An α/β-Hydrolase Fold Subfamily Comprising Pseudomonas Quinolone Signal-Cleaving Dioxygenases
ABSTRACT
The quinolone ring is a common core structure of natural products exhibiting antimicrobial, cytotoxic, and signaling activities. A prominent example is the Pseudomonas quinolone signal (PQS), a quorum-sensing signal molecule involved in the regulation of virulence of Pseudomonas aeruginosa. The key reaction to quinolone inactivation and biodegradation is the cleavage of the 3-hydroxy-4(1H)-quinolone ring, catalyzed by dioxygenases (HQDs), which are members of the α/β-hydrolase fold superfamily. The α/β-hydrolase fold core domain consists of a β-sheet surrounded by α-helices, with an active site usually containing a catalytic triad comprising a nucleophilic residue, an acidic residue, and a histidine. The nucleophile is located at the tip of a sharp turn, called the “nucleophilic elbow.” In this work, we developed a search workflow for the identification of HQD proteins from databases. Search and validation criteria include an [H-x(2)-W] motif at the nucleophilic elbow, an [HFP-x(4)-P] motif comprising the catalytic histidine, the presence of a helical cap domain, the positioning of the triad’s acidic residue at the end of β-strand 6, and a set of conserved hydrophobic residues contributing to the substrate cavity. The 161 candidate proteins identified from the UniProtKB database originate from environmental and plant-associated microorganisms from all domains of life. Verification and characterization of HQD activity of 9 new candidate proteins confirmed the reliability of the search strategy and suggested residues correlating with distinct substrate preferences. Among the new HQDs, PQS dioxygenases from Nocardia farcinica, N. cyriacigeorgica, and Streptomyces bingchenggensis likely are part of a catabolic pathway for alkylquinolone utilization.
IMPORTANCE Functional annotation of protein sequences is a major requirement for the investigation of metabolic pathways and the identification of sought-after biocatalysts. To identify heterocyclic ring-cleaving dioxygenases within the huge superfamily of α/β-hydrolase fold proteins, we defined search and validation criteria for the primarily motif-based identification of 3-hydroxy-4(1H)-quinolone 2,4-dioxygenases (HQD). HQDs are key enzymes for the inactivation of metabolites, which can have signaling, antimicrobial, or cytotoxic functions. The HQD candidates detected in this study occur particularly in environmental and plant-associated microorganisms. Because HQDs active toward the Pseudomonas quinolone signal (PQS) likely contribute to interactions within microbial communities and modulate the virulence of Pseudomonas aeruginosa, we analyzed the catalytic properties of a PQS-cleaving subset of HQDs and specified characteristics to identify PQS-cleaving dioxygenases within the HQD family.
INTRODUCTION
The 4-quinolone ring is a common core structure in synthetic antibacterials as well as in bioactive natural products (1–3). Among the plethora of quinolone alkaloids isolated from plants of the Rutaceae family, a number of (2-alkyl-)4(1H)-quinolone derivatives have been described, including 3-methoxylated derivatives and N-methyl-4(1H)-quinolones bearing saturated or unsaturated hydrocarbon chains at C-2 (2, 3). Quinolone derivatives with antibacterial activities are also produced by Gram-positive (4, 5) and Gram-negative bacteria, especially Pseudomonas, Burkholderia, and Alteromonas spp. (6–14). Organisms described to produce quinolone-type secondary metabolites are distributed globally. The Rutaceae family includes shrubs, trees, and herbaceous perennials that are widespread in temperate and tropical regions. Pseudomonas, Burkholderia, and Alteromonas spp. are considered ubiquitous in the environment, inhabiting soil, freshwater, and marine ecosystems or living in association with plants (15).
Among the more than fifty 2-alkyl-4(1H)-quinolones produced by the opportunistic pathogen Pseudomonas aeruginosa (16), 2-heptyl-3-hydroxy-4(1H)-quinolone, termed the “Pseudomonas quinolone signal” (PQS), and its biosynthetic precursor, 2-heptyl-4(1H)-quinolone (HHQ), have received much attention due to their role as quorum-sensing signaling molecules. PQS and HHQ address the alkylquinolone-based pqs system, which, together with the N-acylhomoserine lactone-based rhl and las systems, form a complex quorum-sensing network in P. aeruginosa, regulating a variety of virulence factors (17, 18). HHQ moreover exhibits bacteriostatic activity against several Gram-negative bacteria. PQS represses the motility of Gram-positive bacteria (19), exhibits iron-trapping, prooxidant, and host immunomodulatory properties (20), and affects mitochondrial respiration (21). Considering the biological activities of quinolones, it is hardly surprising that microorganisms have evolved enzymes or pathways for the detoxification or even biodegradation of these compounds. The marine Gammaproteobacterium Microbulbifer sp. strain HZ11, as well as the Gram-positive opportunistic pathogen Staphylococcus aureus, were found to be capable of HHQ hydroxylation at C-3 (22, 23). An analogous hydroxylation represents the first step of HHQ degradation by Rhodococcus erythropolis BG34 and Mycobacteroides abscessus subsp. abscessus. The PQS formed in this reaction then is cleaved to carbon monoxide and N-octanoylanthranilic acid (Fig. 1). The hydrolysis of the latter yields anthranilic acid and octanoate, which can be funneled into central metabolic pathways.
FIG 1
The cleavage of the heterocyclic ring is the key reaction to inactivation and biodegradation of quinolones. It is mediated by dioxygenases that accept 3-hydroxylated 4(1H)-quinolones as substrates. The first 3-hydroxy-4(1H)-quinolone 2,4-dioxygenases (HQD) were isolated from the 4(1H)-quinolone-degrading bacterium Pseudomonas putida strain 33/1 and the 2-methylquinolone degrader Arthrobacter sp. strain Rue61a and were identified as cofactorless dioxygenases (Qdo and Hod) active toward 3-hydroxy-4(1H)-quinolone (HQ) (Fig. 1) and 2-methyl-3-hydroxy-4(1H)-quinolone, respectively (24). Both enzymes were later identified to belong to the α/β-hydrolase fold superfamily (25, 26). Recently, we described the PQS dioxygenases AqdC1 and AqdC2 from R. erythropolis and AqdC from M. abscessus subsp. abscessus, which catalyze the cleavage of PQS to carbon monoxide and N-octanoylanthranilic acid (NOAA), as further members of the α/β-hydrolase fold superfamily (Fig. 1) (27, 28).
The α/β-hydrolase fold core is defined to be composed of eight β-strands, all parallel except the second one, surrounded by α-helices. Another feature is the canonical nucleophile/histidine/acidic residue catalytic triad, with the nucleophile positioned in the so-called nucleophilic elbow motif. This motif, Sm-X-Nu-X-Sm-Sm (where Sm indicates a small amino acid, X stands for any amino acid, and Nu refers to the nucleophile), forms a sharp turn with the nucleophilic residue on the tip (29). The α/β-hydrolase fold superfamily is defined by structural homology rather than sequence similarity and represents one of the largest families of structurally related proteins. Catalytic members include mainly hydrolases but also enzymes that use HCN, H2O2, or O2 instead of H2O as the cosubstrate. ESTHER, a comprehensive database of α/β-hydrolase fold proteins, comprises more than 60,000 proteins, which are clustered into 200 families (as of 29 January 2020) (30). Based on sequence and structure data, family-specific descriptors can be inferred (30, 31); however, functional annotation still is a challenge.
In this study, we used bioinformatics tools to define motifs that are characteristic among enzymes of the HQD group within the α/β-hydrolase fold superfamily. We defined search criteria for the identification of HQD candidate proteins and analyzed the natural diversity of this group of ring-cleaving dioxygenases. Our data suggest that HQDs are distributed among all domains of life and especially occur in environmental and plant-associated microorganisms. Since the cleavage of PQS is particularly interesting for attenuating quorum sensing and virulence of P. aeruginosa, we analyzed the sequence patterns and kinetic properties of HQDs that are active toward this substrate in more detail.
RESULTS
Construction of a hidden Markov model (HMM) for HQDs.
Only a few ring-cleaving (2-alkyl-)3-hydroxy-4(1H)-quinolone 2,4-dioxygenases are currently known (Table 1, entries 1 to 5). To identify further members of this group of dioxygenases, which we hypothesized to form a distinct subfamily within the α/β-hydrolase fold superfamily, an amino acid sequence alignment was constructed to deduce common features and motifs (see Fig. S1 in the supplemental material).
TABLE 1
| Source organism | Name | No. | ENA ID | PDB ID | Expression plasmid | Reference(s) or source |
|---|---|---|---|---|---|---|
| Pseudomonas putida 33/1 | Qdo | 01 | CAA75082.2 | 3IBT | pRSC774-qdo | 26, 62 |
| Arthrobacter sp. strain Rue61a | HodC | 02 | CAA75080.1 | 2WJ3 | pET23a::hodC | 26, 48 |
| Mycobacteroides abscessus ATCC 19977 | AqdC | 03 | CAM60402.1 | 6RA2 | pET28b::his8-TEV-aqdCI | 27, 33 |
| Rhodococcus erythropolis BG43 | AqdC1 | 04 | AKE01130.1 | pET22b::aqdC1 | 28 | |
| Rhodococcus erythropolis BG43 | AqdC2 | 05 | AKE01142.2 | pET28b::aqdC2 | 28 | |
| Nocardia cyriacigeorgica GUH-2 | HQDN.c. | 06 | CCF65795.1 | pET28b::his8-TEV-HQD_N.c. | This study | |
| Nocardia farcinica IFM 10152 | HQDN.f. | 07 | BAD60071.1 | pET28b::his8-TEV-HQD_N.f. | This study | |
| Streptomyces bingchenggensis BCW-1 | HQDS.b. | 08 | ADI11806.1 | pET28b::his8-TEV-HQD_S.b. | This study | |
| Mycobacteroides abscessus subsp. bolletii 50594 | HQDM.a.b. | 09 | AGM31311.1 | pET28b::his6-TCS-HQD_M.a.b. | This study | |
| Serratia liquefaciens HUMV-21 | HQDS.l. | 10 | AKE12954.1 | pET28b::his8-TEV-HQD_S.l. | This study | |
| Ochrobactrum anthropi W13P3 | HQDO.a. | 11 | EXL03318.1 | pET28b::his6-TCS-HQD_O.a. | This study | |
| Caldiarchaeum subterraneum | HQDC.s. | 12 | BAJ50818.1 | pET28b::his6-TCS-HQD_C.s. | This study | |
| Coccidioides immitis RS | HQDC.i. | 13 | EAS31583.1 | pET28b::his8-TEV-HQD_C.i. | This study | |
| Fusarium oxysporum f. sp. raphani 54005 | HQDF.o. | 14 | EXK80068.1 | pET28b::his8-TEV-HQD_F.o. | This study |
a
ENA IDs (European Nucleotide Archive [63]) of the genes are given. PDB IDs of structures and references describing the particular dioxygenase are indicated if available. Expression plasmid names, unless previously published differently, include information on the length of encoded N-terminal polyhistidine tags (6 or 8 residues) and protease cleavage sites (TEV, tobacco etch virus; TCS, thrombin cleavage site).
Predicting conservation and functionally important residues by building HMM profiles and by using the JSD method resulted in accordant motifs (Fig. S1 and S2). The identification of these motifs as functionally or structurally important is supported by the inclusion of the regions comprising the catalytic triad positions (motifs 3, 4, and 9) (Fig. 2). The nucleophilic elbow motif is defined as Sm-X-Nu-X-Sm-Sm (see the introduction) for the superfamily (32). The equivalent HQD motif 3 comprises a conserved His at position Nu + 1, which in AqdC contributes to the positioning of PQS in the substrate-binding cavity (33). The motif is extended by a conserved Trp at position Nu + 4, which in AqdC is hydrogen bonded to Nδ1 of the His residue at Nu + 1.
FIG 2
Identification and phylogenetic analysis of novel HQDs using defined motifs.
An HMMsearch of the UniProtKB database using the HMM profile, created from the sequence alignment shown in Fig. S1, resulted in 118,475 sequences. Setting the limit to a bit score of >180 restricted the number to 288. The refinement of this subset by the selection of sequences, including the simplified amino acid motifs 3 and 9 (Fig. 2), H-x(2)-W and HFP-x(4)-P, at the correct positions led to a data set of 191 amino acid sequences of putative HQDs. The exclusion of redundant UniProt entries reduced the final data set to 161 sequences. Among this data set, sequence identities ranged from 27.11% (A0A2N5AYV3 [Rhizobium loti] versus A0A1S1QMJ8 [Frankia sp. strain EUN1h]) to 99.63% (e.g., A0A3L6N2W9 [Fusarium oxysporum f. sp. cepae] versus A0A420NU70 [Fusarium oxysporum]) (data not shown).
The organisms harboring the respective genes are distributed among all domains of life (Fig. 3). With 113 sequences, the most represented domain is Bacteria, with 62 sequences originating from Gram-positive and 51 from Gram-negative bacteria. The Gram-positive members comprise Actinobacteria (Actinomadura sp., Arthrobacter sp., Rhodococcus spp., Mycobacteroides spp., Nocardia spp., Mycolicibacterium spp., and Streptomyces bingchenggensis) and some bacilli (Brevibacillus spp.); the Gram-negative members belong to alphaproteobacteria of the Rhizobiales order (Ochrobactrum spp., Rhizobium spp., Mesorhizobium spp., Nitratireductor spp., Aminobacter spp., and Beijerinckia sp.) and to the gammaproteobacteria (e.g., Pseudomonas sp., Myxococcus sp., Serratia sp., and Archangium sp.). All these bacterial genera are known to be widely distributed in terrestrial and aquatic ecosystems (15, 34–36). The eukaryotes and the Archaea are represented by 41 (exclusively fungal; mainly Fusarium spp. and Coccidioides spp.) and 7 sequences, respectively. Most of the archaeal organisms originate from a microbial mat community in a geothermal groundwater stream (37, 38). Interestingly, many of the taxa harboring HQD candidate proteins have the potential to associate with plants. Twenty-four different sequences for the motif, referred to as the “nucleophilic elbow” and featuring a variety of amino acids at positions 1, 2, 5, and 6, were found (Fig. 3 and Fig. S2).
FIG 3
HQD activity of selected members of the subfamily.
For verification of HQD activity, we selected 9 potential members of the proposed HQD subfamily (numbers 6 to 14 in Table 1), originating from organisms belonging to different phylogenetic groups and representing different nucleophilic elbow sequences (Fig. 3), for expression in Escherichia coli. For the species S. liquefaciens, the sequence derived from searching the UniProt database was from strain FDAARGOS_125 (UniProt accession no. A0A0X8SJN7), isolated from petroleum sludge (39). S. liquefaciens strain HUMV-21 contains a protein that, except for being shorter by two amino acids at its N terminus, is identical (no UniProt entry) (Table 1, motif 10). We used the latter for functional analysis, because strain HUMV-21 is a clinical isolate from human skin ulcer with the ability to produce AHL-type quorum-sensing signals and to form biofilms (40, 41), which may interact not only with the human host but also with other cooccurring pathogens. Comparison of the sequences of all 14 HQD (candidate) proteins revealed identities between 29.66% (Caldiarchaeum subterraneum HQD [HQDC.s.] versus Ochrobactrum anthropi HQD [HQDO.a.]) and 71.59% (Nocardia farcinica HQD [HQDN.f.] versus Nocardia cyriacigeorgica HQD [HQDN.c.]) (Table S1).
Biotransformation assays with cultures of HQD-producing E. coli strains verified that all 14 enzymes tested are capable of catalyzing the cleavage of the simplest substrate HQ to form N-formylanthranilic acid and, therefore, are true members of the HQD family. PQS, the preferred substrate of AqdC (33, 42), was not cleaved by HQDC.s., S. liquefaciens HQD (HQDS.l.), and HQDO.s. (Fig. 4). None of the tested dioxygenases was capable of cleaving quercetin, a structurally related flavonol compound known to also undergo 2,4-dioxygenolytic ring cleavage, which, however, was reported to be catalyzed by dioxygenases of the cupin superfamily (43, 44). The levels of solubly expressed HQDs differed considerably, as suggested by SDS-PAGE analysis of cell extract supernatants of HQD expression strains (Fig. 4 and Fig. S3). However, the biotransformation data provide an estimation for the specificity of the selected enzymes for HQ and the 2-heptyl congener PQS. Qdo, Hod, M. abscessus subsp. bolletii 50594 HQD (HQDM.a.b.), HQDS.l., HQDO.a., and HQDC.s. showed a clear preference for the HQ substrate, while the remaining HQDs (AqdC, AqdC1, AqdC2, HQDN.c., HQDN.f., S. bingchenggensis HQD [HQDS.b.], Coccidioides immitis HQD [HQDC.i.], and Fusarium oxysporum HQD [HQDF.o.]) preferred PQS, which was converted to N-octanoylanthranilic acid.
FIG 4
Purification of HQD enzymes.
To further characterize the catalytic properties of HQDs active on PQS, these enzymes were purified from cell extract supernatants of the respective recombinant E. coli strains by immobilized metal affinity chromatography. Except for the HQDs of fungal origin, the purity of the recombinant enzymes was higher than 98% (Table 2 and Fig. S4). The yields of protein varied from 0.06 mg (HQDF.o.) to 5.3 mg (HQDN.c.) (AqdC, 0.8 mg; AqdC1, 2.2 mg; HQDN.f., 2.0 mg; HQDS.b., 1.4 mg; HQDC.i., 0.07 mg) per g of culture biomass.
TABLE 2
| No. | Name | Purity (%) | Sp act (U/mg) | kcat (s−1) | Km (μM) | kcat/Km |
|---|---|---|---|---|---|---|
| 03 | AqdC | 100 | 60.2 ± 2.2 | 41.9 ± 1.1b | 5.8 ± 0.4b | 7.3 ± 0.6b |
| 04 | AqdC1 | 98.4 | 20.7 ± 1.2 | 16.0 ± 0.8 | 7.0 ± 0.2 | 2.3 ± 0.2 |
| 06 | HQDN.c. | 100 | 33.6 ± 2.3 | 22.1 ± 0.7 | 4.9 ± 0.4 | 4.6 ± 0.6 |
| 07 | HQDN.f. | 100 | 73.2 ± 2.8 | 43.9 ± 1.5 | 3.1 ± 0.4 | 14.1 ± 1.8 |
| 08 | HQDS.b. | 99.2 | 34.1 ± 2.3 | 27.4 ± 2.3 | 2.7 ± 0.9 | 9.3 ± 1.1 |
| 13 | HQDC.i. | 82.4 | 20.0 ± 3.2 | 15.2 ± 0.9 | 7.3 ± 1.1 | 2.1 ± 0.4 |
| 14 | HQDF.o. | 79.5 | 8.5 ± 1.8 | 7.6 ± 0.4 | 5.0 ± 0.7 | 1.6 ± 0.3 |
a
Results are represented as means ± standard deviations (from ≥2 biological and 3 technical replicates each). The purity of the protein preparations was determined by densitometric analysis of SDS gels (Fig. S4).
b
Data from reference 33.
Catalytic efficiency of HQDs toward PQS.
Because PQS as a potential target for antivirulence therapies (42, 45) is the most interesting substrate, we aimed at characterizing the catalytic activity of newly identified HQDs, which appear to prefer PQS over HQ, in more detail (Table 2). The highest turnover rates for PQS were exhibited by HQDs of N. farcinica and the M. abscessus protein AqdC. The lowest Km values were observed for the HQDs of S. bingchenggensis and N. farcinica; the latter enzyme showed the highest catalytic efficiency toward PQS.
Characteristics of the HQD subfamily.
By sequence similarity or prediction by PROMALS3D, the same overall fold as that described for AqdC, HodC, and Qdo can be assumed for the enzymes verified as HQDs (Fig. 5) and also for the final data set of 161 sequences (data not shown). This fold is composed of an α/β-hydrolase fold core domain extended by a cap domain formed by four helices (αCap1 to αCap4) (26, 33). In JSD results of the set of 14 HQDs (using the same threshold of >0.72 as that for the initial calculations [5 HQDs]), most of the residues and motifs revealed by the initial search still were identified as highly conserved (Fig. 5). Most of the hydrophobic residues that, in AqdC, enclose the quinolone ring of the substrate (Trp31, Ala96, Phe131, Leu135, Ile138, Leu151, Trp155, Trp180, Ala183, and Ile187 [33]) are also conserved (Fig. 5).
FIG 5
Finally, we compiled the characteristics of HQDs previously reported and checked the final data set of 161 sequences for these features (Table 3). Although most of the characteristics were not applied for the search workflow, they fully apply to the whole data set, including all verified HQDs, supporting the applicability of our search workflow to identify members of the HQD subfamily within the α/β-hydrolase fold superfamily.
TABLE 3
| Characteristic(s)a | Detail/function | Reference(s) |
|---|---|---|
| [H-x(2)-W]97-100 | Simplified nucleophilic elbow motif; motif 3 | |
| [HFP-x(4)-P]246-253 | Motif 9, including triad’s histidine | |
| αCap1-αCap4 | Cap domain formation; contributes to substrate binding, shields oxidation reaction from surrounding | 26, 33, 48 |
| D121 | Triad acidic residue is an Asp positioned between β6 and αCap1 | 26, 33 |
| [W/L]31, H97, [I/V]187 | Oxyanion hole formation and contribution to substrate binding site | 26, 33 |
| [F/L]131, [M/L/V/I]135, [F/L]151, W155, W180 | Contribute to the hydrophobic cavity for the quinolone substrate; alternative hydrophobic residues might be permitted | 26, 33 |
| M172 | Optional; potentially blocks alkyl-tail tunnel; indicator that short-chained substrates are preferred | 33 |
| A96 | Optional; replaces triad nucleophile; indicator for high kcat | 33 |
a
Numbering of amino acids according to AqdC sequence.
To conclude, we propose the use of a combination of HMMsearch- and ScanProSite-based screening to search for motifs 3 and 9 and to check their positions in the predicted α/β-hydrolase fold. HMMSearch will provide sequences with sufficient homology to screen for the motifs and exclude sequences unlikely to belong to HQDs. Additionally, but possibly unnecessary due to preselection using HMMSearch, the position of the acidic triad residue should be checked for the characteristic position at the end of strand β6, and the presence of a putative cap domain should be confirmed. This seems to be a reliable method for the prediction of HQDs.
DISCUSSION
Twenty years ago, the cofactorless dioxygenases Qdo and Hod, catalyzing ring cleavage of HQ and its 2-methyl congener, respectively, were hypothesized to belong to the α/β-hydrolase fold superfamily (25). Subsequent structural and functional studies were mainly motivated by the objective to understand how these enzymes work (26, 46–49); however, the identification of orthologs active toward the quorum-sensing signal molecule PQS triggered further interest in HQDs. In this study, a combination of in silico analysis involving HMMsearch and visual or ScanProSite-based screenings was performed to define search and validation criteria for HQD identification. Applying the proposed search strategy, from an initial data set of 118,475 sequences, 161 were finally selected as putative HQDs. Intercomparison of all sequences included in the data set showed sequence identities as low as 27.11%, demonstrating the extensive sequence variety covered by the strategy applied.
The applicability of the search approach was verified by demonstrating the ability of nine predicted HQDs, which share sequence identities of between 29.66% and 71.59% and which originate from different phyla, to cleave HQ, the most basic HQD substrate.
To identify HQDs and to distinguish those with a likely preference for PQS, there are a number of characteristics to be considered. The crystal structures of AqdC, Hod, and Qdo revealed that amino acids from both the core and the cap domain contribute to the substrate cavity as well as to the tunnel connecting it to the protein surface (26, 33). Therefore, as shown for Hod, the correct positioning of the cap domain, as well as the conservation of certain cap domain residues, is required for optimal enzymatic activity (48). Minor changes of cap domain residues contributing to the active-site cavity can result in impairment of function, as exemplified by Hod, where substitution of a strictly conserved tryptophan (W155 in AqdC numbering) by alanine led to less productive binding of the substrate and weakened the shielding of the active site from solvent (48). Preference for PQS appears to be favored by the presence of an amino acid with a small side chain (e.g., serine, threonine, and alanine) at position 172 (AqdC numbering), as in AqdC, AqdC1, AqdC2, HQDN.c., HQDS.b., and HQDC.i.. Comparison of the AqdC and Hod structures had shown that this position lies at the entry of a tunnel, which, in AqdC, accommodates the heptyl chain of PQS, whereas in Hod a methionine blocks this tunnel (33). Consistent with the notion that a Met at this position is an indicator for a preference of substrates with short-chain alkyl substituents (or lacking a substituent at C-2) rather than PQS, E. coli clones expressing HQDM.a.b., HQDS.l., HQDO.a., and HQDC.s. readily converted HQ but showed no or only very poor activity toward PQS (numbers 9 to 12; conserved M172 is shown in Fig. 5). Interestingly, enzymes with high turnover rates for PQS, as determined for AqdC, HQDN.c., HQDN.f., and HQDS.b. (numbers 3, 6, 7, and 8), share an alanine instead of a serine at the triad’s nucleophile position, supporting our proposal that the active-site alanine is favorable for the dioxygenolytic reaction (33). However, for detoxification of PQS in natural habitats of P. aeruginosa and in clinical settings, high affinity toward PQS is also important, since concentrations detected, e.g., in sputum samples of cystic fibrosis patients are in the low-micromolar, if not nanomolar, range (50, 51). Due to their low Km values of 3.1 μM and 2.7 μM, HQDN.f. and HQDS.b. may be effective PQS quenching enzymes in such settings.
Interestingly, the genes coding for HQDs with high activity toward PQS are part of conserved gene clusters, which, for R. erythropolis BG43 and M. abscessus subsp. abscessus (DSM 44196), were shown to code for a PQS-inducible pathway for biodegradation of HHQ and PQS to the central metabolites anthranilic acid and octanoic acid (see Fig. S5 in the supplemental material) (28). While the synteny of the aqdR-aqdABC cluster is conserved in N. cyriacigeorgica, N. farcinica, and S. bingchenggensis, the hqd gene of O. anthropi is part of a cluster similar to that of Arthrobacter sp. strain Rue61, which codes for methylquinoline degradation and includes the hod gene (52). However, many of the newly verified hqd genes (Fig. S5), as well as other hqd candidate genes (data not shown), are not part of such gene clusters. Thus, while genomic context may in some instances predict a functional role for the hqd ortholog, it is not useful as a general search criterion for the identification of members of the HQD family.
Our study defines and characterizes the family of 3-hydroxy-4(1H)-quinolone-cleaving dioxygenases. The developed search and validation criteria facilitate functional annotation of these enzymes, which, due to their overall fold, are easily mistaken for hydrolases. The definition of the enzyme family will extend the knowledge of cofactorless dioxygenases active toward bioactive 3-hydroxy-4(1H)-quinolones and the α/β-hydrolase fold superfamily of proteins.
MATERIALS AND METHODS
Chemicals, bacterial strains, and plasmids.
3-Hydroxy-4(1H)-quinolone (HQ) and N-octanoylanthranilic acid (NOAA) were synthesized as described before (24, 33). All other chemicals were obtained from commercial sources at the highest purity available. Unless noted otherwise (Table 1), synthetic hqd genes optimized for codon usage of E. coli (BioCat GmbH) (see Table S2 in the supplemental material) were cloned into the vector pET28b. E. coli BL21(DE3) was used as the host for the recombinant plasmids. Source organisms for the genes of interest and plasmids used in this study are listed in Table 1.
Bioinformatic analyses.
Amino acid sequences of currently known (2-alkyl-)3-hydroxy-4(1H)-quinolone 2,4-dioxygenases (Table 1, entries 1 to 5) were aligned using the T-Coffee multiple-sequence alignment server (M-Coffee [53]), and the alignment was complemented by the secondary structure of AqdC (PDB code 6RA2 [33]) using ESPript 3.0 (54). A hidden Markov model (HMM) was developed from this T-Coffee alignment using HMMER v3.2.1 and visualized by creating an HMM logo using the Skylign tool (content above background mode) (http://skylign.org/) (55). Motifs detected by visual inspection were further verified by Jensen-Shannon divergence (JSD) calculation (http://compbio.cs.princeton.edu/conservation/score.html [56]) with default settings, using the initial T-Coffee alignment as the input. Positions with a value of >0.72 were taken as conserved (Fig. 2; see also Fig. S1 in the supplemental material). HMMSearch (https://www.ebi.ac.uk/Tools/hmmer/search/hmmsearch) was carried out in the UniProtKB database (57) using HMM as a template. A subset of sequences showing a bit score of >180 was chosen, since lower scores were associated with poor template coverage. To further exclude incomplete sequences or sequences unlikely to belong to the HQD subfamily, the ScanProsite tool (https://prosite.expasy.org/scanprosite/, option 2 [58]) was applied to the subset. Two amino acid motifs, H-x(2)-W and HFP-x(4)-P (with x as a placeholder for a defined number [in parentheses] of any amino acid), were used as search motifs. Since H-x(2)-W is a very degenerate motif occurring frequently, the position of the motif in the α/β-hydrolase fold candidate proteins was inspected. The UniProt ID mapping tool (https://www.uniprot.org/uploadlists/; from UniProtKB AC/ID to UniRef100) was used to exclude redundant entries. From the amino acid sequences resulting from this search, a maximum likelihood phylogenetic tree was generated using Geneious R7 (Geneious, Auckland, New Zealand).
Structure homology models were constructed using SWISS-Model (https://swissmodel.expasy.org/ [59]). Secondary structures of the whole data set (161 sequences) were predicted by the PROMALS3D online tool (http://prodata.swmed.edu/promals3d/promals3d.php). Visual inspections of protein (homology) structures were performed using PyMOL (PyMOL Molecular Graphics System, version 2.0.4; Schrödinger, LLC.). The genomic environment of hqd genes was examined using EFI-GNT (Enzyme Function Initiative-Genome Neighborhood Tool [60]).
Biotransformation of (2-alkyl-)3-hydroxy-4(1H)-quinolones.
To verify the conversion of (2-alkyl-)3-hydroxy-4(1H)-quinolones by recombinant E. coli strains harboring expression plasmids (Table 1), 5 ml LB was inoculated with 500 μl of an overnight culture to an optical density at 600 nm (OD600) of 0.3 to 0.4, induced with 1 mM isopropyl β-d-thiogalactopyranoside (IPTG), and, after 1 h of cultivation at 25°C (shaking at 150 rpm), 50 μM HQ or PQS was added. Samples of the growing cultures, taken at appropriate time intervals, were extracted with acidified (1 g/liter citric acid) ethyl acetate. Extracts were dried, redissolved in acidified (1 g/liter citric acid), 50% (vol/vol) ethanol, and separated by high-performance liquid chromatography (HPLC) on a Eurospher II 100-5 C18 column (Knauer) at 35°C. The identity of the substrates and products was confirmed by comparing their specific retention times and UV spectra with those of reference compounds.
Expression and purification of 3-hydroxy-4(1H)-quinolone 2,4-dioxygenases.
E. coli BL21(DE3) harboring recombinant plasmids was grown aerobically under orbital shaking (150 rpm) at 37°C in LB medium in the presence of appropriate antibiotics. Expression conditions were optimized for each protein. In brief, cultures were supplemented with 0.2 to 0.5 mM IPTG at an OD600 of 0.5 to 1 and incubated at 15°C to 20°C for 16 to 20 h. Cells were harvested by centrifugation, resuspended in lysis buffer (300 mM NaCl, 20 mM Tris, 0.05% NP-40, pH 8.0), and disrupted by sonication. Proteins were purified by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography (elution buffer, 20 mM Tris-HCl, pH 8, 300 mM NaCl, 300 mM imidazole), and purity was analyzed by densitometric analysis (Image Lab 6.0.1; Bio-Rad) of Coomassie-stained SDS-PAGE gels (12.5%). The elution buffer was exchanged against storage buffer (20 mM Tris, 10% glycerol, pH 8.0) by ultrafiltration, and proteins were frozen in liquid nitrogen and stored at –80°C until further use. As described before, AqdC2 is highly active in recombinant E. coli cells, but all attempts to purify AqdC2 fusion proteins failed due to poor solubility (28).
Enzyme assays.
The catalytic activity of purified enzymes toward PQS was determined spectrophotometrically at 30°C by monitoring substrate conversion. The standard assay contained 50 mM Tris-HCl (pH 8.0), 2 mM EDTA, 10% (wt/vol) polyethylene glycol 1500 (to increase the solubility of the substrate), 4% (vol/vol) dimethyl sulfoxide (DMSO), and 20 μM PQS. The reaction was started by the addition of enzyme. The extinction coefficient of PQS in the assay buffer at the wavelength used for detection (ɛ337) is 10.17 × 103 M−1 cm−1. The apparent steady-state kinetic constants of the enzymes were estimated from fitting the activity data, measured at a series of substrate concentrations, to the Michaelis-Menten equation. Two biological replicates with at least three technical replicates were tested.
ACKNOWLEDGMENTS
We thank A. Kappius for excellent technical assistance, D. Danso (Hamburg) for helpful advice, and P. Weyrauch and L. Steffens for the construction of the plasmids pET23a::hodC and pET28b::aqdC2, respectively.
This project was supported by the Deutsche Forschungsgemeinschaft (FE 383/25-1) and the European Union’s Horizon 2020 research and innovation program under Marie Skłodowska-Curie grant agreement no. 722390.
S.F., together with S.C.W., conceived the project and wrote the manuscript. S.C.W. performed all bioinformatics analyses. A.A.S.M. and S.C.W. performed biochemical assays, biotransformations, and HPLC analyses. All authors contributed to the final version of the manuscript.
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Information & Contributors
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Published In
Applied and Environmental Microbiology
Volume 86 • Number 9 • 17 April 2020
eLocator: e00279-20
Editor: Maia Kivisaar, University of Tartu
PubMed: 32086305
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© 2020 American Society for Microbiology. All Rights Reserved.
History
Received: 4 February 2020
Accepted: 12 February 2020
Published online: 17 April 2020
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Maia Kivisaar
Editor
University of Tartu
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2020.
An α/β-Hydrolase Fold Subfamily Comprising Pseudomonas Quinolone Signal-Cleaving Dioxygenases. Appl Environ Microbiol
86:e00279-20.
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