Crystal structure of alkyl hydroperoxidase D like protein PA0269 from Pseudomonas aeruginosa: Homology of the AhpD-like structural family
© Clarke et al; licensee BioMed Central Ltd. 2011
Received: 21 January 2011
Accepted: 26 May 2011
Published: 26 May 2011
Alkyl hydroperoxidase activity provides an important antioxidant defense for bacterial cells. The catalytic mechanism requires two peroxidases, AhpC and AhpD, where AhpD plays the role of an essential adaptor protein.
The crystal structure of a putative AhpD from Pseudomonas aeruginosa has been determined at 1.9 Å. The protein has an all-helical fold with a chain topology similar to a known AhpD from Mycobacterium tuberculosis despite a low overall sequence identity of 9%. A conserved two α-helical motif responsible for function is present in both. However, in the P. aeruginosa protein, helices H3, H4 of this motif are located at the N-terminal part of the chain, while in M. tuberculosis AhpD, the corresponding helices H8, H9 are situated at the C-terminus. Residues 24-62 of the putative catalytic region of P. aeruginosa have a higher sequence identity of 33% where the functional activity is supplied by a proton relay system of five residues, Glu36, Cys48, Tyr50, Cys51, and His55, and one structural water molecule. A comparison of five other related hypothetical proteins from various species, assigned to the alkyl hydroperoxidase D-like protein family, shows they contain the same conserved structural motif and catalytic sequence Cys-X-X-Cys. We have shown that AhpD from P. aeruginosa exhibits a weak ability to reduce H2O2 as tested using a ferrous oxidation-xylenol orange (FOX) assay, and this activity is blocked by thiol alkylating reagents.
Thus, this hypothetical protein was assigned to the AhpD-like protein family with peroxidase-related activity. The functional relationship of specific oligomeric structures of AhpD-like structural family is discussed.
Alkyl hydroperoxidases play an important role in detoxifying peroxides and other reactive oxygen species in the cell. As part of an effort to identify and characterize potential antimicrobial targets from the pathogenic microorganism Pseudomonas aeruginosa, the hypothetical protein PA0269 from the carboxymuconolactone decarboxylase (CMD) family was selected for structure determination. This protein family includes a few representative members: the first, carboxymuconolactone decarboxylase, is involved in biodegradation of monocyclic aromatic carbon sources, the second, alkyl hydroperoxidase, is related to protecting against oxidative stress and the third has an unknown function. Carboxymuconolactone decarboxylase catalyzes decarboxylation of γ-carboxymuconolactone to β-ketoadipate enol-lactone in the catabolism of aromatic compounds through the protocatechuate branch of the β-ketoadipate pathway (EC 18.104.22.168). Known structures for this family include the trimer Mycobacterium tuberculosis AhpD [1–3], the hexameric protein TTHA0727 from Thermus thermophilus and some additional hexameric hypothetical proteins from thermophilic and methanobacteria from structural genomics consortium efforts.
Important antioxidant protection is provided by the AhpC/AhpD system, where in M. tuberculosis, the proteins have no sequence identity but work under the same promoter and can oligomerize [5, 6]. Mycobacterial peroxiredoxin alkyl hydroperoxide reductase C (AhpC) is a member of the family of non-heme peroxidases which protects heterologous bacterial and human cells against oxidative and nitrosative injuries . AhpC metabolizes peroxides a conserved N-terminal cysteine residue, which undergoes oxidation . At the final step of the catalytic cycle, the cysteine residue must be reduced. AhpD has been shown to play a role as a component of the AhpC/AhpD-dependent system since the mycobacterial lysate supports peroxidase activity of AhpC only in the presence of AhpD . Thus, alkyl hydroperoxidase D is an adaptor protein completing the AhpC/AhpD-dependent system, exhibits peroxidase activity and is involved in the antioxidant defense mechanism.
The mechanism of activity of alkyl hydroperoxidase D is mostly characterized for AhpD from M. tuberculosis. In the thioredoxin-like active site of M. tuberculosis AhpD, the catalytic residues Cys130 and Cys133 within the Cys-Ser-His-Cys sequence are responsible for peroxidase activity [1–3]. The proposed reaction mechanism of AhpD is based on a proton relay system consisting of five residues, Glu118, Cys130, His132, Cys133 and His137, and one structural water molecule . This mechanism was strongly supported by site-directed mutagenesis experiments and enzyme kinetics . The arrangement of the active site residues of the other CMD family member, carboxymuconolactone decarboxylase, have not been determined. In the Thermus thermophilus TTHA0727 structure, the analogue of the essential catalytic residue Cys130 residue of AhpD is replaced with a corresponding Ser70, indicating that this protein lacks peroxidase activity its function is unknown .
Here, we report the crystal structure and putative functional analysis of the hypothetical protein PA0269 from the pathogenic bacteria P. aeruginosa at 1.9Å resolution. Despite low overall sequence identity to M. tuberculosis AhpD, two catalytic sulfhydryl groups of cysteine residues and other functionally important residues around these cysteines form an exact configuration of the proton relay system with a very similar active site. Further examination has revealed the proteins share a conserved structural motif containing all the residues of the active site in a hairpin of two α-helices. This suggests the hypothetical P. aeruginosa protein may function as a weak hydroperoxidase, as verified by the ability to reduce H2O2 in the FOX assay, and possibly as the adaptor protein AhpD in an AhpC/AhpD-dependent peroxidase system. Using the homology of the conservative hairpin motif, we have found five additional related protein structures with yet unknown or uncharacterized functions. These proteins were also assigned to the AhpD-like protein family with probable peroxidase activity.
Protomer protein structure
However, in a particular section of the protein chain containing the key catalytic Cys residues, spanning residues 24-62 of PA0269 and residues 107-145 of M. tuberculosis AhpD, the sequence identity increases to 33%. This section of sequence corresponds to a conserved motif of two α-helices, H3 (residues 31-47) and H4 (residues 48-62) in the P. aeruginosa protein structure and H8 (residues 113-129) and H9 (residues 130-144) in the M. tuberculosis AhpD structure. The hairpin of the conserved two-helical motif is highlighted in somewhat different orientations in the two proteins in Figure 1. The configurations of two helices H3 and H4 for P. aeruginosa and corresponding H8 and H9 for M. tuberculosis structures are similar. However, they are localized at different sections of the protein chain. This might explain why the pair wise sequence identity of these proteins is extremely low. This also suggests that comparing the protein chains by the superposition of Cα atoms would be very difficult and the rmsd-value of Cα atoms cannot be reasonably assessed.
The transposition of the two conserved helix hairpin motifs along the protein chain is an interesting feature. In fact, the sequence motif created by H3, H4 occurs in the N-terminus in the P. aeruginosa structure but the equivalent H8, H9 helices are found at the C-terminus of M. tuberculosis AhpD. The genes coding the two proteins with similar function appear to have been mutated during the evolutionary process. It is important to note that the general all-helical motif of the protein tertiary structures is retained in both.
Establishment of the putative active site of PA0269
A stereoscopic view of the structure of PA0269 is presented in Figure 1c. The possible catalytic residues located in the sequence fragment Cys-Ala-Tyr-Cys were compared with the fragment Cys-Ser-His-Cys in the sequence of M. tuberculosis AhpD. The putative catalytic residues Cys48 and Cys51 of the P. aeruginosa protein are located in helix H4. All five putative catalytic residues of the proton relay system, Glu36, Cys48, Tyr50, Cys51 and His55, are located in two adjacent helices, H3 and H4 at the N-terminus of the protein chain, while in M. tuberculosis AhpD, the five catalytic residues found in the helices H8 and H9 at C-terminus of the protein chain as Glu118, Cys130, His132, Cys133 and His137.
Only one substitution in the series of catalytic residues, P. aeruginosa His132 corresponds in position to Tyr 50 in M. tuberculosis. This may have some weakening effect on the catalytic peroxidase activity of the P. aeruginosa protein mainly due to a difference in pKa of the residues. This value for tyrosine is approximately 10.1, while for the histidine side chain it is about 6.9. However, these values could vary, depending on the local environment of residues inside the protein molecule. In addition, the M. tuberculosis residue His132 (in the position corresponding to P. aeruginosa Tyr50) is not absolutely conserved in AhpD from different species, indicating that its catalytic role could be played by alternative residues .
Testing the ability to reduce hydrogen peroxide
Interestingly, the activity is strongly inhibited by preincubation of the reduced protein with N-ethylmaleimide, confirming that the peroxidase activity is cysteine dependent (Figure 3).
Structural homology of peroxidase-related AhpD-like protein family
Sequence homology of PDB structures of the putative alkyl hydroperoxidase D-like structural family as compared with the P. aeruginosa structure (PDB code 2O4D)
Description of proteins and species
Protein of unknown function from Agrobacterium tumefaciens, putative antioxidant, defense protein AhpD Length, 153 residues
Alkyl hydroperoxidase AhpD core protein uncharacterized peroxidase-related from Ralstonia eutropha Length, 194 residues
Uncharacterized peroxidase-related protein from Deinococcus geothermalis Length, 196 residues
Alkyl hydroperoxidase AhpD core protein from Rhodospirillum rubrum Length, 138 residues
Alkyl peroxidase AhpD from Mycobacterium tuberculosis Length, 177 residues
The crystal structure of protein TTHA0727 from Thermus thermophilus is also partially similar to the structure of AhpD-like proteins. The study of this structure shows the existence of a γ-CMD related structural family with various functional proteins . This protein also has sequence homology to the AhpD-like protein from Moorella thermoacetica, hypothetical protein AF0348 from Archaeoglobus fulgidus and AhpD-like protein from Mezorhizobium sp. with pair wise sequence identity of 35%, 29% and 23%, respectively . The superposition of three other known structures revealed a conserved motif of three α-helices numbered as H4, H5, H6 in TTHA07027 where helices H4, H5 can be compared with H3, H4 in the AhpD-like protein PA0269 from P. aeruginosa. In spite of the similarity in topologies, the AhpD-like protein family and γ-CMD protein family need to be considered as two different families. In fact, the sequence identity motifs are very different in the three helices of the γ-CMD family compared to the two helices of AhpD-like family. It is also significant that the five catalytic residues of the proton relay system responsible for peroxidase activity or the entire catalytic sequence motif Cys-X-X-Cys are not found in these members γ-CMD family members.
Oligomeric structure of alkyl hydroperoxidases D in crystal
The situation is slightly changed when P. aeruginosa AhpD is crystallized in a different space group, C2 (code 2IJC, ref. ). Here, the asymmetric unit contains two oligomers, with a total of 9 protein chains. The asymmetric unit content can be easily divided in two independent oligomers. One oligomer is a hexamer with the shape of a plane hexameric ring and it is exactly the same as described above for the structure 2O4D, while the second oligomer is a trimer, forming an entity with an antiparallel arrangement of associated protein subunits, as shown in Figure 7b.
If a set of similar oligomeric complexes from different species are compared, some complexes have the form of planar rings with a central holes and three-fold rotation symmetry axis as presented in Figure 7a. Complexes of different types are displayed in Figure 7b. Here, examples of asymmetric trimer and symmetrical dimers with two-fold rotation symmetry axis are shown. It should be noted that for the proteins of two structures, 2OYO and 2OUW, the similar hexameric forms also have been observed in the crystalline state. The authors of these structures state the hexameric form is the functioning biological unit, supported by the observation of oligomers in solution. However, in all cases, it is confirmed that the conserved portion of the protein responsible for function is structurally independent of the oligomeric state.
The crystal structure of the hypothetical PA2069 protein allows the recognition of a putative catalytic helix-turn-helix motif containing the sequence Cys-X-X-Cys. Structural analysis shows the conserved arrangement of several side groups of adjacent residues which could be necessary for constructing a proton relay system involving a single molecule of structural water. Unfortunately, in all protein members of the AhpD-like family, the binding sites of possible substrates or inhibitors are not yet known, although a small cavity on the surface of protein near the catalytic cysteines is available.
We have analyzed the crystal structures of a total of six proteins with putative or uncharacterized peroxidase activity. They have rather low sequence identity with the exception of approximately 30-residue spans containing the catalytic cysteines. Nonetheless, all proteins have a common conserved motif of two α-helices which include five residues of the proton relay system. This allows us to suggest the functional activity of the proteins is similar to AhpD. Analysis of the functional assignment of PA0269 was done by comparison with the functionally characterized M. tuberculosis AhpD structure. The organization of ahpD and ahpC genes in M. tuberculosis and ahpD, ahpC and ahpF genes in P. aeruginosa are different. In M. tuberculosis, the ahpD and ahpC genes are located in one operon with some other genes of metabolic proteins (GenBank accession NZ_ADAB 01000068). In P. aeruginosa, the gene for PA0269 (ahpD) is located in one operon while the genes for PA0139 (ahpC) and PA0140 (ahpF) are located in another operon (GenBank accession NC_002516). This difference could be one of the reasons for weak peroxidase activity which we have observed in the simplified biochemical assay.
An analysis of the AhpD-like family homology shows that the structure and sequence of the conserved domain are very similar but the remainder of each protein varies significantly. We could consider such a phenomenon as a case of molecular convergent evolution while maintaining the functional activity of the peroxidase as the leading factor. The conservation of activity is provided by retaining the conserved motif of two hairpin α-helices. Among the members of the AhpD-like family, we have observed an unusual situation where the conserved motif in the M. tuberculosis AhpD structure belongs to a topologically distinct region of the protein compared to P. aeruginosa AhpD and other members of family. It means this particular protein is built with a different folding pattern and different topology than all other members of the AhpD-like family. Even though all members of the protein family belong to one class of globin-like α-proteins, different chain topologies are allowed. A transposition of part of the gene sequence is possible as long as the peroxidase activity is retained. Maintaining the common structural class of an all-helical fold is very favorable because it seriously simplifies the folding mechanism and increases the speed of evolution, as in the case of M. tuberculosis AhpD.
A variety of oligomeric forms has been observed between the AhpD-like protein family members. Most often they exist in a hexamer form, although they may appear as a trimer or dimer. Large contact areas between pairs of adjacent molecules suggest that oligomeric complexes could occur not only in a crystalline state but also in solution. For several proteins, this is observed experimentally, allowing some authors to define the biological unit as an oligomer. However, the biologically relevant assembly may be influenced by the presence of AhpC in the cell. Nevertheless, the functionally important conserved two-helical motif is always structurally independent which suggests that the peroxidase activity is retained both for separate monomers and their oligomers.
Recent progress in expressing recombinant proteins and producing Se-methionine isomorphous crystals, as well as successes in various methods of protein crystallography, have resulted in a growing of number of protein structure files deposited in the Protein Data Bank. Structural homology can be used successfully to classify proteins to protein families only when sequence residue identity is higher than 30-35% along the majority of the protein chain. Also, a number of structures with putative or unknown function are likely related to a known protein family but have limited sequence homology and are not fully functionally characterized . In this communication, a similar problem to identify the functional activity of related protein structures has been resolved by searching for specific conserved structural motifs responsible for the functional activity. As a result a total of five uncharacterized proteins from various species have been assigned to the AhpD-like protein family.
Cloning, Expression and Purification
The target gene PA0269 (encoding the full length protein of 145 aa) was amplified from P. aeruginosa genomic DNA (strain 633, ATCC# 17933D) with the forward primer 5'-GCGGCGGCCCATATGACCACCCGCCTCGAATG-3' and the reverse primer 5'-GCGCGGATCCTTATCATTCCGGTTGCATGCCC-3' and inserted into a pET15b vector (Novagen, USA) using standard methods. Protein for crystallization experiments was expressed in E. coli BL21 (DE3) cells using the M9 selenomethionine high-yield media kit (Medicilon, CA, USA), harvested and flash-frozen. Expression of native protein for the assay was carried out in Terrific Broth using the same host. Cells were thawed and lysed by sonication (Branson, VWR) on ice in the presence of 0.5% CHAPS, 0.2 mM PMSF, 0.5 mM benzamidine and 300 units of benzonase (Novagen, WI) in binding buffer (50 mM HEPES pH 7.5, 0.5 M NaCl, 5 mM imidazole and 5% glycerol). The mixture was clarified at ~69 000 g (24 000 rpm in a Beckman J-25I, JA-25.50 rotor) for 40 min and applied to a DE52 column (Whatman, UK) pre-equilibrated with binding buffer. The flow-through was applied to a column of Ni-NTA Superflow (Qiagen, USA), washed with binding buffer followed by wash buffer (binding buffer with 0.03 M imidazole) and elution buffer (binding buffer with 0.25 M imidazole). The fractions containing the protein were pooled and concentrated. The selenomethionine sample and half of the native sample were immediately subjected to gel filtration. The remainder of the native sample was treated with thrombin to cleave the polyhistidine tag and passed through a Ni-NTA column to remove uncleaved protein prior to size exclusion chromatography. Gel filtration of all samples was performed on a Superdex 200 26/60 column (GE Healthcare, USA) equilibrated with gel filtration buffer (10 mM HEPES pH 7.5, 0.25 M NaCl, 0.5 mM TCEP). Fractions of the pure protein were pooled and concentrated to 94 mg/ml (SeMet sample) or 50 mg/ml (native protein sample with cleaved His tag). The protein was flash-frozen and stored at -80°C. MALDI-ToF mass spectrometric analysis of tryptic fragments confirmed the identity of the protein and incorporation of SeMet.
The selenomethionine labeled protein was thawed and diluted to 15 mg/ml with gel filtration buffer. The protein was centrifuged at 14 000 rpm for 10 minutes at 20°C. Crystals were obtained after a few days by sitting drop vapor diffusion against a reservoir solution containing 0.1 M Tris at pH 7.6, 0.2 M sodium chloride, 0.4 M sodium dihydrogen phosphate and 1.6 M dipotassium hydrogen phosphate in CrystalClear strips (Hampton Research, CA, USA) at 20°C. Crystals were flash-frozen in a mixture of reservoir and 20% ethylene glycol.
X-ray data collection and structure determination
Summary of X-ray Data and Structure Statistics
Unit cell dimensions (Å)
92.72, 92.72, 65.30
Observations (unique reflections)
* Numbers in parentheses are for the highest resolution shell.
Refinement and structure statistics:
RMS Deviations from ideal geometry
Bond length (Å)
Bond angle (°)
Number of atoms
Protein non-hydrogen atoms
Water oxygen atoms
Mean B-factor (Å2) for protein atoms
Mean B-factor (Å2) for water
Ramachandran plot statistics (%)
Residues in most favoured regions
Residues in allowed regions
The ferrous oxidation-xylenol orange (FOX) assay
Ten μM of reduced protein and 100 μM DTT were mixed in 50 mM potassium phosphate buffer pH 7.0 at room temperature. In one set of experiments, reduced protein was preincubated with 80 mM NEM (N-ethylmaleimide) to alkylate free thiols in the protein prior to the FOX assay. The reaction was initiated by the addition of 60 μM hydrogen peroxide (H2O2). The concentration of remaining hydrogen peroxide was determined at various time points up to 120 min by a xylenol orange-iron reaction. Briefly, 35 μl of the reaction was removed from the reaction mixture and added to 665 μl of FOX reagent (250 μM ammonium ferrous sulfate, 125 μM xylenol orange, 100 mM sorbitol, and 25 mM sulfuric acid). The FOX mixture was incubated for at least 20 min at room temperature and the absorbance at 560 nm was then monitored. Residual H2O2 concentration in the reaction was calculated using a standard curve. The assay was performed in triplicate.
Structural homology search
A structural similarity search was carried out using the DALI server . Protein secondary structure comparison was performed with the use of the service at the European Bioinformatics Institute . Sequence alignment and homology analysis were performed with the program suites protein BLAST  and advanced search PDB from the Protein Data Bank . Three-dimensional protein structures were superposed with Swiss PDB-Viewer . PyMOL was used to examine the protein structures and present figures .
alkyl hydroperoxidase D
ferrous oxidation-xylenol orange assay, used for detection hydrogen peroxide.
The authors acknowledge the grant sponsors: Ontario Research and Development Challenge Fund (99-SEP-0512), National Institutes of Health (Grant NIH RO1 GM50389). We thank Dr. Kevin Battaile of IMCA-CAT beam line at Argonne National Laboratory for his help in conducting experiments. We also thank Prof A. Efimov from the Institute of Protein Research, Russian Academy of Sciences, and Prof V. Lunin from the Institute of Mathematical Problems of Biology, Russian Academy of Sciences, for valuable discussions about the way of identifying putative functional activity of enzymes. We thank Dr V. Ksenzenko from the Institute of Protein Research, Russian Academy of Sciences for his help in analyzing genomic construction of bacteria. We also would like to thank Affinium Pharmaceuticals Inc. for their contribution at earlier stages of the project, and Kimberly J. Nelson for useful experimental suggestions.
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