Biochemical Journal

Research article

Structure and activity of the Pseudomonas aeruginosa hotdog-fold thioesterases PA5202 and PA2801

Claudio F. Gonzalez , Anatoli Tchigvintsev , Greg Brown , Robert Flick , Elena Evdokimova , Xiaohui Xu , Jerzy Osipiuk , Marianne E. Cuff , Susan Lynch , Andrzej Joachimiak , Alexei Savchenko , Alexander F. Yakunin


The hotdog fold is one of the basic protein folds widely present in bacteria, archaea and eukaryotes. Many of these proteins exhibit thioesterase activity against fatty acyl-CoAs and play important roles in lipid metabolism, cellular signalling and degradation of xenobiotics. The genome of the opportunistic pathogen Pseudomonas aeruginosa contains over 20 genes encoding predicted hotdog-fold proteins, none of which have been experimentally characterized. We have found that two P. aeruginosa hotdog proteins display high thioesterase activity against 3-hydroxy-3-methylglutaryl-CoA and glutaryl-CoA (PA5202), and octanoyl-CoA (PA2801). Crystal structures of these proteins were solved (at 1.70 and 1.75 Å for PA5202 and PA2801 respectively) and revealed a hotdog fold with a potential catalytic carboxylate residue located on the long α-helix (Asp57 in PA5202 and Glu35 in PA2801). Alanine residue replacement mutagenesis of PA5202 identified four residues (Asn42, Arg43, Asp57 and Thr76) that are critical for its activity and are located in the active site. A P. aeruginosa PA5202 deletion strain showed an increased secretion of the antimicrobial pigment pyocyanine and an increased expression of genes involved in pyocyanin biosynthesis, suggesting a functional link between PA5202 activity and pyocyanin production. Thus the P. aeruginosa hotdog thioesterases PA5202 and PA2801 have similar structures, but exhibit different substrate preferences and functions.

  • crystal structure
  • hotdog fold
  • Pseudomonas aeruginosa
  • pyocyanin
  • thioesterase


Thioesterases (thioester hydrolases, EC 3.1.2) are ubiquitous and diverse enzymes with essential roles in various processes including fatty acid, polyketide and non-ribosomal peptide biosynthesis, acyl-CoA turnover, bioluminescence, signal transduction and removal of acyl chains from post-translationally modified proteins [17]. These enzymes are widespread in eukaryotes, bacteria and archaea (4092 sequences in the InterPro database, accession IPR001031).

Thioesterases are classified into two families (I and II) on the basis of their distinct structural folds, amino acid sequence and different catalytic mechanisms. Classical α/β-hydrolase-fold thioesterases belong to family I [8], whereas the family II thioesterases have a ‘hotdog’ fold, and their active sites usually contain either an aspartic acid or glutamic acid residue that functions as a general base [912]. The hotdog fold is described as an anti-parallel β-sheet (a ‘bun’) wrapped around a five-turn α-helix (a ‘sausage’) [13]. Most of the characterized hotdog-fold proteins possess thioesterase or thiol ester dehydratase activity [14,15]. The hotdog-fold thioesterases hydrolyse acyl-CoA thioesters to free fatty acids and CoASH, as well as the acyl-ACPs [acyl thioesters of ACPs (acyl-carrier proteins)]. A comprehensive sequence analysis of 1357 hotdog-fold proteins clustered them into 85 groups [14]. For 17 subfamilies, a conserved sequence motif was identified and some functional or structural information was found, whereas the remaining groups contained predominantly uncharacterized proteins [14].

The biochemically and structurally characterized hotdog-fold thioesterases includes: 4HBA-CoA (4-hydroxybenzoyl-CoA) thioesterases from Pseudomonas sp. strain CBS-3 [16,17] and Arthrobacter sp. strain SU [12], phenylacetyl-CoA thioesterases PaaI from Escherichia coli [18] and Thermus thermophilus [19], Rv0098 from Mycobacterium tuberculosis [20], TesB (thioesterase II) from E. coli [9,10,21], and hTHEM2 (human thioesterase superfamily member 2) [22]. The characterized hotdog thioesterases have either a hexameric (trimer of dimers) or tetrameric (dimer of dimers) organization, with two dimers connected through the interaction of their β-sheets (a ‘back-to-back’ association, as in the Arthrobacter 4HBA-CoA thioesterase) or their α-helices (a ‘face-to-face’ association, as in the Pseudomonas 4HBA-CoA thioesterase) [2325]. The structures of the complexes of several hotdog thioesterases with their substrates or inhibitors have been determined and revealed the location of the active-site residues and substrate-binding sites [12,17,19,22]. There are two models that have been proposed for the catalytic mechanism of hotdog thioesterases. Structural studies with the Pseudomonas 4HBA-CoA thioesterase revealed that the catalytic Asp17 is located close to the thioester carbonyl carbon and therefore might function as a nucleophile in the reaction, which proceeds via an acyl-enzyme intermediate [17]. However, the structures of the TtPaaI (T. thermophilus PaaI) and hTHEM2 complexed with the substrate analogues indicate that the catalysis probably proceeds via a general base-catalysed hydrolysis with the catalytic carboxylate residue acting as a general base to activate the attacking water [19,22]. For the TtPaaI thioesterase, an asymmetric induced-fit mechanism was proposed where only two of the four active sites of the tetramer bind the substrate (‘half-of-the-sites reactivity’) [19]. However, there is no evidence for the presence of this mechanism in other hotdog thioesterases.

Presently, the vast majority of hotdog proteins remain uncharacterized and their substrate specificities remain unknown. Both prokaryotic and eukaryotic organisms produce numerous acyl-CoA thioesters (over 40 acyl-CoAs are listed on the E. coli database EcoCyc, which are intermediates of various biosynthetic and catabolic pathways and represent potential substrates for thioesterases. The genome of the opportunistic pathogen Pseudomonas aeruginosa encodes at least 23 predicted hotdog-like proteins (Supplementary Table S1 at, all of which remain biochemically uncharacterized. In the present paper, we present the results of the structural and biochemical characterization of two hotdog-fold proteins from this organism, PA5202 and PA2801, which demonstrated significant thioesterase activity in vitro. Their crystal structures were determined, and PA5202 was further characterized using site-directed mutagenesis and gene knock-out approaches revealing the first thioesterase with a preference to HMG-CoA (3-hydroxy-3-methylglutaryl-CoA) and glutaryl-CoA.


Gene cloning, mutagenesis and protein purification

Chromosomal DNA was isolated using a DNeasy miniprep kit (Qiagen), whereas plasmids and PCR products were purified using miniprep and Qiaquick purification kits (Qiagen). The genes encoding PA1835, PA2801, PA5026, PA5185 and PA5202 were PCR amplified and cloned into a modified pET15b vector (Novagen) containing an N-terminal His6 tag followed by a tobacco etch virus protease cleavage site (ENLYFQ:G). Overexpression plasmids were transformed into the E. coli BL21 (DE3) Gold strain (Stratagene). Site-directed mutagenesis (alanine residue replacement) was performed using the QuikChange™ site-directed mutagenesis kit as described previously [26]. The P. aeruginosa mutant strain PA8379 (obtained by transposon insertion) was generously provided by the UWGC (University of Washington Genome Center, Seattle, WA, U.S.A.). The transposon insertion site was verified using the UWGC protocol [27]. PA1835, PA2801, PA5026, PA5185 and PA5202 were overexpressed in E. coli and purified using metal-chelate affinity chromatography on nickel affinity resin (Qiagen) with high yield (>100 mg/l of culture) and homogeneity (>95%) as described previously [26]. The oligomeric state of the purified proteins was determined by gel filtration on a Superdex 200 10/300 column (GE Healthcare) equilibrated with 50 mM potassium Hepes buffer (pH 7.5) and 250 mM NaCl using an ÄKTA FPLC (GE Healthcare). Retention time of the proteins was used to estimate the relative molecular mass of the proteins via linear regression using ribonuclease A (13.7 kDa), ovalbumin (43 kDa) and aldolase (158 kDa) as standards.

Enzymatic assays

Purified hotdog-fold proteins were screened for the presence of thioesterase activity against a set of 27 commercially available acyl-CoA thioesters (Supplementary Table S2 at; Sigma). The screening reactions were performed in 96-well plates at 37°C using a previously described method [28]. Thioesterase activity of PA5202 against HMG-CoA and other substrates was measured spectrophotometrically in triplicates in 96-well plates at 37°C in a reaction mixture (100 μl final volume) containing 50 mM potassium Hepes buffer (pH 8.0), 2 mM EDTA, 0.3 mM DTNB [5,5′-dithiobis-(2-nitrobenzoic acid) or Ellman's reagent], 0.6–1.0 mM substrate and 0.1–0.4 μg of protein [28]. Reactions were continuously monitored by absorbance at 412 nm over 10 min; the amount of thiol groups hydrolysed was determined using DTNB ∊412nm=13.6 M−1·cm−1. For determination of the Km and Vmax values, thioesterase activity was determined over a range of substrate concentrations (between 0.05 and 1 mM for HMG-CoA or glutaryl-CoA and up to 2 mM for other substrates). Kinetic parameters were calculated by non-linear regression analysis of raw data fit to the Michaelis–Menten function using GraphPad Prism software (version 4.00 for Windows).

Growth experiments and pigments production

Growth kinetics and pigment production were evaluated using 250 ml Erlenmeyer flasks containing 25 ml of LB (Luria–Bertani) medium and incubated at 37°C with aeration (250 rev./min). Cell growth was monitored by measuring absorbance at 600 nm (D600). Cultures were inoculated with cells from overnight-grown LB cultures (the starting D600=0.1). Pyocyanin was quantified spectrophotometrically by absorbance at 700 nm (∊=4310 M−1·cm−1) and purified as described previously [29,30]. The ability of cells to grow in a variety of carbon sources was evaluated using Mops-buffered minimal medium [31] supplemented with selected carbon sources (0.2% final concentration). The experimental cultures were inoculated with cells from an overnight LB-grown culture (initial D600=0.05) and grown in 250 ml Erlenmeyer flasks containing 25 ml of culture at 37°C with aeration (220 rev./min).

qRT-PCR (quantitative real time PCR)

The expression of genes encoding selected enzymes related to ketone metabolism was quantified by qRT-PCR using protocols and procedures described previously [26]. Briefly, culture samples were collected from exponentially growing cells (D600~0.6–0.7), mRNA extraction was carried out using a Ribopure® extraction kit (Ambion), and cDNA synthesis was performed with a Superscript first-strand synthesis kit (Invitrogen). The qRT-PCR experiments were carried out on an Applied Biosystems 7300 apparatus (Applied Biosystems) using SYBR Green qPCR SuperMix UDG (Invitrogen). All samples were normalized to recA (recombinase A) expression as a control. The relative expression values were estimated by determination of mRNA abundance compared with the wild-type strain under the same culture conditions described previously [30].

Protein crystallization

Crystals of SeMet (selenomethionine)-substituted PA2801 were grown at 22°C using the hanging drop vapour diffusion method. A 2 μl protein sample (21.6 mg/ml) was mixed with an equal volume of reservoir solution as described previously [32]. Crystals appeared after several days in the presence of 0.2 M calcium chloride (pH 5.1) and 28% PEG [poly(ethylene glycol)]-3350. The crystals were transferred into a reservoir solution containing 16% glycerol as a cryoprotectant and then mounted on the beam. Crystals of SeMet-substituted PA5202 were grown using crystallization solution containing 0.1 M magnesium acetate, 0.1 M Tris/HCl (pH 8.5), 20% PEG-3350 and 2% MPD (2-methyl-2,4-pentanediol). As a cryoprotection agent 25% ethylene glycol was used.

Structure determination

Diffraction data were collected using the SBC-Collect program at the 19-BM and 19-ID beamlines of the Structural Biology Center at the Advanced Photon Source [33]. Data were integrated and scaled using the HKL2000 software package [34]. The structures of PA5202 and PA2801 were determined by MAD (multi-wavelength anomalous diffraction) or SAD (single wavelength anomalous diffraction) phasing respectively, using the HKL2000 suite incorporating the following programs: SHELXC, SHELXD, SHELXE, MLPHARE and SOLVE/RESOLVE [34,35]. The initial protein models were further processed with ARP/wARP software [36], with final models built using the program COOT [37] and refined with the REFMAC5 program of the CCP4 suite [38,39]. Full-length PA5202 was used as one TLS (Translation/Libration/Screw motion) group, whereas for PA2801, five TLS groups were determined by the TLSMD server [40]. The quality of the structures was checked using the validation tools of COOT [37] and Molprobity [41]. All residues with the exception of Asp27 in chain B of PA5202 were within acceptable regions of the Ramachandran plot. A summary of data collection and refinement statistics is shown in Table 1.

View this table:
Table 1 Data collection and refinement statistics for the crystal structures of PA5202 and PA2801

Values in parentheses are for the highest-resolution shell.

PDB accession codes

Co-ordinates and structure factors have been deposited to the PDB with the accession codes 1ZKI (PA5202) and 3QY3 (PA2801).


Thioesterase screen of five purified P. aeruginosa hotdog-fold proteins

As the first step for the biochemical characterization of P. aeruginosa hotdog-fold proteins, we selected the five proteins for which the crystal structures were solved by the Midwest Center for Structural Genomics: PA1835 (PDB code 1YOC), PA2801 (PDB code 2ALI), PA5026 (PDB code 1SH8), PA5185 (PDB code 2AV9) and PA5202 (PDB code 1ZKI). To reveal the biochemical activity and in vitro substrates of these proteins, they were purified and screened for thioesterase activity against a set of 27 various CoA thioesters (Supplementary Table S2). In these screens, PA5202 was the most active enzyme (specific activity over 2 μmol·min−1 per mg of protein) and showed thioesterase activity against glutaryl-CoA, HMG-CoA and succinyl-CoA, as well as towards several other substrates (Figure 1A). PA2801 exhibited high thioesterase activity against octanoyl-CoA and glutaryl-CoA, and significant activity towards lauroyl-CoA, hexanoyl-CoA and hydroxybutyryl-CoA (Figure 1B). PA5185 showed significant thioesterase activity only against propionyl-CoA (Figure 1C), whereas no hydrolytic activity against the available thioester substrates was found in PA1835 and PA5026 (results not shown).

Figure 1 Substrate profiles of PA5202 (A), PA2801 (B) and PA5185 (C) thioesterase activity against 27 different CoA thioesters (only substrates with detectable activity are shown)

Each bar represents an average of the results from at least two independent determinations, with S.D. indicated by the error bars.

PA5202, PA2801 and PA5185 share a low overall amino acid sequence similarity (14–31% sequence identity) and belong to two different hotdog subfamilies (Figure 2). PA5202 is a member of the PaaI subfamily (subfamily-11; predicted thioesterases), whereas PA2801 and PA5185 belong to the YbaW subfamily (subfamily-16, unknown proteins) [14,25]. However, it has been mentioned that the consensus sequence motifs are not well conserved in hotdog-like proteins [25]. Sequences of PA5202, PA2801 and PA5185 show the presence of a potential catalytic carboxylate (Asp57 in PA5202, Glu35 in PA2801 and Asp41 in PA5185) located on the longest α-helix (α4 in PA5202 and α2 in PA2801) (Figure 2). Recent bioinformatic and experimental studies of hotdog-fold proteins disclosed several quaternary associations including dimers, tetramers with helix–helix interactions (‘face-to-face’), tetramers with β-sheet–β-sheet interactions (‘back-to-back’) and three types of hexamers [14,22,25]. Analysis of the oligomeric state of the P. aeruginosa proteins using gel filtration revealed a tetrameric organization of active hotdog-fold proteins (PA2801, PA5185 and PA5202), whereas the proteins without thioesterase activity (PA1835 and PA5026) were found to exist as dimers in solution (Supplementary Table S3 at PA1835 and PA5026 belong to the hotdog subfamily-13 (YiiD-like), which includes the E. coli YiiD protein with the acetyltranferase and hotdog domains fused together, but no enzymatic activity has been reported for its hotdog domain [25]. Thus the hotdog-fold proteins PA2801, PA5185 and PA5202 from P. aeruginosa are thioesterases with different in vitro substrate preferences. Of the three proteins, PA5202 showed the highest specific activity under the screening conditions, whereas PA2801 exhibited the highest level of substrate promiscuity. Although the three proteins showed different preferred in vitro substrates, they shared at least two secondary substrates: hydroxybutyryl-CoA and acetyl-CoA (Figure 1).

Figure 2 Structure-based sequence alignment of PA5202 (A) and PA2801 (B) with other hot-dog thioesterases with solved structures of the PaaI subfamily proteins (A) and the YbaW subfamily proteins (B)

The secondary structure elements of PA5202 and PA2801 are shown above and those of TtPaaI and EC TesC below the alignment. Residues conserved in all aligned proteins are highlighted in grey, and the predicted catalytic carboxylate residues are boxed. PA5202 residues mutated to alanine in the present study are marked with black triangles above the alignment and numbered. The compared proteins are: (A) PA5201 (Q9HTY7), E. coli PaaI (P76084) and T. thermophilus PaaI (TTHA0965; Q5SJP3); and (B) PA2801 (Q9I042), PA5185 (Q9HU04) and E. coli TesC (P77712).

To our knowledge, PA5202 represents the first hotdog-fold thioesterase with a preference for the polar aliphatic acyl-CoAs: glutaryl-CoA and 3HMG-CoA. Glutaryl-CoA has been shown to be an intermediate of the degradation of benzoate, pyridines, tryptophan or lysine [42,43]. HMG-CoA has been shown to be a key intermediate of the mevalonate pathway, which produces precursors of various isoprenoids (sterols, cholesterol, dolichols, triterpenes and ubiquinone) and prenyl groups for the C-prenylation of proteins or various aromatic natural products including phenazines [44]. Presently, it is unclear if the mevalonate pathway is present in P. aeruginosa [45]. HMG-CoA is also an intermediate of the synthesis and degradation of ketone bodies (acetoacetate, β-hydroxybutyrate and acetone) and several amino acids (L-valine, L-isoleucine and L-leucine) [46]. Fifty years ago, the presence of thioesterase activity against HMG-CoA was demonstrated in extracts from E. coli, Neurospora crassa, Tetrahymena pyriformis and mammalian tissues [47]. However, a HMG-CoA-specific thioesterase has never been purified and characterized.

To determine if the hydrolytic activity of PA5202 against HMG-CoA or glutaryl-CoA is physiologically pertinent, its thioesterase activity was further characterized. This enzyme showed maximal activity at alkaline pH (8.5–9.5) and was stimulated by low concentrations of Mg2+, Mn2+ and Ca2+ (0.1 mM; 80%, 60% and 45% stimulation respectively), whereas low concentrations (0.01 mM) of Cu2+ and Zn2+ were inhibitory (20% of residual activity). In addition, the thiol inhibitors N-ethylmaleimide and iodoacetate (1 mM) completely inhibited the activity of PA5202. Under optimal reaction conditions, PA5202 exhibited classical Michaelis–Menten kinetics with hyperbolic saturation curves for all positive substrates (Supplementary Figure S1 at The steady-state kinetic constants measured for the acyl-CoA and aryl-CoA thioesters as substrates are summarized in Table 2. The protein demonstrated high catalytic activity towards glutaryl-CoA (kcat=26.8 s−1) and HMG-CoA (kcat=3.9 s−1), but substrate affinity was higher to HMG-CoA (Km=0.1 mM). Substrate affinity of PA5202 was higher to stearoyl-CoA (Km=5 μM) or palmitoyl-CoA (Km=23 μM), but the catalytic activity was much lower than that with HMG-CoA (Table 2). Therefore PA5202 exhibits a high catalytic efficiency with glutaryl-CoA and HMG-CoA in vitro (kcat/Km=0.3–0.4×105 M−1·s−1). Both thioesters are known to be metabolized by HMG-CoA reductase (EC; Km range 3–60 μM), HMG-CoA hydrolase (EC; Km range 7–10 μM), HMG-CoA lyase (EC; Km range 0.02–3.1 mM) and glutaryl-CoA dehydrogenase (EC; Km range 10–50 μM) (BRENDA database, No information is presently available on the intracellular concentration of acyl-CoA thioesters in Pseudomonas, but in E. coli it is has been shown to vary in the range of 4–600 μM depending on growth conditions [4850]. Thus both HMG-CoA and glutaryl-CoA thioesterase activities of PA5202 are likely to be physiologically significant.

View this table:
Table 2 Kinetic parameters of the wild-type and mutant PA5202 with various substrates

WT, wild-type. Bold indicates the two preferred substrates of PA5202.

Crystal structures of PA5202 and PA2801

PA5202 and PA2801 were crystallized using a hanging-drop method, and their crystal structures were determined at 1.7 Å (1 Å=0.1 nm) and 1.75 Å resolution respectively, using MAD (for PA5202) or SAD (for PA2801) methods (Table 1). The crystal structure of PA5185 was reported previously [51]. Both PA5202 and PA2801 protomers have a classical hotdog-like fold with an anti-parallel β-sheet (a ‘bun’) wrapped around a long α-helix (a ‘sausage’) (Figures 3A and 3B). In the PA5202 structure, three additional, short α-helices (α-1, α-2 and α-3) cover the long α-helix α-4, whereas in PA2801 two short α-helices (α-1 and α-3) are positioned near two opposite ends of the β-sheet, close to the ends of the long α-2 helix (Figures 3A and 3B). The order of the secondary structure elements in the PA5202 protomer (ααββααββββ) is the same as in the Arthrobacter 4HB-CoA thioesterase (PDB code 1Q4U) and human hTHEM2 thioesterase (PDB code 3F5O), but is different from that in PA2801 (βααβββββα).

Figure 3 Overall structure of the P. aeruginosa hot-dog proteins PA5202 and PA2801

The Figure shows PA5202 monomer (A), PA2801 monomer (B), PA5202 dimer (C), PA2801 dimer (D), PA5202 tetramer (E) and PA2801 tetramer (F). In (A) and (B), the helices and strands are labelled and shown in magenta and green respectively. Protein subunits are shown in different colours in (CF). Note that in the PA5202 tetramer (E) two dimers interact by their β-sheets (a ‘back-to-back’ orientation), whereas in the PA2801 tetramer (F) the dimers are connected by their α-helices (a ‘face-to-face’ orientation).

Although PA5202 and PA2801 belong to different hotdog-fold subfamilies, they dimerize in a similar way through the formation of a large common β-sheet with additional interactions between the ends of two long α-helices, as well as between the protein loops (Figures 3C and 3D). The structures also suggest a tetrameric organization of both proteins that was confirmed by gel-filtration studies. In these experiments, PA5202 had a molecular mass of ~54.0 kDa (predicted monomer size=13.8 kDa), whereas PA2801 had a molecular mass of 87.6 kDa (predicted monomer size=17.7 kDa). Presently, two tetrameric hotdog protein clades are known with the ‘back-to-back’ (sheet-to-sheet) or ‘face-to-face’ (helix-to-helix) association of dimers [14,22,25]. As shown in Figures 3(E) and 3(F) the structure of the PA5202 tetramer revealed a ‘back-to-back’ dimer association, whereas PA2801 has a ‘face-to-face’ oligomerization structure. Like PA2801, the structure of PA5185 (also from the YbaW subfamily) showed a hotdog-like tetramer with the ‘face-to-face’ association of two dimers [51].

A Dali search identified several structural homologues of PA5202 in the PDB: TtPaaI [PDB code 1WN3; Z-score=20.7, r.m.s.d. (root mean square deviation)=1.1 Å] and the uncharacterized protein Q7W9W5_BORPA from Bordetella parapertussis (PDB code 3DKZ; Z-score=21.0, r.m.s.d.=1.2 Å). For PA2801, the structurally similar proteins were the E. coli YbgC (PDB code 1S5U; Z-score=18.6, r.m.s.d.=1.7 Å) and PA5185, another biochemically uncharacterized hotdog-like protein from P. aeruginosa (PDB code 2AV9; Z-score=18.1, r.m.s.d.=1.5 Å). The last protein shares 31% sequence identity with PA2801 and also belongs to the YbaW hotdog subfamily (Figure 2).

Site-directed mutagenesis of PA5202 and potential active site

We used site-directed mutagenesis (alanine residue replacement) to assess the residues of PA5202 important for its enzymatic activity. A total of 17 conserved or semi-conserved residues located in the large cavity with the long α-helix at the bottom were mutated to alanine, and the mutant proteins were purified and tested for thioesterase activity against HMG-CoA (Figure 4). The Y14A mutant protein was expressed in an insoluble form, whereas the other proteins were found to be soluble. Enzymatic assays revealed that four proteins (N42A, R43A, D57A and T76A) showed negligible activity, five proteins (R41A, H48A, S54A, Y83A and R104A) exhibited a greatly reduced activity (25% and lower), whereas other proteins retained at least 50% (R86A and R103A) or wild-type level (S12A, F53A, S65A, S105A and K117A) activity (Figure 4). Except for S54A, the active mutant proteins demonstrated a reduced substrate affinity to HMG-CoA with the strongest effect in R103A (Table 2).

Figure 4 Alanine replacement mutagenesis of PA5202 thioesterase activity of the purified mutant proteins against HMG-CoA

Reaction mixtures contained 0.6 mM HMG-CoA and 0.2 μg of purified protein. Results are means ± S.D. from at least two independent determinations.

Our attempts to obtain a crystal structure of PA5202 in complex with HMG-CoA or other substrate have been unsuccessful so far. Therefore we compared the PA5202 apo-structure with the structure of TtPaaI (PDB code 1WN3) complexed with a substrate analogue (hexanoyl-CoA) to assign the potential roles for the PA5202 residues. The ligand-binding pocket and active site of TtPaaI are formed by the residues from three subunits of the protein tetramer [19,22], and its structure can be quite well superimposed with the PA5202 structure (r.m.s.d.=1.11 Å for the Cα atoms of 115 aligned residues) (Supplementary Figure S2 at We propose that these proteins have similar binding sites for the CoA part of their substrates, but use different residues for the co-ordination of the substrate acyl part (hexanoyl in TtPaaI and HMG in PA5202). Since TtPaaI and PA5202 have the same (‘back-to-back’) dimer–dimer association and share significant sequence and structure similarity, it is probable that PA5202 also has a similar active-site organization. For both TtPaaI and hTHEM2, a general base catalytic mechanism has been proposed which involves activation of a catalytic water molecule by a conserved carboxylic residue located on the long α-helix (Asp65 in hTHEM2 and Asp48 in TtPaaI) [19,22]. The homologous carboxylate residue Asp57 is also critical for the activity of PA5202 (Figure 4), suggesting that it probably functions as a general base in this enzyme. In addition, this residue makes a hydrogen bond to the side chain of the semi-conserved Thr76 (2.7 Å), which is also required for PA5202 activity (Figures 4 and 5A). This interaction is expected to maintain the Asp57 side chain in an unprotonated state [5254], important for the co-ordination of a catalytic water molecule. There is a similarly positioned serine residue in the active site of hTHEM2 (Ser83, 3.6 Å from Asp65), which has been proposed to co-operate with Asp65 in the co-ordination of the catalytic water [22]. This might also be true for the PA5202 Thr76, since alanine replacement of this residue has a dramatic effect on PA5202 thioesterase activity (Figure 4). The E. coli thioesterase II (from the TesB-like family) has Thr228 at a similar position, whereas in the crystal structure of the 4HBA-CoA thioesterase from Arthrobacter sp. strain SU (the 4HBT-II subfamily) the catalytic Glu73 is hydrogen bonded to the side chain of Asn96 [10,12]. However, structures of TtPaaI (PDB code 1WN3) and several other hotdog thioesterases including PA2801 (PDB code 3QY3) and E. coli PaaI (PDB code 2FS2) have revealed no presence of a hydroxy or polar side chain near the catalytic carboxylate, suggesting some differences in their catalytic mechanisms.

In the PA5202 active site there are three hydrophilic amino acids located near the catalytic Asp57 (Asn42, His48 and Ser54) whose replacement to alanine produced mutant proteins with a greatly reduced activity, suggesting that these residues are also important for PA5202 activity (Figures 4 and 5A). The homologous mutant proteins N50A and H56A of hTHEM2 and N33A of TtPaaI also showed low thioesterase activity [19,22]. In these two proteins and PA5202, the histidine residue is part of the highly conserved HGG motif (His56-Gly57-Gly58 in hTHEM2) present in many hotdog thioesterases (Figure 2). For hTHEM2 and TtPaaI, it has been proposed that the side chain of Asn50 (Asn33 in TtPaaI) and the backbone amide of Gly57 (Gly40 in TtPaaI) bind and polarize the carbonyl oxygen of the substrate thioester, stabilizing the negatively charged tetrahedral intermediate of the thioesterase reaction and facilitating hydrolysis of the thioester bond (an oxyanion hole) [19,22]. In PA5202, the side chain of Asn42 and the main chain amide of Gly49 probably play the same role and function as an oxyanion hole. The role of the His48 and Ser54 side chains in PA5202 activity is presently unclear.

Similar to TtPaaI, the phosphopantetheine part of the acyl-CoA substrate probably interacts with the side chain of the highly conserved Tyr83 in the PA5202 active site, hence the very low activity of the Y83A protein (Figure 4). The CoA adenosine 3′,5′-diphosphate moiety appears to be co-ordinated by the side chains of Arg85, Arg103, Arg104, Ser105 and Lys117 with the phosphate-binding residues (Arg85, Arg103, Arg104 and Lys117) located on the loops connecting the last three β-strands. Accordingly, the PA5202 R85A, R103A and R104A mutant proteins showed reduced activity and substrate affinity, whereas S105A and K117A exhibited wild-type level activity and reduced substrate affinity (Figure 4 and Table 2). Similar results were obtained with hexanoyl-CoA as substrate except for the wild-type level activity in the R43A protein (Supplementary Figure S3 at, which was almost inactive against HMG-CoA (Figure 4) suggesting that this residue might be involved in the co-ordination of the HMG carboxyl.

The structure of PA2801 also revealed the presence of a potential catalytic carboxylate (Glu35) located on the long α-helix in the active site, and the side chain of Tyr65 seems to be appropriately positioned to interact with the phosphopantetheine part of the substrate (Figure 5B). In addition, there are three positively charged residues (Arg83, Lys111 and Arg121) which can co-ordinate the substrate adenosine 3′,5′-diphosphate moiety (Figure 5B). However, in contrast with PA5202, the PA2801 active site contains two aromatic side chains (Trp16 and Tyr22) and the positively charged Arg38 near the catalytic Glu35, which might determine a different substrate preference of PA2801 (Figure 5B). Overall, the PA2801 tetramer organization (‘face-to-face’ dimer–dimer association) and active-site residues are similar to that of the Pseudomonas 4HBA-CoA thioesterase (PDB code 1BVQ), whose active site is formed by the residues from two protomers [16,17,19]. This also appears to be true for PA2801. Thus PA5202 and PA2801 have the same structural fold, but evolved a different substrate preference, and future structural studies will reveal the molecular details of the thioester-bond hydrolysis in their active sites.

Figure 5 Close-up stereo view of the active sites of PA5202 (A) and PA2801 (B)

The side chains of the active site residues are shown as sticks along with the protein ribbon. The protein monomers are coloured in different colours: cyan, purple, green and blue. Note that the residues from three monomers (cyan, purple and green) contribute to one active site in PA5202 (A), whereas the PA2801 active site is formed by the residues from two monomers (B). In (A), R103* and R104* denote two arginine residues which are part of another active site, but are located close to the first active site shown on the picture, suggesting that the second active site might be affected by the substrate binding to the first active site.

Growth and gene expression experiments with the PA5202 deletion strain

To provide insight into the physiological function of the PA5202 thioesterase in P. aeruginosa, we performed growth experiments with the P. aeruginosa PA5202 deletion strain using 26 different carbon sources (Supplementary Table S4 at This strain showed wild-type growth rates on rich (LB) medium with a doubling time of 37–40 min. However, the PA5202 deletion strain showed a delayed growth (a 1–2 h lag phase) on minimal medium with TCA (trichloroacetic acid) cycle intermediates as carbon sources (results not shown). The growth delay was even more prominent (3–6 h) when glutarate, propionate or acetate was used as a carbon source (Figure 6A). One of the possible reasons for the observed growth delay of the PA5202 deletion strain might be the toxic effect of the temporal accumulation of the physiological substrate of PA5202 (HMG-CoA or glutaryl-CoA). This is supported by a recent observation that intracellular accumulation of HMG-CoA inhibits the growth of metabolically engineered E. coli cells expressing the heterologous HMG-CoA synthase [50]. In E. coli, this toxic effect was eliminated by co-expression of the HMG-CoA reductase. It has been suggested that high intracellular levels of HMG-CoA might inhibit enzymes in the early steps of the E. coli type II fatty acid biosynthetic pathway [50].

Figure 6 Growth and pyocyanin production by the wild-type and PA5202 deletion strains of P. aeruginosa

(A) Growth on minimal medium with the addition of different carbon sources (0.2%): propionate (●,○), acetate (▲,Δ) or glutarate (■,□). Experiments were carried out in triplicates using the inoculum grown on LB medium at 37°C (16–18 h). The flasks were inoculated (D600=0.05) with the P. aeruginosa wild-type (closed symbols) or ΔPA5202 (open symbols) strains, and cellular growth was monitored by measuring the culture absorbance (OD600). (B) Growth (■,●) and production of pyocyanin (□,○) by the wild-type (■,□) and PA5202 deletion (●,○) strains on rich medium (LB) at 37°C. Pyocyanin concentration was estimated by absorbance measurements of culture supernatants at 700 nm. Results are means ± S.D. from at least two independent determinations.

In addition, the PA5202 deletion strain showed a greatly increased production of the extracellular pigment pyocyanin in the stationary growth phase on rich medium (LB) (Figure 6B and Supplementary Figure S3). Pyocyanin (1-hydroxy-5-methyl-phenazine) is a blue antibiotic pigment and a known quorum sensing-dependent virulence factor produced by P. aeruginosa, which kills mammalian and bacterial cells through the generation of reactive oxygen species [5557]. Although pyocyanin represents one of the P. aeruginosa virulence factors and has broad biological importance, the biochemistry of the biosynthesis of the tricyclic phenazine ring system is not well understood [29,58,59]. After 5 h of growth, the culture of the PA5202 deletion strain developed a characteristic greenish colour, and spectrophotometric analysis of culture supernatants demonstrated a greatly increased (6-fold) production of pyocyanin (Figure 6B). The observed increase in pyocyanin production by the PA5202 deletion strain can be attributed to the increase in the intracellular level of the PA5202 in vivo substrate (HMG-CoA or glutaryl-CoA), which might have a direct or indirect effect on the pyocyanin synthesis. It is known that, in Pseudomonas, HMG-CoA and glutaryl-CoA are intermediates of the degradation pathways of several amino acids (tryptophan, lysine and leucine), which eventually produce acetyl-CoA [46,60]. These reactions are not directly connected to the known reactions of pyocyanin biosynthesis (which do not involve any acyl-CoA intermediate), suggesting that the increase in the pyocyanin production is not likely due to the direct effect of the PA5202 gene deletion, but probably mediated by presently unknown metabolic reactions. Recently, a redox homoeostasis maintenance mechanism based on the pyocyanin production has been proposed for P. aeruginosa stationary-phase cultures suggesting that pyocyanin may influence the intracellular redox state by decreasing carbon flux through central metabolic pathways [29]. In addition, although hotdog thioesterases are mainly known to use acyl-CoAs as substrates, several enzymes can also hydrolyse acyl-ACPs [14,15,25,61]. Therefore we cannot rule out that the increase in the pyocyanin production in the PA5202 deletion strain reflects a balancing of the P. aeruginosa intracellular redox state or acyl-ACP pool.

We also determined the effect of the PA5292 gene deletion on the expression of three groups of P. aeruginosa genes involved in ketone-body metabolism (PA0266, PA0447, PA1999, PA2003, PA2011 and PA5015), pyocyanin biosynthesis (PA4209, PA4210, PA4211 and PA4214) and pyocyanin-induced shock response (PA2274, PA4205 and PA4206) (Table 3). After 3 h of growth, the total RNA was extracted from P. aeruginosa cells (1 h before the visible production of pyocyanin) and the selected mRNAs were quantified using real-time PCR as described in the Experimental section. As shown in Table 3, the deletion of PA5202 had a small effect on the expression of the group-I genes, suggesting that the enzymes of ketone-body metabolism have a limited role in the utilization of the PA5202 substrates (HMG-CoA or glutaryl-CoA). In contrast, expression of the genes involved in pyocyanin biosynthesis or pyocyanin-induced stress response was greatly increased in the PA5202 deletion strain compared with the wild-type cells (Table 3). In P. aeruginosa, the synthesis of pyocyanin is known to be induced by the quorum-sensing quinolone signalling molecule PQS (2-heptyl-3-hydroxy-4-quinolone), whose precursors are derived from chorismate or through the degradation of tryptophan and include anthranilate-CoA, β-ketodecanoyl-CoA and β-ketodecanoyl-ACP, as well as glutaryl-CoA as intermediates [6264]. Potentially, the in vivo activity of PA5202 might reduce the intracellular level of PQS, thereby suppressing the induction of the pyocyanin synthesis.

View this table:
Table 3 Relative expression of selected genes involved in ketone body metabolism determined by qRT-PCR in the ΔPA5202 mutant background

KBA, ketobutyric acid.

Thus biochemical and structural characterization of two hotdog proteins from P. aeruginosa identified two new thioesterases with similar structures, but different substrate preferences and active-site residues. PA2801 is most active towards octanoyl-CoA, whereas PA5202 is the first thioesterase with a preference for HMG-CoA and glutaryl-CoA. Growth experiments with the PA5202 deletion strain revealed an increased production of pyocyanin and increased expression of genes involved in pyocyanin biosynthesis, suggesting a functional link between the PA5202 activity and pyocyanin synthesis in vivo. Future biochemical and structural studies of the P. aeruginosa hotdog proteins will identify novel thioesterases and provide further insight into their activity and function.


Claudio Gonzalez, Anatoli Tchigvintsev, Greg Brown and Robert Flick carried out the biochemical studies; Elena Evdokimova and Xiaohui Xu performed the crystallization experiments; Susan Lynch provided the P. aeruginosa PA5202 deletion strain; Jerzy Osipiuk, Marianne Cuff, Andrzej Joachimiak and Alexei Savchenko solved and refined the crystal structures; and Claudio Gonzalez and Alexander Yakunin designed the research, analysed the data and wrote the paper.


This work was supported by the Government of Canada through Genome Canada and the Ontario Genomics Institute [grant number 2009-OGI-ABC-1405], the Protein Structure Initiative of the National Institutes of Health (Midwest Center for Structural Genomics) [grant number GM074942], and the Institute of Food and Agricultural Sciences, University of Florida. Use of the Advanced Photon Source was supported by the U.S. Department of Energy, Basic Energy Sciences, Office of Science, and the use of Structural Biology Center beamlines was supported by the Office of Biological and Environmental Research, under contract DE-AC02-06CH11357.


We thank all members of the SPiT (Centre for Structural Proteomics, Toronto, Canada) for help in conducting experiments.


  • The co-ordinates and structure factors of PA5202 and PA2801 have been deposited in the PDB under codes 1ZKI and 3QY3 respectively.

Abbreviations: ACP, acyl-carrier protein; acyl-ACP, acyl thioester of ACP; DTNB, 5,5′-dithiobis-(2-nitrobenzoic acid); 4HBA-CoA, 4-hydroxybenzoyl-CoA; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; hTHEM2, human thioesterase superfamily member 2; LB, Luria–Bertani; MAD, multi-wavelength anomalous diffraction; PEG, poly(ethylene glycol); PQS, 2-heptyl-3-hydroxy-4-quinolone; qRT-PCR, quantitative real time PCR; r.m.s.d., root mean square deviation; TesB, thioesterase II; TLS, Translation/Libration/Screw motion; TtPaaI, Thermus thermophilus PaaI; SAD, single wavelength anomalous diffraction; SeMet, selenomethionine; UWGC, University of Washington Genome Center


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