the formation of 3-oxo-hexanoylhomoserine lactone [3]; LasI fromPseudomonasaeruginosa,whose product is 3-oxo-dodecanoyl homoserine lactone [4];and TofI from Burkholderia glumae, which catalyzes theformation of octanoyl homoserine lactone (C8-HSL) [5]. Thesynthases exhibit an a-b-a fold that is similar to thatobserved in GCN5-related histone acetyltransferases(GNATs), although no sequence similarity between AHL

behavior is often mediated by communication systems that rely on the

production and detection of small diffusible signaling molecules such as AHLs.

10.001 Trends in Biochemical Sciences, January 2012, Vol. 37, No. 1 7

Box 1. Chemical diversity of quorum-sensing signals

N-Acyl-L-homoserine lactones represent just one of the ‘‘dialects’’

used for quorum sensing. Bacteria use a chemically diverse range of

signaling molecules for communication, each with different biosyn-

thetic pathways, chemical properties, binding proteins and degrada-

tion mechanisms. A sample of this diversity is represented in Figure I

by the chemical structures of N-(3-oxo-hexanoyl)-L-homoserine

lactone (3-oxo-C6-HSL) from Photobacterium fischeri [36], p-coumar-

oyl-L-HSL (pC-HSL) from Rhodopseudomonas palustris [37], diffusi-

ble signal factor (DSF) from Xanthomonas campestris [38], cholera

autoinducer-1 (CAI-1) from Vibrio cholerae [39], 2-heptyl-3-hydroxy-4-

quinolone (pseudomonas quinolone signal or PQS) from P. aerugi-

nosa [40], autoinducer-2 (AI-2) from Vibrio harveyi [41] and AgrD1

thiolactone (autoinducer peptide I, or AIP-I) from Staphylococcus

aureus [42].[(Figure_I)TD$FIG]

O

3-oxo-C6-HSL

O

O

O

HO

HO

OHNH

OH

O

DSF

NH

PQS

pC-HSL

O

OHO

HO

HO

OH

NH

O

OOH OH

B

CH3

AI-2

O

HCAI-1

O

S

AIP-I

-Tyr-Ser-Thr-Cys-Asp-PheNH+

3

IIeMet

TiBS

Figure I. Selected chemical structures of interbacterial signaling molecules. For abbreviations, see the text.

Review Trends in Biochemical Sciences January 2012, Vol. 37, No. 1

synthases and GNATs is evident. The recently determinedstructure of TofI is a major advance, because it provides thefirst view of ligands bound to an AHL synthase. Previouspredictionsof substratebindingmodes,whichwerebasedonthe structural similarity between AHL synthases andGNATs [5], are largely borne out by the TofI structure, inwhich the product MTA and the inhibitor J8-C8 (which is astructural analog of the product C8-HSL) are bound(Figure 2). The binding pocket for the acyl chain of thesubstrate and product forms a deep cavity so that the[(Figure_1)TD$FIG]

S

OO

ACP ++H3N

O O

LuxI

+S

AHL

OO

HN

O

O

Figure 1. Chemical reaction catalyzed by LuxI. All known LuxI-type AHL synthases utilize

used as the second substrate. Abbreviations: AHL, acyl homoserine lactone; SAM, S-ad

8

hydrophobic acyl chain is completely removed from theaqueous solvent. MTA is bound in a cleft, but remainspartially exposed to the solvent. There is a stacking inter-action between the ribose ring of MTA and Trp33, a residuethat is conserved in AHL synthases; functional analysisrevealed that substitution of Trp33 with non-aromatic resi-dues abrogates TofI activity [5]. In the absence of ligands,residues 32–40 are disordered, but they form part of anordered loop when Trp33 interacts with MTA (and presum-ably SAM).

-

+S O

N

N

N

N

N N

N N

NH2

NH2

OH OH

SAM

OH OH MTA

HSO ACP+

TiBS

SAM as a substrate; product diversity comes from the different acyl-ACPs that are

enosyl methionine; ACP, acyl carrier protein; MTA, methylthioadenosine.

[(Figure_2)TD$FIG]

J8-C8 O

NH

O

MTA

TiBS

Figure 2. Structure of AHL synthase TofI from Burkholderia glumae (Protein Data

Bank code 3p2h). The inhibitor J8-C8, whose structure is given in the inset, is bound

in the center of the protein with the C8 acyl chain extending into a hydrophobic

pocket. Methylthioadenosine (MTA) is bound in the pocket at the top of the protein.

Residues 32–40 are shown in red; this portion of the protein is disordered in the

absence of ligands, but becomes stabilized through interactions with MTA.

Box 2. Quorum-sensing systems as drug targets

Antibiotics currently in use are either bactericidal (bacteria-killing) or

bacteriostatic (growth inhibitory). As resistance to these drugs

continues to increase, new strategies for treating bacterial infections

are being sought. One approach that has attracted interest is

targeting of the components of bacterial physiology that are

required for infectious activity, but not for viability. Quorum-sensing

systems regulate the expression of virulence factors, among other

things, so inhibition of quorum sensing should attenuate virulence.

The observation that the alga Delisea pulchra produces halogenated

furanones (i.e. a,b-unsaturated five-membered lactones) that inter-

fere with AHL-mediated signaling [43] stimulated the search for

other compounds that inhibit quorum sensing [44,45].

Most of the compounds that have been identified to date target

the transcription factor to which the signal molecule binds, rather

than inhibit the synthase responsible for its synthesis. Investigation

of LuxR expressed in E. coli revealed that furanones increased the

rate of LuxR turnover, although furanone binding to LuxR was not

demonstrated [43].

In mouse models of P. aeruginosa infections, furanones increase

bacterial clearance and decrease tissue damage [46,47]. Interestingly,

halogenated furanones were also found to inhibit Staphylococcus

epidermidis biofilm formation, even though this process is coordi-

nated by a different signal, autoinducer-2 (Box 1), not AHL [48].

Despite these promising observations, the road to clinical use of

quorum-sensing inhibitors is not without obstacles. The stability

and toxicity of halogenated furanones may make them unsuitable

for use in humans. Furthermore, compounds that bind to the

receptor proteins are not guaranteed to be inhibitors of quorum

sensing, but may instead act as activators [49].

Review Trends in Biochemical Sciences January 2012, Vol. 37, No. 1

A wide variety of AHLs with different acyl chains arefound in nature. Chain lengths from C4 to C18 have beenobserved; a ketone is frequently found at C3, which issometimes reduced to a hydroxyl group; and in some casesthe acyl chain is branched or unsaturated [6]. AHLsynthases typically exhibit strict, but not absolute, sub-strate specificity. For example, the best substrate for RhlI,an AHL synthase from P. aeruginosa, is butyryl-ACP,but it can catalyze the slow formation of N-hexanoyl-homoserine lactone [7]. The structures of EsaI and TofIsuggest that substrate specificity is determined by the sizeof the tunnel in which the acyl chain of acyl-ACP binds.Substitution of two residues of an AHL synthase fromErwinia carotovora to increase the volume of the acylchain tunnel altered the specificity so that it producedN-(3-oxooctanoyl)-L-homoserine lactone rather than itsnormal product N-(3-oxohexanoyl)-L-homoserine lactone[8]. However, the binding pocket for acyl-ACP in LasI isopen-ended, so no such clear structural basis for the spec-ificity of LasI has been observed. Another potential sourceof specificity for the reaction in vivo that has not beenexplored fully is ACP itself. The P. aeruginosa genomeencodes three ACPs, and in vitro kinetic studies indicatethat only two are good substrates for RhlI [9]. Given themyriad proteins with which ACPs interact [10], it is possi-ble that organisms that express more than one ACP dis-tribute the chore of acylation specificity determinationbetween holo-ACP synthases and the AHL synthases forwhich the acyl-ACPs are substrates.

One of the most surprising findings in recent work wasthe discovery that some bacteria produce p-coumaroyl-homoserine lactone when they are grown in the presenceof p-coumarate [11]. Because p-coumarate is not a bacterialmetabolite, but is a component of lignin in plants, theimplication of this finding is that the bacteria rely on a

plant host to supply the side chain needed to form thesignaling molecule. This observation extends the interspe-cies cross-talk that AHLs can mediate to the synthesis ofthe AHLs themselves. Although not yet demonstrated,bacteria presumably express the enzymes necessary toconvert p-coumarate to p-coumaroyl-ACP. It has beenshown that a strain of Bradyrhizobium sp. produces cin-namoyl-homoserine lactone [12], so N-aroyl-homoserinelactones might be more common than has been recognizedto date.

An early question about the mechanism of AHLsynthases was whether the acyl group was transferredfrom acyl-ACP to the enzyme to form a covalent interme-diate in the course of the reaction, following a ping-pongmechanism. This hypothesis was initially supported byobservations of the RhlI reaction when the concentrationsof butyryl-ACP and SAMwere varied [7]. However, productinhibition studies ruled out a ping-pong mechanism andestablished that SAM was the first substrate to bind toRhlI and that MTA was the last product to dissociate. Bycontrast, studies of TofI established that the inhibitorJ8-C8, which binds to the site that the acyl group ofacyl-ACP must occupy, can bind to the enzyme in theabsence of SAM, so some uncertainty remains about theorder of substrate binding in AHL synthases. Consider-ation of the relative locations of the ligand binding sitesrevealed in the TofI structure suggests that acyl-ACP andSAM must bind from opposite faces of the enzyme to beconsistent with the order of substrate binding observed inthe RhlI reaction; otherwise, SAM binding would blockacyl-ACP from reaching its binding site.

Interest in developing compounds that disrupt quorum-sensing systems into pharmacological agents provides ur-gency to the task of defining the chemical mechanism of

9

Review Trends in Biochemical Sciences January 2012, Vol. 37, No. 1

AHL synthases (Box 2). Structural characterization of theproteins provides one route to designing inhibitors, butcharacterization of the chemical mechanism can provideinsights into the nature of the intermediates that formduring the catalytic cycle and the transition states for thechemical transformations. This important information canbe leveraged to design potent specific inhibitors. Currently,the general features of the chemical transformations thatoccur in the AHL synthase reaction are clear, but manydetails remain to be defined.

One of the interesting features of the AHL synthasereaction is that two distinct chemical reactions are cata-lyzed, acylation and lactonization. A priori, neither reac-tion would seem to be a chemical prerequisite for the other,so either reaction could occur first. However, N-butyryl-SAM was detected as an intermediate in rapid-mixingchemical quench experiments, which demonstrates thatacylation precedes lactonization [13].

Lactonization occurs via nucleophilic attack of the car-boxylate oxygen on the methylene carbon adjacent to thepositively charged sulfur of SAM. Alternative mechanismsin which S-methylthioadenosine is eliminated before lac-tone formation in the RhlI reaction can be discountedbased on the fact that no solvent protons are incorporatedinto the product, and the predicted intermediate, N-butyr-ylvinylglycine, is not turned over by the enzyme [9]. Themechanistic possibilities for lactonization span a continu-um from a purely SN2mechanism, inwhich the carboxylateoxygen adds to carbon concomitant with expulsion of MTA,to an SN1 mechanism, in which MTA departs first, leavingan electropositive carbon to which the carboxylate oxygen[(Figure_3)TD$FIG]

+ AHL

-OH - O

O

His106

His104

His169

Zn1 Zn2His235

Asp108

His109Asp191

- Product

+ HOH

- H+

O -

Zn1 Zn2

Try194

HOOO -

NH

O

Asp108

O

Zn

OH

Zn1

- O

O

NH

Zn

Zn1Zn1 Zn1Zn2

Zn1

Zn2Zn1

O

Figure 3. Chemical mechanism of AHL lactonase. A dinuclear zinc ion cluster at the acti

AHL substrates with diverse N-acyl substitutions.

10

adds. Computational studies suggest that the latter is abetter description of the reaction, which proceeds withconsiderable electropositive character at the methylenecarbon in the transition state [9].

The roles played by individual amino acid residues inthe AHL synthase reaction are unclear, although muta-genesis studies of conserved and random residues haveidentified crucial residues in RhlI [14,15]. The pH depen-dence of kinetic parameters in the RhlI reaction shows thatthe ionization states of two residues are important forcatalysis and binding [9]. The acylation reaction isexpected to require catalysis by acidic and basic enzymeside chains, but structural studies of GNAT superfamilyacetyltransferases have demonstrated that this can beaccomplished in different ways. More detailed interpreta-tion of the pH kinetics would be greatly aided by structuresof AHL synthases with bound substrates or products.

Bacterial censorship: AHL degradationWhenever an organism evolves a competitive advantage, itis almost an inevitable corollary that competing organismswill develop interfering strategies. This seems to be thecase for quorum sensing, because several differentenzymes capable of disrupting AHL-based quorum sensinghave been discovered. The majority of these ‘‘quorum-quenching’’ enzymes can be categorized into two distinctgroups: AHL lactonases, which catalyze hydrolytic ringopening of the lactone to form an N-acyl-homoserine prod-uct, and AHL acylases, which catalyze hydrolytic cleavageof the amide bond to form homoserine lactone and freefatty acid. Although the role of these enzymes in

Try194

Asp108

1 Zn2

O- O

OH

O

NH

Zn2

Zn1

Asp108

O

H

Asp108

O

NH

- OOH

OHO

Zn1

Zn21

Zn2

O

- O

- O

O

O

- O

Zn2

TiBS

ve site uses both zinc ions (Zn1 and Zn2) to catalyze the ring-opening hydrolysis of

Review Trends in Biochemical Sciences January 2012, Vol. 37, No. 1

their native environments is not always clear [16], theirquorum-quenching abilities and their utility as biochemi-cal tools, as well as in potential industrial and therapeuticapplications, are quite promising.

AHL lactonases

AHL lactonases can be further divided into three catego-ries that show homology to different protein superfamilies:amidohydrolases, paraoxonases and metallo-b-lactamases[17–19]. We focus on examples of AHL lactonases in themetallo-b-lactamase superfamily, which are arguablythe most active based on available kcat/KM values, andthe best defined in terms of catalytic mechanism.

The first quorum-quenching enzyme discovered was theautoinducer inactivator A (AiiA) from the Gram-positivebacterium Bacillus thuringiensis, later found to be wide-spread among Bacillus isolates [19,20]. AiiA was originallyidentified as a member of the metallo-b-lactamase super-family through recognition of the conserved metal-bindingmotif HXHXDH. Although a few initial reports questionedthe necessity of metal ions for catalysis, AiiA contains abinuclear zinc ion cluster at its active site that is essentialfor both proper folding and catalytic activity, as shown bymetal analysis, extended X-ray absorption fine structure(EXAFS), NMR and a series of X-ray crystal structures

[(Figure_4)TD$FIG]

(a) (b)

(d)

Tyr194

Asp108

Zn1Zn2

(c)

Figure 4. Structures of AHL-degrading enzymes. Substrate binding and hydrolysis occu

cut-away view of the AHL lactonase AiiA from Bacillus thuringiensis in complex with th

that the lactone ring, now opened, binds deep within the protein, with the N-acyl chai

Conversely, a cut-away view of the AHL acylase PvdQ from Pseudomonas aeruginosa in

binds deep within a hydrophobic cavity, leaving the lactone ring, not seen here in the pr

The b-chain is shown in blue and the a-chain in purple (Protein Data Bank code: 2wyc).

shown in panels (c,d). (c) The active site of AiiA (in green) has specific interactions bet

3DHB). (d) The active-site serine b1 of PvdQ (in blue) forms a covalent ester with the AH

(Protein Data Bank code: 2wyb). Abbreviation: Wat, water molecule.

[21,22]. The direct participation of the metal ions incatalysis was first demonstrated by comparison of hydro-lysis kinetics for AHLs and sulfur-substituted analogs for aseries of different AiiA metalloforms [23]. These measure-ments revealed a substantial effect on the relative kcatvalues with respect to the identity of the metal ion sub-stitutions, which reflects a kinetically significant interac-tion that occurs between the ring leaving-groupheteroatom and the metal ion center during substrateturnover.

The determination of product-bound structures shedlight on the position of the substrate at the active siteand its interactions with themetal center and other nearbyresidues, which led to proposal of the following mechanism(Figure 3) [24,25]. The AHL ring binds at the active site,placing its less hindered face toward themetal center, withits carbonyl carbon interacting with one of the zinc atoms(zinc-1, which is ligated by three histidines) and the leavinggroup oxygen placed over the other zinc atom (zinc-2, whichis ligated by a histidine and two aspartates). The hydroxideion that bridges both metal ions attacks the carbonylcarbon of the lactone, which leads to formation of a tetra-hedral adduct that is stabilized by zinc-1 and the phenolside chain of the neighboring Tyr194. This tyrosine is notconserved in the metallo-b-lactamase superfamily, but is

Asnβ260

Valβ70Wat Serβ1

TiBS

r via very different mechanisms in AHL lactonase compared to AHL acylase. (a) A

e bound product N-hexanoyl-L-homoserine (Protein Data Bank code: 3DHB) shows

n making weaker non-selective interactions along the surface of a wide cavity. (b)

complex with the bound product 3-oxo-dodecanoate shows that the N-acyl chain

oduct complex, to make less selective interactions with the surface of a wide cavity.

To illustrate mechanistic differences, close-up views of reaction intermediates are

ween each zinc ion and the product carboxylate (in grey) (Protein Data Bank code:

L substrate (in grey), which allows visualization of a trapped reaction intermediate

11

Review Trends in Biochemical Sciences January 2012, Vol. 37, No. 1

conserved in all of the AHL lactonases identified to date.The expected structure of the tetrahedral adduct parallelsthat of an observed phosphate ligand found at the activesite of the related lactonase AiiB [26]. Collapse of thetetrahedral adduct leads to expulsion of the oxygen leavinggroup, which is stabilized, possibly as the anion, by zinc-2.The Asp108 residue, originally ligated to zinc-2 and thebridging hydroxide in the resting enzyme, takes an alter-native conformation in which zinc-2 is released and theside chain is repositioned to shuttle a proton from thenewly formed carboxyl group to the leaving group. Thisproton transfer results in formation of the ring-openedN-acyl-homoserine product, which can now form a biden-tate bridge between themetal ions through its carboxylate,which results in the structure observed by X-ray crystal-lography (Figure 4). Product release, followed by regener-ation of the starting enzyme, completes the catalytic cycle.Stable isotope incorporation studies support this addi-tion–elimination mechanism [21]. One early mechanisticproposal placed the lactone ring in the opposite orienta-tion, with the carbonyl ligated instead to zinc-2 [27];however, this proposal is inconsistent with reactionmodeling and the product-bound structures, and probablyreflects the non-productive binding mode of the inhibitorcomplex from which the proposal was derived. The AHLlactonase mechanism shares many similarities withthat of the more distantly related metallo-b-lactamaseenzymes, notably in the use of zinc-2 to stabilize theleaving group [28]. These mechanistic similarities further

[(Figure_5)TD$FIG]

HOHOH

O

- Product

Serβ1 Serβ1NH2

NH3+

+ AHL

O -

O

n NH

H

O

O

n OH OH

HOH HO

OH

NH2

O O

O

NH

NH3+

Serβ1 Serβ1

Valβ70

O

H2N

O -

n

Figure 5. Chemical mechanism of the AHL acylase PvdQ. An N-terminal serine residue

formation of a covalent intermediate (which can be visualized using X-ray crystallograph

catalytic cleavage of the amide bond in AHL substrates.

12

support the inclusion of AHL lactonases in this diverseenzyme superfamily.

The substrate preference of AiiA is primarily deter-mined by selective recognition of the chiral (S)-homoserinelactone moiety, around which the enzyme is observed toclamp down on ligand binding [24]. TheN-acyl chain of thesubstrate does not seem to bind tightly, but rather binds ina relatively unconstrained manner via multivariate inter-actions with a wide hydrophobic groove along the surface ofthe protein (Figure 4). These interactions are reflected inthe strict selectivity of AiiA for the chiral lactone moietyand its broad substrate tolerance for the acyl chain, whichallows facile hydrolysis of acyl chains that are 4–12 carbonslong (or possibly longer) and tolerance for 3-oxo substitu-ents [21,29]. In fact, the recently identified N-aroyl-HSLquorum-sensing signals are two of the best substrates forAiiA, which reflects the diversity of N-substitutions thatare effectively processed [30].

AHL acylases

The second category of quorum-quenching enzymes, AHLacylases, are members of the N-terminal nucleophile(NTN) hydrolase superfamily. Other family members in-clude penicillin G acylase, penicillin V acylase, glutaryl-7-aminocephalosporanic acid acylase and aceulin acylase;each catalyzes an amide hydrolysis reaction. Themost fullycharacterized AHL acylase is PvdQ from P. aeruginosa[31]. Much like other NTN hydrolases, PvdQ is producedas an inactive precursor protein containing a signal peptide

OH

O

O

- OHH

- HSL+ HOH

HOH

NH

NH

NH3+

Serβ1

Valβ70

H2N

H2N

Asnβ269 Asnβ269Valβ70

Serβ1NH3

+

O -

O

O

n O

O

O

OH2N

O

O

O

Asnβ269

O

O

NHn

TiBS

(Serb1), unmasked by an autoprocessing reaction, serves as a nucleophile for the

y, as shown in Figure 4). Subsequent hydrolysis of the intermediate completes the

Review Trends in Biochemical Sciences January 2012, Vol. 37, No. 1

that directs export into the periplasmic space. Export isfollowed by intermolecular cleavage to remove the signalpeptide. The resulting protein undergoes two autoproces-sing events in which a short linker sequence is excised fromthe middle of the protein, which leaves an N-terminalpeptide (a-peptide) and a C-terminal peptide (b-peptide).The a- and b-peptides associate non-covalently to form themature heterodimeric enzyme. Autoprocessing is essentialfor enzyme activation because the newly formedN-terminalamine of Ser1 on theb-peptide (Serb1) plays an integral rolein catalysis.

NTN hydrolases have been described as having a single-amino-acid catalytic centre, but a number of PvdQ residuesother than Serb1 are suspected to facilitate turnover, ashighlighted in the following proposed mechanism(Figure 5) [32,33]. The AHL substrate binds close to Serb1,which uses its N-terminal amine as a general base todeprotonate, through an intervening water molecule, theside-chain hydroxyl. The deprotonated side chain of Serb1attacks the carbonyl carbon of the AHL amide. Formationof the tetrahedral adduct, stabilized by the backbone amideof Valb7 and the Asnb26 side chain, is followed by collapseand expulsion of the homoserine lactone moiety, withprotonation of the leaving amine by a bound water mole-cule. This forms a covalent ester intermediate with the sidechain of Serb1 (Figure 5), for which subsequent hydrolysiscan lead to formation and release of the fatty acid productand reformation of the resting enzyme. Most of the steps inthis proposed mechanism are based on analogy to therelated NTN hydrolases, and several mechanistic detailsspecific to PvdQ are not firmly established. However, theproposed mechanism is consistent with the recent PvdQstructures reported for product-bound complexes, as wellas the structure of the covalent ester intermediate that wastrapped at acidic pH [33].

Substrate recognition by PvdQ sharply contrasts withthat of AHL lactonase. As described above, AHL lactonaseclamps down over the lactone ring of the substrate andonly has weak non-selective interactions with the N-acylchain. Conversely, PvdQ binds the N-acyl chain of thesubstrate and does not have any obvious binding site forthe lactone ring (Figure 4). The deep hydrophobic pocket ofPvdQ, inwhich theN-acyl chains of the substrate bind, hasextensive and specific hydrophobic interactions that en-force stricter selectivity for acyl chains that are�8 carbonslong (Figure 4) [31]. By contrast, the lactone ring is notobserved in the current PvdQ structures, but is predictedto be located within a large solvent-filled cleft of theheterodimer [33]. There are no obvious residues placedfor selective interaction with the homoserine lactone moi-ety; thus, broad selectivity for this part of the substratethat is also often observed with other related NTN hydro-lases can be predicted. This prediction is also consistentwith a proposed alternative non-AHL substrate of PvdQthat would carry a much larger moiety linked to a fattyacid chain [34].

Concluding remarksThe recent structural determinations of enzymes capableof AHL synthesis and degradation have allowed amore detailed understanding of the basis of their catalytic

mechanisms and substrate selectivity. These studies pro-vide a foundation for understanding the native functions ofthe enzymes, for choosing the proper enzyme as a tool tounderstand quorum sensing, and for more directed engi-neering efforts to optimize their functions. The homology ofthese enzymes to others in their respective superfamilies,most notably to b-lactam-processing enzymes, highlightsintriguing parallels between how bacteria interact withg-lactones and b-lactams. Given that cell-to-cell signalinghas been proposed as the primary function of many naturalproducts that are used clinically as antibiotics [35], thehomology between antibiotic-inactivating enzymes andquorum-regulating enzymes suggests a possible common‘‘etymology’’ for these elements of bacterial communication.

AcknowledgementsWork in the authors’ laboratories was supported in part by a grant fromthe Robert A. Welch foundation (F-1572 to W.F.) and a grant from theNIH (GM59653 to P.A.T.).

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