PqsBC, a condensing enzyme in the biosynthesis of the pseudomonas aeruginosa quinolone signal: Crystal structure, inhibition, and reaction mechanism

JBC Papers in Press. Published on January 25, 2016 as Manuscript M115.708453

The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M115.708453

Structure and function of the condensing enzyme PqsBC

PqsBC, a condensing enzyme in the biosynthesis of the Pseudomonas aeruginosa

quinolone signal: crystal structure, inhibition, and reaction mechanism

Steffen Lorenz Drees¹, Chan Li², Fajar Prasetya², Muhammad Saleem², Ingrid Dreveny², Paul

Williams³, Ulrich Hennecke⁴, Jonas Emsley², and Susanne Fetzner¹

¹Institute for Molecular Microbiology and Biotechnology, University of Münster, D-48149 Münster,

Germany

²Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, Nottingham,

NG7 2RD, U.K.

³Centre for Biomolecular Sciences, School of Life Sciences, University of Nottingham, Nottingham,

NG7 2RD, U.K.

⁴Organic Chemistry Institute, University of Münster, D-48149 Münster, Germany

*Running title: Structure and function of the condensing enzyme PqsBC

To whom correspondence may be addressed:

Susanne Fetzner, Institute for Molecular Microbiology and Biotechnology, University of Münster, D-

48149 Münster, Germany. Tel.: +49 251 8339824; Fax: +40 251 8338388; E-mail: fetzner@uni-

muenster.de.

Jonas Emsley, Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham,

Nottingham, NG7 2RD, U.K. Tel: +44 1158467092, Fax: +44 1158468002; E-mail:

jonas.emsley@nottingham.ac.uk.

Keywords: Pseudomonas aeruginosa, biosynthesis, secondary metabolism, quorum sensing, crystal

structure; 2-alkyl-4(1H)-quinolones, Pseudomonas quinolone signal, condensing enzyme, FabH.

two subunits are organized with respect to the

active site. In this study, the PqsBC structure was

determined to 2Å resolution, revealing that PqsB

and PqsC have a pseudo 2-fold symmetry that

unexpectedly mimics the FabH homodimer. PqsC

has an active site comprised of Cys129 and

His269, and the surrounding active site cleft is

hydrophobic in character and approximately twice

the volume of related FabH enzymes which may

be a requirement to accommodate the aromatic

substrate 2-ABA. From physiological and kinetic

studies, we identified 2-aminoacetophenone as a

pathway-inherent competitive inhibitor of PqsBC,

whose fluorescence properties could be used for in

vitro binding studies. In a time-resolved setup, we

demonstrated that the catalytic histidine is not

ABSTRACT

Pseudomonas aeruginosa produces a number of

alkylquinolone-type secondary metabolites best

known for their antimicrobial effects and involve-

ment in cell-cell communication. In the alkylquin-

olone biosynthetic pathway, the β-ketoacyl-(acyl

carrier protein) synthase III (FabH) like enzyme

PqsBC catalyzes the condensation of octanoyl-co-

enzyme A and 2-aminobenzoylacetate (2-ABA) to

form the signal molecule 2-heptyl-4(1H)-quino-

lone. PqsBC, a potential drug target, is unique for

its heterodimeric arrangement and an active site

different from that of canonical FabH-like

enzymes. Considering the sequence dissimilarity

between the subunits, a key question was how the

1

Structure and function of the condensing enzyme PqsBC

involved in acyl-enzyme formation, but

contributes to an acylation-dependent increase in

affinity for the second substrate 2-ABA. Introduc-

tion of Asn into the PqsC active site led to signifi-

cant activity toward the desamino substrate analog

benzoylacetate, suggesting that the substrate 2-

ABA itself supplies the asparagine-equivalent

amino function that assists in catalysis.

alyzes the activation of anthranilic acid to an-

thraniloyl-CoA (18). A subsequent condensation

reaction with malonyl-CoA, catalyzed by PqsD,

yields the highly unstable intermediate 2-amino-

benzoylacetyl-CoA (2-ABA-CoA), however,

PqsD also uses malonyl-acyl carrier protein (ACP)

as substrate (19). While 2-ABA-CoA is highly

susceptible to spontaneous cyclization to form 2,4-

dihydroxyquinoline (DHQ) (19–21), in vivo this is

counterbalanced by the activity of PqsE, which

acts as 2-ABA-CoA thioesterase to release 2-

aminobenzoylacetate (2-ABA) (21). 2-ABA is an-

other branching point in the pathway and can

undergo decarboxylation to 2-aminoacetophenone

(2-AA) (20), a secondary metabolite reported to

promote chronic infection phenotypes of P.

aeruginosa (22, 23) and to modulate the host

innate immune response (24, 25). Alternatively, it

can be channeled into HHQ biosynthesis by the

activity of PqsBC, which catalyzes the condensa-

tion of 2-ABA and octanoyl-CoA to form HHQ

(20, 21) (Figure 1). Hydroxylation of HHQ to PQS

is catalyzed by the flavin monooxygenase PqsH

(26).

INTRODUCTION

Pseudomonas aeruginosa is a pathogenic Gram-

negative bacterium that inhabits soil and aquatic

environments. P. aeruginosa infections are often

nosocomial, and usually associated with

compromised host defenses (1). The production of

many P. aeruginosa virulence factors is controlled

by quorum sensing (QS⁵), a mechanism where

bacteria communicate by sensing self-generated

signaling molecules. QS systems allow bacterial

populations to synchronize their behavior and so

to act cooperatively (2). Because QS controls the

virulence of many pathogenic bacteria,

interference with QS receptor function or

inhibition of QS signal generation have been

proposed as alternative targets for anti-virulence

drug development (3–7).

PqsD is a member of the FabH family of

condensing enzymes (27). PqsC and PqsB, which

form a tight complex (20, 21), also have been

annotated to belong to this family. FabH (β-ke-

toacyl-ACP synthase III), a key enzyme in fatty

acid biosynthesis, catalyzes the decarboxylating

condensation of acetyl-CoA and malonyl-ACP to

form acetoacetyl-ACP (Figure 1). The active site

of E. coli FabH is comprised of residues Cys112,

His244 and Asn274, which are required to cata-

lyze the complete condensation reaction. While

Cys112 is loaded with the acetyl residue in the

initial step of the reaction, His244 and Asn274 are

involved in the subsequent decarboxylation reac-

tion of malonyl-ACP (reviewed by Heath and

Rock (28)). To identify the determinants of cataly-

sis, to gain insight into the PqsBC reaction mecha-

nism, and to probe possible effects of AQ metabo-

lites on PqsBC activity, we performed the first

structural and mechanistic investigation of this

unique condensing enzyme. FabH-like enzymes

are usually homodimers in solution, however, the

PqsBC crystal structure we report here reveals that

PqsB and PqsC form a novel heterodimer. PqsC

lacks the conserved asparagine and PqsB lacks all

three conserved residues of the FabH catalytic

The QS network of P. aeruginosa is high-

ly complex, comprising several signaling circuits

that are interconnected (for a recent review, see

(8)). The Las and Rhl circuits use specific N-

acylhomoserine lactones as signal molecules,

whereas the Pqs circuit employs the alkylquino-

lone (AQ) signals 2-heptyl-4(1H)-quinolone

(HHQ) and 2-heptyl-3-hydroxy-4(1H)-quinolone

(Pseudomonas quinolone signal, PQS). AQ signal-

ing influences biofilm development and the pro-

duction of a number of virulence factors, such as

pyocyanin, siderophores, rhamnolipid biosurfac-

tant, the cytotoxic lectin LecA, and elastase LasB

(reviewed in: (9, 10)). PQS moreover induces

membrane vesicle formation (11), acts as ferric

iron chelator (12, 13) and pro-oxidant (14), and

exerts host immune modulatory and pro-apoptotic

activities (15–17).

Biosynthesis of HHQ and PQS from an-

thranilic acid and fatty acid precursors is initiated

by the coenzyme A (CoA) ligase PqsA, which cat-

2

Structure and function of the condensing enzyme PqsBC

triad, raising the question of how PqsBC mediates

the condensation reaction.

ny) and applied onto a 5 ml Ni-NTA sepharose

column (Qiagen, Hilden, Germany) equilibrated

with a wash buffer containing 20 mM Tris, pH 8.0,

150 mM NaCl and 10 mM imidazole. The column

was washed with 10 column volumes (CV) of

wash buffer before a 10 CV gradient against an

elution buffer containing 20 mM Tris, 150 mM

NaCl and 150 mM imidazole was applied. Protein-

containing fractions were pooled and dialyzed at

room temperature against a buffer containing 10

mM Tris and 0.5 mM dithiothreitol (DTT) (Carl

Roth, Karlsruhe, Germany) using a 14 kDa molec-

ular weight cut off (MWCO) dialysis tube (Serva-

por, Serva electrophoresis, Heidelberg, Germany).

After 2 h, TEV protease (see below; 1 µg TEV

protease per 20 µg total protein), arginine and glu-

tamate (final concentration 50 mM, Sigma-

Aldrich, St. Louis, MO, USA) were added directly

to the dialysis tube, and the tube was transferred

into fresh buffer. Dialysis was continued over

night at 4°C with gentle stirring. Precipitated

material was removed by centrifugation at

50,000× g (20 min, 4°C) and the supernatant was

passed through a 1 ml HisTrap column (GE

Healthcare Life Sciences) equilibrated with 20

mM Tris-HCl, pH 8 to separate PqsBC from the

uncleaved fusion protein, TEV protease, and His-

tag peptides. The flowthrough was collected and

subsequently applied to a 6 ml Resource Q column

(GE Healthcare Life Sciences) equilibrated with a

wash buffer containing 15 mM MOPS pH 8.1

(Carl Roth, Karlsruhe, Germany) and 1 mM DTT.

The column was washed with 20 CV wash buffer

before a linear 20 CV gradient against 15 mM

MOPS pH 8.0, 400 mM NaCl, 1 mM DTT was

started. PqsBC containing fractions were pooled

and passed through a second 1 ml HisTrap column

equilibrated with a buffer containing 15 mM

MOPS, pH 8.0, 150 mM NaCl, to remove residual

impurities. The flowthrough was collected and

concentrated to a volume of approx. 1.5 ml using a

10 kDa MWCO centrifugal concentrator (Sar-

torius, Goettingen, Germany). The protein solution

was applied onto a HiLoad26/60 Superdex200 gel

filtration column (GE Healthcare Life Sciences)

equilibrated with a buffer containing 40 mM

MOPS, pH 8.1 and 150 mM NaCl. For molecular

weight estimation, a calibration run using a

BioRad Gel filtration Standard was used. Elution

fractions containing the PqsBC protein were

EXPERIMENTAL PROCEDURES

Expression and purification of PqsBC

The pqsB and pqsC genes of P. aeruginosa PAO1

were cloned into plasmid pET28b using restriction

free (RF) cloning (29). In the process, pqsC was

fused with the sequences encoding a short linker

(Ser-Ala-Gly), a recognition site for tobacco etch

virus (TEV) protease, and an N-terminal octahisti-

dine tag. The vector-internal sequences for affinity

tags were not used. The coding sequence of the re-

sulting plasmid pET28b::pqsBC-NHis₈was veri-

fied using commercial sequencing services. Site-

specific mutations within the pqsC gene were in-

troduced using RF-cloning (29). For protein over-

production, E. coli Rosetta2(DE3)[pLysS] carry-

ing the respective expression plasmid was grown

in TB medium (Carl Roth, Karlsruhe, Germany)

supplemented with 150 µg ml^-1kanamycin (Appli-

chem, Darmstadt, Germany). Cells were cultivated

in baffled flasks at 37°C until reaching a cell den-

sity (OD₆₀₀) of 0.8, then cooled to 20°C before

gene expression was induced with 0.2 mM isopro-

pyl-β-D-thiogalactopyranosid (IPTG, Thermo

Scientific, Waltham, MA, USA). After 8 to 12 h,

cells were harvested by centrifugation, flash-

frozen in liquid N₂and stored at −80°C until use.

For the preparation of PqsBC, cells were

resuspended in 20 mM Tris-HCl buffer (pH 8.0)

containing 150 mM NaCl, 10 mM imidazole, 5

mM EDTA, 0.5 mM β-mercaptoethanol and 0.1%

Nonidet P40 (all Carl Roth, Karlsruhe, Germany).

Cells were disrupted by sonication (Hielscher

UP200S Hielscher, Germany), and subsequently

cell debris was removed by centrifugation for 1 h

at 50,000 × g and 4°C. Purification of PqsBC was

performed using immobilized metal affinity

chromatography, anion exchange and gel filtration

chromatography. All chromatographic procedures

were carried out using either Äkta PrimePlus (GE

Healthcare Life Sciences, Pittsburgh, PA, USA) or

BioLogic (BioRad, Hercules, CA, USA) chroma-

tography systems. To this end, cell extract super-

natants were filtered (0.45 µM polyvinylidene

fluoride membrane, Carl Roth, Karlsruhe, Germa-

3

Structure and function of the condensing enzyme PqsBC

pooled and 2 mM DTT was added. The solution

was concentrated to > 20 mg ml^-1and applied onto

two serially connected 5 ml HiTrap buffer ex-

change columns (GE Healthcare Life Sciences)

equilibrated with the storage buffer containing 40

mM MOPS, pH 8.1, and 50 mM NaCl. Eluted pro-

tein fractions were pooled, aliquoted, flash-frozen

and stored at −80°C until required for further use.

Protein concentration was determined by UV

spectroscopy according to Gill and Hippel (30)

using a calculated extinction coefficient of 72,880

M^-1cm^-1. Purification of PqsBC protein variants

followed the same protocol.

exchange of H against D). ¹³C-NMR (150 MHz,

D₂O, 299 K): δ = 200.0, 175.9, 150.5, 135.3,

132.1, 118.0, 117.9, 116.7, 49.9-49.4 (m, overlay

of signals of CH₂-, -CHD- and -CD₂- groups). MS-

ESI(+): m/z = 403 (30) [2M-H+2Na]⁺, 381 (60)

[2M+Na]⁺, 218 (25) [M+K]⁺, 202 (100) [M+Na]⁺,

180 (55) [M+H]⁺, 162 (30) [M-H₂O+H]⁺. MS-

ESI(+)-EM: m/z = 202.04746 calculated for

C₉H₉NO₃Na [M+Na]⁺, found: 202.04705.

Spectrofluorimetric determination of substrate

and inhibitor binding to PqsBC

Binding equilibria as well as octanoylation

of PqsBC were measured by spectrofluorimetric ti-

tration using Jasco FP-6500 or Jasco FP-8300 in-

struments (Jasco, Tokyo, Japan) and 100 µl quartz

cuvettes. All measurements were performed in

ratio mode with 5 nm excitation/emission band-

width, 1s response and manually adjusted photo-

multiplier sensitivity. Titrants were added with an

eVol digital analytical syringe (SGE/Trajan scien-

tific, Melbourne, Australia). For determination of

dissociation constants for the PqsBC-2-ABA and

PqsBC-2-AA complexes, the fluorescence proper-

ties of the substrates were utilized (λ_Ex= 359 nm,

λ_Em= 470 nm). Assays were carried out in 50 mM

HEPES, pH 8.2 and 50 mM NaCl at 22°C, titrating

2-ABA or 2-AA (0-3 mM) into PqsBC (1-10 µM),

or vice versa (0-300 µM PqsBC, 1-10 µM 2-ABA

or 2-AA). After every titration step, the mixture

was equilibrated for 1 min before the measurement

was taken. Recorded spectra were corrected for

dilution and inner filter effects as described

elsewhere (35). For detection of covalent

octanoate binding, 2-AA was used as fluorescence

probe. The same conditions as above were used,

but 2-AA (0-50 µM) and PqsBC (0-50 µM) were

premixed and octanoyl-CoA was added while

fluorescence was monitored over time (0.5 s

resolution, 10 s mixing time). All experiments

were carried out with at least three technical

replicates. To exclude variance between protein

preparations, four different batches of PqsBC were

assayed. Any influence of the affinity tag on sub-

strate or 2-AA binding could be excluded, because

titrations with tagged and protease-processed pro-

teins gave similar binding constants.

Expression and purification of tobacco etch vi-

rus protease

The L56V/S135G/S219V variant of TEV protease

was overproduced and prepared as described by

Cabrita et al. (31).

Synthesis of 2-aminobenzoylacetate (2-ABA)

In a 250 ml flask, 2-nitrobenzoylacetate (32–34)

(84 mg, 0.40 mmol) was dissolved in water (10

ml) and aqueous ammonia (0.1 ml, ca. 25wt% in

water). Palladium on charcoal (75 mg, 5% Pd/C,

Sigma-Aldrich) was added and the flask was

placed under a hydrogen atmosphere (atmospheric

pressure, by three cycles of brief evacuation and

backfilling with hydrogen from a balloon). The

suspension was rapidly stirred for 40 min under

hydrogen and then filtered through a pad of celite.

The solvent was removed under high vacuum to

give the product (56 mg, 0.31 mmol, 78%) as a

yellow-green gum. ¹H-NMR spectra were

recorded on an Agilent DD2 600 spectrometer

(600 MHz). ¹³C-NMR spectra were recorded on an

Agilent DD2 600 spectrometer (150 MHz).

Chemical shifts δ are given in ppm and are refer-

enced according to IUPAC recommendations on a

unified chemical shift scale for all nuclides based

on the proton resonance of tetramethylsilane as

primary reference. MS-ESI(+) spectra were

recorded on a Thermo Scientific Orbitrap LTQ XL

1

using nanospray methods. Data: H-NMR (600

MHz, D₂O, 299 K): δ = 7.67-7.62 (m, 1H), 7.26-

7.21 (m, 1H), 6.72-6.68 (m, 1H), 6.65-6.60 (m,

1H), 3.71/3.69 (s, the intensity of these signals,

which represents the -CH₂- and -CHD- group

depends strongly on the time between dissolution

and measurement of the spectrum due to the slow

Equilibrium kinetic data from spectrofluo-

rimetric titrations were fitted using Matlab R2014b

with optimization toolbox (The Mathworks). For

4

Structure and function of the condensing enzyme PqsBC

titrations of 2-ABA or 2-AA with PqsBC, the con-

centration of the enzyme in complex with

substrate or substrate analog was calculated from

the law of mass action

Enzyme activity assays with 2-ABA and oc-

tanoyl-CoA

Catalytic activity of PqsBC was measured

with 2-ABA and octanoyl-CoA (Sigma-Aldrich).

Assays were performed at 25°C in buffer contain-

ing 50 mM HEPES, pH 8.2, 50 mM NaCl and 100

µM DTT (all Carl Roth, Karlsruhe, Germany).

Discontinuous assays were based on the quantifi-

cation of HHQ by HPLC formed in the reaction as

described previously (21). Continuous spectro-

scopic assays were based on measuring either 2-

ABA consumption or HHQ formation. The extinc-

tion coefficient of 2-ABA, quantified by the

amount of DHQ formed upon acidification, was

5,025 M^-1cm^-1at 360 nm (in assay buffer, pH 8.2).

For setting up a spectroscopic assay detecting

HHQ, defined amounts of 2-ABA were fully con-

verted by PqsBC with an excess of octanoyl-CoA,

and the resulting HHQ was quantified by HPLC.

UV spectra were recorded before and after the

reaction (Jasco V-650 spectrophotometer), which

in combination with the known UV-visible spec-

tral properties of HHQ and 2-ABA allowed the

estimation of a differential extinction coefficient

for the HHQ formation of ε_{313 nm}= 6,520 M^-1cm^-1.

However, the value at concentrations of more than

10 µM HHQ is meaningless due to scattering

effects caused by precipitating HHQ. The apparent

steady-state kinetic constants of PqsBC were esti-

mated by fitting the initial velocities measured at

different substrate concentrations with the Michae-

lis Menten equation. The assays contained 0.02

µM PqsBC, and 2-ABA was varied from 10 to 500

µM at 100 µM octanoyl-CoA, whereas octanoyl-

CoA was varied at concentrations between 1 and

200 µM at 500 µM 2-ABA. For testing the in-

hibitory potential of 2-AA (Sigma Aldrich), DHQ

(Ferak, Berlin, Germany), PQS and HHQ (both

Sigma Aldrich) on 2-ABA conversion by PqsBC,

the assays contained 50 µM octanoyl-CoA and

varying concentrations of 2-ABA and the potential

inhibitor. EC₅₀values were calculated by non-

linear fitting using the appropriate logistic func-

tion. Inhibition constants were either determined

graphically from the Dixon plot (smallest Euclide-

an distance between regression lines) or calculated

by least squares fit over all measured initial veloci-

ties using equation 4.

Equation 1

퐸푆

(퐸_푡+ 퐾_퐷+ 푆_푡) − √((퐸_푡+ 퐾_퐷+ 푆_푡)²− 4퐸_푡∙ 푆_푡)

=

2

Where ES is the concentration of the enzyme-sub-

strate complex, E_tis the total enzyme concentra-

tion, K_Dis the dissociation constant and S_tis the

total substrate concentration. Fluorescence data

was converted to binding ratios using the expres-

sion

Equation 2

퐸푆

퐹 = 퐹₀+

∙ 퐹

푎푚푝

푆_푡

when S_twas kept constant or

Equation 3

퐸푆

퐹 = 퐹₀+

퐸_푡

∙ 퐹

푎푚푝

when E_twas kept constant in the assay. F is the

measured fluorescence, F₀is the starting fluores-

cence value and F_ampis total amplitude of fluores-

cence change.

Transient kinetics of PqsBC octanoylation

Stopped flow fluorimetry was used to

measure the transient kinetics of PqsBC oc-

tanoylation. Experiments were conducted using a

SFM-3 stopped-flow instrument (Biologic, Claix,

France) coupled to a Jasco FP-6500 fluorimeter. 2-

AA or 2-ABA (120 µM) were mixed with PqsBC

protein (30 µM), octanoyl-CoA (120 µM) and 2-

AA or 2-ABA (120 µM) from separate reservoirs.

Fluorescence changes of 2-AA or 2-ABA upon

protein binding were used to probe octanoylation.

Samples were excited at 360 nm and emission was

measured at 450 nm (bandwidth 10 nm). The

minimal detector response time was 10 ms and the

stopped flow unit was operated at maximum speed

(dead time 1.8 ms). 10 to 20 experiments were av-

eraged for data collection. Data were fitted using a

single exponential function and rate constants

were calculated assuming a first order reaction.

5

Structure and function of the condensing enzyme PqsBC

Equation 4

on the in vivo HHQ synthesis, the recombinant

strain was grown as described previously (37) in

the presence of different concentrations of 2-AA.

Culture samples were withdrawn in 30 to 120 min

intervals and analyzed by HPLC for HHQ con-

tents. Possible effects of 2-AA on the production

of the biosynthetic enzymes were monitored by

Western blotting and immunodetection of the

His₆-tagged PqsD protein using a pentahistidine-

specific monoclonal antibody (Qiagen, Hilden,

Germany).

푣_푚푎푥∙ 푆

푣 =

퐼

퐾_푚∙ (1 + ) + 푆

퐾

푖

Where v is the measured initial reaction velocity,

v_maxis the maximum velocity, S is the initial sub-

strate concentration, K_mis the apparent Michaelis

constant without inhibitor added, I is the inhibitor

concentration and K_iis the competitive inhibition

constant. All data processing, fitting and calcula-

tion were performed with Matlab R2014A with

curve fitting and optimization toolboxes (The

Mathworks, Natick, MA, USA).

Crystallization and structure determination of

PqsBC^C129A

Activities of PqsBC with substrate analogs

Protein samples of wild type PqsBC and

the active site protein variant PqsBC^C129Ain 40

mM MOPS buffer pH 8.1, 100 mM NaCl, 0.3 mM

DTT at protein concentrations of 31.5 and 36.5 mg

ml^-1respectively were diluted with 100 mM NaCl

to give 3 mg/ml. These samples were subjected to

screens including Pi-PEG, Procomplex, classics

lite, Mbclass, Index, PACT, Morpheus, JCSG+,

MIDAS using the sitting drop vapor diffusion

method (Mosquito robot, TTP Labtech,

Melbourne, UK). Crystals of PqsBC^C129Aresulted

from the conditions of 0.2 M NaCl, 0.1 M Tris, pH

8.5, 25% PEG 3350 within 24 h whereas crystals

resulted from neither octanoylated nor apo-forms

of wild type PqsBC. Optimization was performed

using 24 well sitting drop plates varying PEG in

1% increments, 23%,-27% as well as Tris pH 8.3-

8.7. The final optimized crystallization condition

was 25% PEG 3350, 0.1 M Tris pH 8.3, and 0.2 M

NaCl with 3 mg ml^-1protein concentration and a

total volume of 4 μl. PqsBC^C129Acrystals were

cryoprotected in a solution of 27% PEG 3350, 0.1

M Tris pH 8.3, 0.2 M NaCl, 25% glycerol, and

flash-cooled in liquid nitrogen prior data collection

at the Diamond Light Source synchrotron facility

beamline I02 to 2.0Å resolution with spacegroup

P2₁2₁2₁and a unit cell of a=78.5Å, b=115.0Å,

c=289.7Å, α=90, β=90, γ=90 (Table 1). This

unit cell was predicted to have 4 PqsB and 4 PqsC

polypeptides in the asymmetric unit with

approximately 50% solvent content. Molecular

replacement was performed with PHASER using a

variety of templates generated using MrBUMP

(38) from pdb codes 3h76, 3h77, 3h78 of the PqsD

structure (39). Using different resolution cut-offs

this resulted in an incomplete solution from which

Since the reactivity of PqsBC with oc-

tanoyl-CoA and substrate analogs benzoylacetate

or malonyl-CoA cannot be analyzed by spectral

changes upon product formation, a colorimetric

assay based on the reaction of Ellman’s reagent

with free thiols originating from transacylation of

octanoyl-CoA was employed (36). For assaying

the activity of PqsBC with octanoyl-CoA and ben-

zoylacetate, 2.5 mM of benzoylacetate and 250

µM of octanoyl-CoA were mixed in a buffer con-

taining 50 mM HEPES, pH 8.2 and 50 mM NaCl

before PqsBC (concentration between 0.001 and 1

µM) was added. The sample volume was 50 µl.

Samples were incubated between 1 and 30 min

before reactions were stopped by adding 48 µl of

200 mM Tris, pH 7.4, 1 % SDS. The chromogenic

reaction was started by adding 2 µl of 50 mM 5,5'-

dithiobis-(2-nitrobenzoic acid) (DTNB). Sample

absorbances were immediately measured at 412

nm (ε_DTNB= 13,700 cm^-1M^-1(36)). All sample

data were corrected against equivalently treated

samples in which the enzyme was inactivated with

0.5% SDS before adding it to the assay mixture.

Assays with octanoyl-CoA and malonyl-CoA were

conducted analogously, using 100 µM octanoyl-

CoA and 250 µM malonyl-CoA. Samples were

incubated for 1 h before reactions were stopped

and free thiol content was measured.

Analysis of the in vivo inhibition of HHQ bio-

synthesis by 2-AA

P. putida KT2440 [pBBR-pqsABCD-his],

when grown in mineral salts medium supplement-

ed with anthranilic acid, produces substantial

amounts of HHQ (37). To test the effect of 2-AA

6

Structure and function of the condensing enzyme PqsBC

a partial model was built using BUCCANEER

(40) and electron density maps were subsequently

improved using 4-fold non-crystallographic

symmetry and solvent flattening with the CCP4

programs DM (41) and PARROT (42). The

resulting density modified 2Å electron density

map was of high quality allowing 95% of the

model to be built with BUCCANEER. The model

was completed manually using COOT and refined

with REFMAC5 (43) giving 2474 residues in total

and crystallographic statistics are listed in Table 1

(pdb code 5DWZ). Values for surface area buried

were calculated using PISA (44).

lographic R-factors of R_work=0.190 and R_free=0.237

(Table 1). The crystallographic asymmetric unit

contains four copies of PqsB and four copies of

PqsC. An analysis of the interfaces reveals a large

~2238ꢀ²surface area buried by interactions

formed between the PqsB and PqsC subunits as

shown in Figure 2A. This is consistent with gel fil-

tration data indicating that PqsB and PqsC form a

tight complex with an apparent molecular mass of

51 kDa. The structure is thus referred to as the

PqsBC^C129Aheterodimer. This large interface be-

tween PqsB and PqsC is observed to be almost

identical in the four copies observed in the asym-

metric unit (values of buried surface area are

2277ꢀ², 2240ꢀ², 2223ꢀ², 2212ꢀ²averaged to

2238ꢀ²) and the heterodimers superimpose onto

each other well with an average r.m.s.d. of 0.243ꢀ

(8182 atoms).

RESULTS

Synthesis and stability of 2-ABA and ben-

zoylacetate

Quantitative investigation of the PqsBC

The arrangement of secondary structural

reaction requires highly purified substrates

applicable at defined concentrations, however, 2-

ABA was previously described as relatively

unstable in solution, decomposing to 2-AA and

also to DHQ (20, 34). After testing a series of

conditions we discovered that hydrogenation in an

alkaline aqueous solution is the key to obtain 2-

ABA in high yield and purity. Using dilute

ammonia solution as medium for hydrogenation of

the 2-nitrobenzoylacetate precursor, we obtained

2-ABA in 78% yield with approximately 95%

purity (as indicated by HPLC and NMR data). In

aqueous solution at pH above 8, it is stable for

several days at room temperature (in the dark), and

can be stored at −80°C for months. When

solutions of 2-ABA were acidified, the compound

decomposed to DHQ. However, when exposed to

organic solvents such as methanol, acetonitrile or

dimethyl sulfoxide, rapid decarboxylation to 2-AA

as sole decomposition product was observed. Ben-

zoylacetate (BA), synthesized as previously de-

scribed (21), was used as an analog of 2-ABA in

the PqsBC reaction. It turned out to be unstable in

aqueous solution at neutral pH and decarboxylated

within minutes to hours, yielding acetophenone.

elements colored from the N- to C-terminus shows

PqsB and PqsC assemble into a dimer with the N-

termini (blue) co-localizing close to the pseudo 2-

fold axis and the C-termini (red) are placed

adjacent directed away from the axis (Figure 2B).

The fold of PqsC and PqsB is closely related to the

FabH enzymes and PqsD with two subdomains

each comprised of a four stranded β-sheet

surrounded by α-helices. The structural superposi-

tion of PqsB and PqsC shown in Figure 2C is

largely driven by similarities in the N-terminal

subdomain (Figure 2D). Substantial differences

occur in the C-terminal subdomains with PqsB

having a shorter sequence (124 amino acids) with

only two α-helices and PqsC (160 amino acids)

having four α-helices (Figure 2E). The large

difference in the amino acid sequence of the PqsB

C-terminal region compared to PqsC means that it

is difficult to align the primary sequences and

there is no identifiable catalytic triad or dyad in

the PqsB structure indicating it likely contributes a

supporting structural role to the enzyme func-

tionality.

PqsC^C129Astructure defines a catalytic dyad

The active site residues His269 and the

mutated Ala129 (replacing Cys129) are clearly

defined in the electron density (Figure 3A). In a

noticeable difference with other members of the

FabH enzyme family an asparagine amino acid

does not form a catalytic triad with His269,

PqsBC^C129Acrystal structure reveals a heterodi-

mer

PqsBC^C129Acrystallized in spacegroup

P2₁2₁2₁and the structure was determined to 2ꢀ

resolution with the final model refined to crystal-

7

Structure and function of the condensing enzyme PqsBC

Cys129 and in its place a valine (Val299) is

present (Figure 3B shows the superposition of

PqsC with the PqsD catalytic triad). Figure 4A

shows PqsC^C129Asuperposed onto the PqsD

structure (pdb code: 3H78) viewed in the area of

the active site cleft illustrating significant struc-

tural differences. The area of the structure known

as the flap in the FabH enzymes (45) is conserved

as a short α-helix in PqsD whereas in PqsC it is

formed by a short antiparallel β9-β10 sheet. Di-

rectly C-terminal to this, further differences are

apparent as the PqsC helix α5 is shorter than the

equivalent α-helix in PqsD/FabH. Residue Pro242

breaks the helix α5 in PqsC and directs an

alternate path of the polypeptide compared to

FabH/PqsD forming an irregular loop structure,

termed loop A which effectively opens up the

active site cleft. Another difference between PqsC

and FabH/PqsD structures is an insertion of 14

amino acids in PqsC between strand β4 and helix

α3 (Figure 5; PqsC residues 88-102). This

insertion is termed loop B and forms a network of

interactions with the PqsC flap and contributes to

the dimer interface with PqsB.

using metapocket (47) and rendered with Pymol.

The PqsC pocket surrounding the catalytic dyad

extends towards the interface with PqsB and is

bifurcated with one branch formed by loop B and

residues from PqsB and a second branch remains

within PqsC in the area of the flap (Figure 6B).

Figure 6B and C show the decyl-formate ligand

from the FabH-decyl formate complex crystal

structure (pdb: 2QNZ (45)) which has been

superposed onto the PqsBC^C129Astructure

illustrating the active site pocket extends in two

directions

from

Ala129

as

in

FabH

(Supplementary movie 1). Figure 6D schematical-

ly shows the residues lining the area of the pocket.

The binding site of coenzyme A has been charac-

terized in both FabH (pdb: 1hnj) and PqsD (pdb:

3h77) complex crystal structures (27, 48, 49). Two

PqsC residues Trp35, Arg168 homologous to the

PqsD (Trp32, Arg151) coenzyme A binding site

are conserved (27, 50). In PqsD these form

interactions on either side of the coenzyme A

nucleobase suggesting a similar interaction may

occur in PqsC (Figure 6D). However no other

similar residues are conserved due to the large

changes associated with loss of the N-terminal re-

gion of helix α5 in PqsC (Figure 4A).

Figure 4B illustrates the structure of the

PqsBC^C129Aheterodimer compared to the PqsD ho-

modimer below viewed with the flap region lying

close to the PqsD dimer axis (Figure 4C). Figure

4D shows two orientations of a C-α trace of

PqsBC polypeptide backbone superposed on the

PqsD homodimer. The superposition is driven

largely by the similarity between PqsC and PqsD

and shows the close relative positioning of the

PqsBC subunits compared to the PqsD

homodimer. The PqsB β-sheets are displaced by 2-

3ꢀ when overlaid with PqsD (Figure 4E). The

PqsBC active site cavity is elongated in the di-

rection of the heterodimer interface where it nar-

rows and extends into PqsB. The gap in the inter-

face is not observed in FabH/PqsD and in part ac-

counts for the smaller ~2238ꢀ²surface area buried

by formation of the PqsBC heterodimer compared

with the larger value of 3000ꢀ²for the FabH/PqsD

homodimer.

Steady-state kinetics suggest competitive inhibi-

tion of PqsBC by 2-AA

Using a continuous spectrophotometric as-

say, initial reaction rates were determined for the

PqsBC reaction at substrate concentrations of up

to 1 mM 2-ABA and 200 µM octanoyl-CoA. Be-

cause a scattering band is emerging at higher con-

centrations of HHQ (Figure 7A), initial rates were

evaluated before the solubility limit of HHQ was

reached. Under the conditions of the assay, a k_cat

of 6.8 ± 0.5 s^-1was observed, and apparent K_m

values of PqsBC were 6.0 ± 0.5 µM for octanoyl-

CoA, and 105 ± 12 µM for 2-ABA. The His-

tagged and untagged protein showed no difference

in their kinetic parameters.

To determine whether the various products

of the AQ biosynthetic pathway have an effect on

PqsBC activity, enzyme assays were performed in

the presence of HHQ, PQS, 2-AA, or DHQ. HHQ

and PQS as well as DHQ at concentrations up to

50 µM did not affect PqsBC activity. 200 µM

DHQ, a concentration which is not reached in P.

aeruginosa cultures (51), caused an about 10%

The PqsC^C129Aactive site volume is twice

as large as those of FabH/PqsD with a calculated

volume of 761Å³compared to 389Å³for PqsD

(pdb:3H76) and 367Å³for FabH (pdb:2QNZ) (cal-

culated using 3vee (46)). Figure 6A highlights in-

ternal cavities in the PqsBC structure calculated

8

Structure and function of the condensing enzyme PqsBC

drop in activity. In contrast, 2-AA is an efficient

inhibitor of PqsBC with an apparent EC₅₀of 46

µM in the in vitro assay. It increased the apparent

K_mof the enzyme for 2-ABA while k_catremained

unaffected (Figure 7B−D), indicating a competi-

tive binding mode with an estimated inhibition

constant K_ibetween 54.5 µM (graphic determina-

tion from Dixon plot, Figure 7D) or 56 ± 3.1 µM

(global fit according to eq. 4, denoted error is the

SECV).

to the K_mvalues of PqsA, PqsD and PqsH (18, 19,

26). This led us to analyze the binding equilibria

of PqsBC with 2-ABA, as well as with its com-

petitive inhibitor 2-AA. Usefully, it turns out that

the fluorescence characteristics of 2-AA and 2-

ABA, both containing a 2-aminobenzoyl moiety,

are strongly influenced by their molecular

environment such that the increase in fluorescence

upon protein binding could be used for de-

termination of the binding affinities to PqsBC.

The K_Dvalue of about 370 µM determined

for the complex of PqsBC with 2-ABA (Table 2)

suggests that the affinity of non-octanoylated

PqsBC for 2-ABA is even lower – more than

three-fold – than expected from the corresponding

apparent K_mvalue. Exchange of Cys129 by Ala or

Ser had an only minor effect on K_D, indicating that

this position does not play a crucial role in the

binding of 2-ABA. K_Ds of the PqsBC/2-AA com-

plex were roughly two fold lower, which matches

the observed ratio of K_m(2-ABA):K_i(2-AA) of 2:1.

The exceedingly high K_Dvalue of the

PqsBC/2-ABA complex and the discrepancy be-

tween equilibrium binding and kinetic data led us

to hypothesize that octanoylation at Cys129 of

PqsC (20), which, based on the current under-

standing of FabH-like reactions is proposed to be

the initial step of the reaction, modulates the affin-

ity of the enzyme for 2-ABA. Assuming that 2-AA

binds to PqsBC and to octanoyl-PqsBC in the

same way as 2-ABA, we used 2-AA for probing

the effect of PqsBC acylation on the affinity for

the second substrate. As illustrated in Figure 9B,

octanoyl-PqsBC has an approximately 35-fold

higher affinity for 2-AA than the unloaded

enzyme. However, such a change in affinity was

not observed for the PqsBC^C129Sand PqsBC^C129A

proteins incubated with octanoyl-CoA, supporting

the notion that covalent binding of the octanoyl

moiety to Cys129 increases the binding affinity of

PqsBC for 2-AA and, most probably, also for 2-

ABA. Octanoylation of PqsBC^C129Swas not

observed.

2-AA inhibits biosynthesis of HHQ in vivo

In view of the observed in vitro inhibition

of PqsBC activity by 2-AA, which is produced by

planktonic P. aeruginosa cultures in concentra-

tions of up to 200 µM (52), we used a recombinant

strain of Pseudomonas putida constitutively ex-

pressing the pqsABCD genes for testing whether

exogenously added 2-AA can inhibit AQ biosyn-

thesis in vivo. Any measured inhibitory effect on

HHQ biosynthesis in this strain would be caused

by inhibition of the biosynthetic proteins. Immu-

nodetection of PqsD, the last protein translated

from the polycistronic mRNA, in total cell pro-

teins demonstrated that exposure to 2-AA did not

alter the production of Pqs proteins by the cells. 2-

AA inhibited HHQ biosynthesis by P. putida

KT2440 [pBBR-pqsABCD], with an EC₅₀value of

319 ± 38 µM (Figure 8).

Inhibition of HHQ synthesis by 2-AA was

observed before for P. aeruginosa and was in part

attributed to downregulation of pqsABCDE ex-

pression by inhibition of the transcriptional regula-

tor PqsR (MvfR). However, post-transcriptional

interference of 2-AA with HHQ biosynthesis was

also proposed (22). Our in vivo and in vitro data

suggest that competitive inhibition of PqsBC

accounts for the post-transcriptional inhibition.

Because the IC₅₀of 2-AA of approximately 370

µM in P. aeruginosa PA14 (indicated by the

relative activity of the pqsA promoter (22)) is in a

similar range as the EC₅₀value determined for the

recombinant P. putida strain used in this study, the

effect of 2-AA on PqsBC likely dominates over its

effects on pqsABCDE transcription.

His269 of PqsC modulates 2ABA/ 2-AA binding

Interestingly, replacement of His269 in

PqsC by alanine resulted in significantly altered

binding properties of the PqsBC^H269Aprotein for

both, 2-ABA and 2-AA. The affinity of the (non-

acylated) mutant protein was 1.5 to 2-fold higher

Octanoylation of PqsBC has a major effect on

binding of the second substrate

The apparent Michaelis constant of PqsBC

for 2-ABA is rather high, at least when compared

9

Structure and function of the condensing enzyme PqsBC

than that of wild-type PqsBC for both compounds,

while octanoylated PqsBC^H269Adid not show the

increase in affinity for 2-AA that was observed for

the octanoylated wild-type enzyme (Figure 9B,

Table 2). Again assuming that the interaction with

2-AA reflects 2-ABA binding, these findings

suggest that His269 acts as modulator which

depending on the absence or presence of an oc-

tanoyl moiety in the active site has restricting or

promoting effects, respectively, on binding of the

second substrate. However, when comparing

different structures of cysteine-loaded and

unloaded FabH-like enzymes, no distinct structural

change of the catalytic histidine is observable,

suggesting that electronic rather than spatial

changes drive the observed affinity shift.

shown to interactively mediate the decarboxyla-

tion of the second reactant (malonyl-ACP in FabH

and PqsD) (19, 48). In E. coli FabH, both residues

are required for decarboxylation activity (48).

Interestingly, exchange of His269 to alanine in

PqsBC nearly abolished catalytic activity (k_cat=

0.0005 s^-1, corresponding to a more than 10,000

fold decrease in activity). This is in striking

contrast to FabH, where replacement of the

catalytic histidine resulted in only a 10-fold

decrease in activity (48). Because the PqsBC^H269A

protein still undergoes the acylation step (see

above), the histidine clearly is essential for a later

step of the PqsBC-catalyzed reaction, which

apparently does not depend on participation of an

Asn residue.

2-ABA, the substrate of PqsBC, in con-

trast to malonyl-CoA/-ACP features an amino

group in close proximity to the β-keto carbonyl

which – as suggested by the increased stability of

2-ABA over its desamino-analog BA in aqueous

solutions – influences the chemical properties of

the β-keto acid moiety. We hypothesized that in

the PqsBC catalytic cycle, the amino group of 2-

ABA might functionally replace the amide group

of the missing active-site asparagine. To test this

hypothesis, we analyzed the conversion of BA by

PqsBC. Using octanoyl-CoA consumption as a

read-out for the reaction, a greatly diminished

reaction velocity of 0.02 s^-1was observed.

Octanoylation of Cys129 of PqsC is independ-

ent of His269

In previous studies of FabH and FabH-like

proteins, a possible involvement of the active-site

histidine in deprotonation of the cysteine to act as

a nucleophile in the initial acylation step has been

discussed (28, 49). In case of PqsBC, the observed

competitive inhibition of the reaction by 2-AA and

the high affinity of 2-AA for the octanoylated pro-

tein suggested that this compound could be used as

fluorescence probe to monitor the acylation step in

a time-resolved setup (Figure 10A). The binding

velocity of 2-AA to PqsBC was faster than

resolvable by the stopped-flow instrument

(approx. 5 ms detection limit), which was a

prerequisite for using 2-AA as probe for

determination of the octanoylation rate. From sin-

gle turnover stopped-flow fluorimetric measure-

As a complementary approach, we reintro-

duced the asparagine into the active site of PqsBC.

The crystal structure reveals Val299 occupies an

identical position to the corresponding asparagine

residue in PqsD (see Figure 3B), and no steric hin-

drance could be predicted for the amide group ex-

tension. PqsBC^V299Nhas a very low activity toward

2-ABA (k_cat= 0.013 s^-1). Because the observed K_D

of the complex with 2-AA was 20-fold higher

compared to the wild type enzyme (Table 2), bind-

ing of the 2-aminophenyl moiety possibly is spa-

tially impaired, leading to a substrate orientation

unfavorable for catalysis. However, BA was

converted with a 14-fold increased k_catof 0.18 s^-1

by PqsBC^V299N. Taken together, the data strongly

support a model in which 2-ABA itself supplies

the asparagine-equivalent amino function that

assists in catalysis of the decarboxylation step.

ments (Figure 10B-D), rate constants of k_WT

=

86.9 ± 2.6 s^-1, k_H269A= 76.6 ± 1.9 s^-1and k_H269A(2-

ABA) = 59.4 ± 5.1 s^-1were deduced for the

octanoylation reaction in wild-type PqsBC, the

PqsBC^H269Avariant, and PqsBC^H269Awhen probed

with 2-ABA instead of 2-AA, respectively. If the

His269 residue were important for activation of

the catalytic Cys129, more drastic differences in

the acylation rates would have been expected.

The amino group of 2-ABA functionally re-

places the canonical catalytic asparagine

While the catalytic cysteine accepts the

acyl moiety, the histidine and asparagine residues

in the active site of FabH-like enzymes have been

10

Structure and function of the condensing enzyme PqsBC

is unexpectedly similar to the homodimer in

FabH-like proteins and it is likely that PqsB has a

scaffolding role in formation of the heterodimer.

The structure of PqsC reveals major struc-

tural alterations in the area of the entry to the

active site. The shortened PqsC helix α5, a

prominent difference to PqsD and FabH, widens

the entry to the catalytic site and provides an

extended cavity that could accommodate a bicyclic

molecule such as the reaction product HHQ.

Substitution of Val299 by asparagine impacts

substrate specificity

Besides condensing anthraniloyl-CoA and

malonyl-CoA or malonyl-ACP, PqsD can also me-

diate the condensation of anthraniloyl-CoA and 3-

ketodecanoic acid to form HHQ (53). If PqsBC

catalyzed the decarboxylative condensation of oc-

tanoyl-CoA and malonyl-CoA, 3-ketodecanoyl-

CoA would be formed, so PqsBC might provide a

substrate for PqsD-mediated HHQ synthesis. We

therefore analyzed PqsBC and PqsBC^V299Nwith re-

spect to their activity toward malonyl-CoA. While

the turnover number of wild-type PqsBC with

malonyl-CoA was only 0.0005 s^-1and the

PqsBC^H269Aprotein was even less active, the

V299N variant showed a 5-fold higher activity.

Thus, substitution of the active-site Asn by Val,

amongst other adaptions, not only sets PqsBC

apart from the canonical FabH-like enzymes, but

also reduces the cross-reactivity with malonyl-

CoA. With respect to physiology, however, the

activity of both PqsBC and PqsBC^V299Nwith mal-

onyl-CoA is far too low to be considered an effi-

cient alternative path for providing a 3-

ketodecanoyl moiety for PqsD-supported synthesis

of HHQ.

Within the catalytic cavity of PqsC, the

distance and the conformation of Cys129 and

His269, the two remaining canonical catalytic

residues of FabH-like enzymes, are conserved,

pointing toward conserved functions in the cata-

lytic mechanism. Compared with malonyl-ACP,

the second substrate of FabH, the equivalent sub-

strate 2-ABA of PqsBC (Fig. 1) has the advantage

of UVA-absorbing and distinct fluorescent proper-

ties that allowed a series of experiments that pro-

vided insight into the reaction mechanism.

Moreover, the spectroscopic properties of 2-AA,

which we identified as a potent competitive in-

hibitor of PqsBC, enabled us to separately address

the initial acylation step. Octanoylation of PqsBC

induced a significant increase in affinity for 2-AA,

which, albeit less pronounced, could also be meas-

ured for 2-ABA binding by the octanoyl-

PqsBC^H269Aprotein. Even though similar effects

have not been demonstrated for other FabH-like

enzymes, such an affinity shift caused by the

loading step could be a common trait of FabH-like

enzymes to prevent premature binding of the sec-

ond substrate. Equilibrium titrations suggested that

His269 is a major factor for the acylation-depend-

ent affinity shift.

DISCUSSION

PqsBC, a key enzyme in the biosynthesis

of the quorum sensing signal molecules HHQ and

PQS and a member of the β-ketoacyl-ACP-syn-

thase III family of condensing enzymes, has

unique features of a heterodimeric subunit

composition and the lack of the canonical active-

site asparagine. The data presented provide the

first crystal structure of PqsBC and by combining

structural data with kinetic and spectroscopic

experiments we moreover were able to

characterize individual reaction steps.

With respect to the role of the catalytic

histidine in the acylation reaction, contradictory

models on its influence on the catalytic cysteine in

FabH (48, 49) and chalcone synthase (54, 55), a

closely related polyketide synthase, have been

discussed. In PqsBC, the velocity of octanoylation

was virtually the same for the wild-type protein

and the PqsBC^H269Avariant, indicating that the

histidine is not required for activation of the acyl-

accepting cysteine. Given the similar confor-

mations and distances between the catalytic histi-

dines and cysteines in PqsC and FabH and the fact

that Cys129 of PqsC, like Cys112 in FabH, is lo-

cated at the amino terminus of an α-helix (Figure

While catalytic involvement of the PqsC

subunit has been demonstrated in a previous study

by identifying the acylation of Cys129 (20), the

role of PqsB was enigmatic. The crystal structure

of PqsBC^C129Ashows that PqsB has no catalytic

residues and large differences occur in the overall

structure compared to PqsC and FabH. The

orientation of PqsC and PqsB subunits forms a

novel dimer with a pseudo 2-fold symmetry which

11

Structure and function of the condensing enzyme PqsBC

5), it is likely that the α-helix dipole effect reduces

the pK_aof Cys to facilitate formation of the

thiolate ion necessary for transacylase activity, as

suggested by Davies et al. (48) for FabH. Also, a

proton relay mechanism as hypothesized by Qiu et

al. (49) seems unlikely for PqsC.

for nucleophilic attack at the acyl-cysteine (Fig.

11). The reaction product of the subsequent

decarboxylative Claisen condensation would be 1-

(2-aminophenyl)decane-1,3-dione,

which

continues to react to form HHQ via intramolecular

enamine formation. Even though the active site

offers enough space for this consecutive step, the

way in which the putative intermediate probably is

bound (Fig. 11) renders an on-site ring closing re-

action unlikely, because activation of the carbonyl

for the subsequent cyclization and elimination step

should rather involve a general acid that provides a

proton to the keto group that is distal to the

aromatic ring. The cyclization would moreover

require a conformational change within the sub-

strate to bring the amino group in proximity to the

distal carbonyl carbon. Given a spatial proximity

of the amine and the 3-carbonyl, ring closure

could also occur non-enzymatically. Taken

together this work significantly increases our

knowledge of the structure, enzyme mechanism

and inhibition of PqsBC and in a broader

dimension, the work has implications for the

development of novel therapeutics which control

P. aeruginosa infection by attenuating virulence

gene expression.

The major question of how PqsBC is func-

tional without the catalytic asparagine was ad-

dressed with the desamino-substrate analog BA,

and by reintroducing the asparagine into the pro-

tein. Whereas for the wild-type protein the activity

toward BA was about 300-fold lower than toward

2-ABA, the PqsBC^V299Nvariant performed signifi-

cantly better with BA than with 2-ABA. As previ-

ously demonstrated for 2-AA (56), the amino

group of 2-ABA is hydrogen bonded to its β-keto

group (Figure 11). This intramolecular hydrogen

bond replaces the hydrogen bond formed by the

active-site Asn of FabH with the thioester

carbonyl of malonyl-ACP. Taken together with the

observed His269-dependent binding effect, which

(as suggested by equilibrium binding experiments)

does not seem to distinguish between 2-ABA and

2-AA, we propose that the amine of the substrate

and the histidine’s imidazole activate the β-keto

oxygen by hydrogen bonding which promotes

decarboxylation and stabilizes the enolate required

Acknowledgments: This work was supported by the Deutsche Forschungsgemeinschaft (grant no. FE

383/23-1 to SF), and by a PhD studentship to FP from the Directorate General of Indonesia Higher

Education. We acknowledge the Diamond light source for provision of synchrotron radiation source in

using the beamline I04.

Conflict of interest: The authors declare that they have no conflicts of interest with the contents of this

article.

Author contributions

SF conceived the study. SF and JE coordinated the study. SLD, SF and JE wrote the paper. SLD opti-

mized the synthesis of 2-ABA, prepared PqsBC and designed, performed and analyzed all spectroscopic,

kinetic and physiological experiments (Figures 7 to 11 and Table 2). FP, SM performed crystallization,

ID, CL performed data collection, JE performed molecular replacement calculations and density

modification, CL performed data processing and structure refinement. UH prepared 2-

aminobenzoylacetate and its precursor, performed all MS and NMR analyses and contributed to the

mechanistic discussion. All authors reviewed the results and approved the final version of the manuscript.

12

Structure and function of the condensing enzyme PqsBC

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Footnotes:

⁵The abbreviations used are:

2-AA – 2-Aminoacetophenone

2-ABA – 2-Aminobenzoylacetate

2-ABA-CoA – 2-Aminobenzoylacetyl-CoA

AQ – 2-Alkyl-4(1H)-quinolone

BA – Benzoylacetate

CoA – Coenzyme A

CV – Column volume

DHQ – 2,4-Dihydroxyquinoline

DTNB – 5,5'-Dithiobis-(2-nitrobenzoic acid)

DTT – Dithiothreitol

HHQ – 2-Heptyl-4(1H)-quinolone

MWCO – Molecular weight cutoff

PQS – Pseudomonas quinolone signal

QS – Quorum sensing

SECV – Standard error of cross-validation

SDS – Sodium dodecylsulfate

TEV – Tobbaco etch virus

FIGURE LEGENDS

Fig. 1. Reactions of FabH (shaded box) and the FabH-like proteins PqsD and PqsBC involved in 2-alkyl-4(1H)-

quinolone biosynthesis. The PqsD- and PqsBC-catalyzed reactions proceed in a similar manner to that of the model

enzyme FabH: In the initial step, the acyl moiety of an activated carboxylic acid is transferred to a strictly conserved

active-site cysteine residue. Subsequently, a β-ketoalkanoic acid (malonyl-ACP in case of FabH, malonyl-CoA or

malonyl-ACP in PqsD, 2-ABA in PqsBC) is decarboxylated and the resulting reactive enolate intermediate (not

shown) attacks the thioester bond of the acyl-enzyme to form the reaction product. Intermediates and products of the

alkylquinolone biosynthetic pathway: Ant-CoA − anthraniloyl-coenzyme A, 2-ABA-CoA − 2-aminobenzoylacetyl-

CoA, 2-ABA – 2-aminobenzoylacetate, HHQ – 2-heptyl-4(1H)-quinolone, PQS – Pseudomonas quinolone signal. .

Fig. 2. Cartoon diagrams of the PqsBC^C129Astructure. A The PqsBC^C129Aheterodimer is shown with PqsC (cyan)

and PqsB (red) covered with a transparent molecular surface illustrating the interface. B The PqsB and PqsC^C129A

structures are colored in a rainbow from N (blue) to the C-terminus (red) with secondary structure elements labeled

for PqsC^C129Ain all panels. C A superposition of PqsB and PqsC^C129A. D N-terminal sub-domains of PqsB (1-154)

and PqsC (1-184) superposed with catalytic dyad residue A129 is shown as sticks (purple). E C-terminal sub-

domains of PqsB (155-279) and PqsC (188-348) arranged side by side viewed with the same orientation showing

PqsC^C129Ahelix α6 is missing in PqsB. PqsC residue His269 is shown as sticks (purple).

16

Structure and function of the condensing enzyme PqsBC

Fig. 3. Stereo figures to illustrate the PqsBC^C129Acatalytic dyad. A A 2.0 Å electron density map is shown (gray)

contoured at 2.0 sigma level calculated using the final refined weighted 2Fo-Fc co-efficients (REFMAC) and

rendered with Pymol showing PqsC residues Ala129, Ala130, His269, Gln270, Val299, Met300 as sticks in green. B

The PqsBC^C129AAla129, His269 catalytic dyad is shown in cyan superposed with the PqsD catalytic triad (gray).

Fig. 4. Comparison of PqsD and PqsBC^C129A. A The PqsC^C129Astructure is shown as a cartoon (cyan) superposed

onto PqsD (gray). Residues from PqsC shown as sticks in purple are Ala129, His269, Val299 and Pro242 (orange).

PqsD residues Ala112, His257, Asn287 are shown as sticks (gray). B Cartoon diagram of PqsBC (red, cyan) with

the flap (PqsC residues 207 to 227) in green shown in the same orientation as the PqsD homodimer below (C). D A

superposition of the PqsBC structure (red, cyan) with the PqsD homodimer (gray) illustrated as Cα- backbone

representations with two views shown. E Same as D, but only strands for PqsB (red) and PqsD (gray) are shown

as cartoon.

Fig. 5. Alignment of the PqsC amino acid sequence with related enzymes showing the assignments of the PqsC^C129A

structure at the top (blue). The catalytic residues are marked by red triangles. The sequences shown are PqsD (P.

aeruginosa) and FabH (E. coli).

Fig. 6. The PqsBC^C129Aactive site pocket. A Two views showing the internal pockets calculated using metapocket

shown as mesh (gray) together with cartoon representations of the PqsBC^C129Astructure. B The PqsBC structure is

shown with the view clipped such that the PqsC flap is removed to show the relationship between the PqsC catalytic

dyad (purple) and the PqsC cavity (yellow mesh). For perspective a decyl formate molecule is shown as sticks (or-

ange) derived from a superposition of the Mtb FabH-decyl formate complex structure (pdb:2QNZ). C A transparent

surface representation of the PqsC active site pocket is show (light cyan) with residue A129 in purple. For perspec-

tive the decyl formate molecules showing the orientation of the proposed entry and exit portals from the FabH-decyl

formate complex structure are shown (orange). D Graphical representation of the PqsBC active site cleft. Residues

conserved with CoA binding sites of FabH/PqsD enzymes are W35 and R168.

Fig. 7. Steady state kinetics and competitive inhibition of PqsBC by 2-AA. A Conversion of 2-ABA (λ_maxat 360

nm) and octanoyl-CoA (absorbing at 260 nm) to HHQ (λ_maxat 313 nm). 20 nM PqsBC was mixed with 50 µM of

each substrate. Spectra were measured every 60 s with a bandwidth of 1 nm. HHQ formation could be observed at

313 nm with a differential extinction coefficient of 6,520 M^-1cm^-1. The remaining absorption above 340 nm after

full conversion of the substrates (bold trace) is due to scattering and can be eliminated by diluting to 50% in 2-

propanol/HCl (dashed trace). Inset: conversion of 10 µM substrates, monitored at 313 nm (HHQ) and 360 nm (2-

ABA) with the determined extinction coefficients. Data interval is 1 min.

B-D Inhibition of PqsBC activity by 2-AA. B Nonlinear fits of measured initial rates. The Lineweaver Burk plot (C)

shows the characteristic pattern of competitive inhibition. Using the Dixon plot (D), the inhibitor constant K_iof the

competitive reaction could be determined graphically. Experiments were performed with 50 µM octanoyl-CoA and

20 nM PqsBC in HEPES buffer, pH 8.2 and 25°C; arrows indicate ascending concentrations.

Fig. 8. In vivo inhibition of HHQ biosynthesis by 2-AA. Cultures of P. putida KT2440 [pBBR::pqsABCD-his] were

grown in the presence of 1 mM anthranilic acid (as precursor for HHQ synthesis) and various concentrations of 2-

AA. 2-AA does not affect growth of P. putida KT2440, and it is not utilized as carbon source. A HHQ contents of

culture samples, as determined by HPLC. Black solid line – DMSO control, black dashed line – 50 µM, black dash-

dotted line – 100 µM, grey solid line – 200 µM, grey dashed line – 500 µM, grey dash-dotted line – 1 mM. Errors

reflect standard errors (SEM) of three biological replicates. B Dose-response plot, using cultures sampled 3.5 h after

inoculation. The arrow indicates the calculated EC₅₀of 319 ± 38 µM. C Immunodetection of PqsD-His₆in Western

blots of cell extract supernatants confirms synthesis of the Pqs proteins. Cell extracts were from cultures

(independent duplicates) harvested after 7 h of incubation. Each lane contained 50 µg of total protein.

Fig. 9. Equilibrium binding of 2-AA to PqsBC proteins in apo- and octanoylated forms. A Fluorescence excitation

and emission spectra of 2-AA (1 µM) in unbound (dashed line) and protein-bound (20 µM PqsBC, continuous line)

form. B Titration data and curve fittings of 5 µM PqsBC (continuous lines) or PqsBC^H269Avariant (dashed lines)

with 2-AA in the presence (circles) or absence (dots) of 10 µM octanoyl-CoA. Data suggests increase of binding

17

Structure and function of the condensing enzyme PqsBC

affinity to 2-AA of the acyl-protein in the wild type but not in the H269A variant (Numerical data provided in Table

3)

Fig. 10. Role of His269 in octanoylation of PqsBC, monitored by transient kinetics. A Time dependent measurement

of 2-AA fluorescence (λ_Ex= 400 nm, λ_Em= 460 nm, 0.5 s time resolution) in complexes with PqsBC proteins.

Octanoylation (addition of 10-fold molar excess of octanoyl-CoA indicated by arrow) shifts the binding equilibrium

of 2-AA and PqsBC (solid black line). The effect was reduced in PqsBC^H269A(dashed black line), and addition of

octanoyl-CoA to PqsBC^C129Adid not change 2-AA fluorescence (gray line). Enzyme and 2-AA concentrations were

varied between 1-20 µM and 1-100 µM respectively. B-D, Octanoylation of PqsBC proteins, analyzed with stopped-

flow fluorimetry: B PqsBC octanoylation probed with 2-AA, C PqsBC^H269A, probed with 2-AA, D PqsBC^H269A

,

probed with 2-ABA. Assays were conducted at 6°C, detector response time was 10 ms, enzyme concentration was

varied to achieve sufficient signal to noise ratio. Rate constants were calculated assuming a first order reaction

characteristic. Denoted errors are standard errors of cross-validation (SECV).

Fig. 11. Proposed catalytic mechanism of PqsBC toward the formation of the 1-(2-aminophenyl)decane-1,3-dione

intermediate, and its reaction to HHQ. For details, see text.

18

Structure and function of the condensing enzyme PqsBC

TABLES

Table 1. Data collection and refinement statistics.

PqsBC^C129A

Data collection

Space group

P2₁2₁2₁

78.5, 115.0, 289.7

90, 90, 90

0.97949

2.0

a, b, c (Å)

α, β, γ (°)

wavelength

Resolution (Å)

Rmerge^a*

0.069

I / σI

12.5 (1.9)

99.2 (96.7)

4.5 (4.6)

0.998 (0.661)

166566

Completeness (%)

Redundancy

CC 1/2^b

Unique reflections

c

R_work

0.190 (0.291)

0.237 (0.321)

38.1

R_free

Overall B factor (A²)

Bond lengths (Å)

Bond angles (°)

Most favored (%)^d

Allowed (%)

0.016

1.81

97.7

2.3

Outliers (%)

0

a

*Values in parenthesis correspond to the highest resolution shell. R_merge= Σh Σi |Ii(h) - <I(h)> / | Σh Σi Ii(h), where I

is the observed intensity and <I> is the average intensity of multiple observations from symmetry-related reflections

b

c

calculated with XDS. Correlation co-efficient value calculated using XDS to determine the resolution cutoff. All

values calculated using REFMAC. R_work= Σh ||Fo|h - |Fc|h| / Σh|Fo|h, where Fo and Fc are the observed and calcu-

lated structure factors, respectively. R_freecomputed as in R_work, but only for (5%) randomly selected reflections,

which were omitted in refinement. ^dRamachandran plot.

19

Structure and function of the condensing enzyme PqsBC

Table 2. Dissociation constants of PqsBC in complex with 2-ABA or 2-AA.

K_Dvalues were deduced from fluorimetric titrations (1-10 µM PqsBC, 0-3 mM 2-AA or 2-ABA). Errors reflect

standard errors of mean (SEM, n ≥ 3). Titrations were carried out in the absence (−) or presence (+Oct-CoA) of a

10-fold excess of octanoyl-CoA over PqsBC. The H269A and C129S values are possibly affected by minor residual

activity (k_cat(H269A) = 0.0005 s^-1, k_cat(C129S): below detection limit of the spectrophotometric assay).

K_D(2-ABA) (µM)

+Oct-CoA

K_D(2-AA) (µM)

+Oct-CoA

Protein

PqsBC

−

a

368±19

198±11

330±30

411±43

nb^b

-

161±8

117±6

158±12

170±10

nb^b

5±1

88±5

141±19

177±8

89±4

PqsBC^H269A

PqsBC^C129A

PqsBC^C129S

PqsBC^V299N

166±12

312±38

420±45

a

-

^aExperiment not possible due to turnover; ^bno binding or K_Dabove detection limit (estimated to > 1 mM)

20

Structure and function of the condensing enzyme PqsBC

FIGURES

Figure 1

21

Structure and function of the condensing enzyme PqsBC

Figure 2

22

Structure and function of the condensing enzyme PqsBC

Figure 3

23

Structure and function of the condensing enzyme PqsBC

Figure 4

24

Structure and function of the condensing enzyme PqsBC

Figure 5

25

Structure and function of the condensing enzyme PqsBC

Figure 6

26

Structure and function of the condensing enzyme PqsBC

Figure 7

Figure 8

27

Structure and function of the condensing enzyme PqsBC

Figure 9

Figure 10

28

Structure and function of the condensing enzyme PqsBC

Figure 11

29

PqsBC, a condensing enzyme in the biosynthesis of the Pseudomonas aeruginosa

quinolone signal: crystal structure, inhibition, and reaction mechanism

Steffen Lorenz Drees, Chan Li, Fajar Prasetya, Muhammad Saleem, Ingrid Dreveny, Paul

Williams, Ulrich Hennecke, Jonas Emsley and Susanne Fetzner

J. Biol. Chem. published online January 25, 2016

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PqsBC, a condensing enzyme in the biosynthesis of the pseudomonas aeruginosa quinolone signal: Crystal structure, inhibition, and reaction mechanism

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