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Article

Crystal Structure and Sequence Analysis of N5, N10-Methylenetetrahydrofolate Dehydrogenase/Cyclohydrolase Enzyme from Porphyromonas gingivalis

by
Sehyeok Im
1,2,†,
Hackwon Do
1,2,†,
Jisub Hwang
1,2,
Youn-Soo Shim
3,* and
Jun Hyuck Lee
1,2,*
1
Research Unit of Cryogenic Novel Material, Korea Polar Research Institute, Incheon 21990, Republic of Korea
2
Department of Polar Sciences, University of Science and Technology, Incheon 21990, Republic of Korea
3
Department of Dental Hygiene, Sunmoon University, Asan 31460, Republic of Korea
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Crystals 2023, 13(10), 1489; https://doi.org/10.3390/cryst13101489
Submission received: 5 October 2023 / Revised: 12 October 2023 / Accepted: 12 October 2023 / Published: 13 October 2023
(This article belongs to the Special Issue X-ray Protein Crystallography)
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Abstract

:
The methylenetetrahydrofolate dehydrogenase–cyclohydrolase (FolD) enzyme has a dual activity of N5,N10-methylenetetrahydrofolate dehydrogenase and cyclohydrolase. This enzyme plays a critical role in the chemical modification of tetrahydrofolate, which is an important coenzyme involved in the synthesis of DNA, RNA, and amino acids. Therefore, bacterial FolD has been studied as a potential drug target for the development of antibiotics. Here, we determined the crystal structure of FolD (PgFolD) from the oral pathogen Porphyromonas gingivalis at 2.05 Å resolution using the molecular replacement method. The crystal structure of PgFolD was successfully refined to a crystallographic R-factor of 21.4% (Rfree = 23.8%). The crystals belong to the space group of P4322 with the unit cell parameters of a = 110.7 Å, b = 110.7 Å, and c = 69.8 Å, containing one subunit in the asymmetric unit. Our analytical size-exclusion chromatography results indicated that PgFolD forms a stable dimer in solution. Additionally, structural and sequence comparison studies with previously known FolDs revealed that PgFolD has a different substrate-binding site residue composition. These findings provide valuable insights for the structure-based development of specific inhibitors against the Porphyromonas gingivalis pathogen.

1. Introduction

Tetrahydrofolate (THF) and THF derivatives, including 5-methyl-THF, 5,10-methylene-THF, and 10-formyl-THF, serve as co-factors in various biological reactions for DNA synthesis, amino acid metabolism, and other critical cellular processes [1,2,3,4,5]. THF is a folic acid compound modified by adding four hydrogen atoms. Various enzymes are involved in the chemical modification of THF. Methylenetetrahydrofolate dehydrogenase–cyclohydrolase (FolD) is a dual-function enzyme that converts N5, N10-methylene-THF into N10-formyl-THF in a two-step reaction. The first step is the NADP+- or NAD+-dependent oxidization of N5, N10-methenyl-THF by FolD via its methylenetetrahydrofolate dehydrogenase activity, and the second step is the hydrolysis of N5, N10-methenyltetrahydrofolate into N10-formyl-THF via the cyclohydrolase activity [6]. FolD knockout studies have shown that it is essential in both Gram-positive and Gram-negative bacteria [7,8,9].
To date, several bacterial FolDs have been characterized both biochemically and structurally. The crystal structures of the apo form of FolD from Escherichia coli (EcFolD; PDB codes 5O28 and 1B0A) have been previously determined [10,11]. Using structural information, methylene-tetrahydrofolate- and NADP-bound structures were modeled, and key amino acid residues important for substrate and co-factor binding were predicted [11]. In 2012, the crystal structure of FolD from Acinetobacter baumannii (AbFolD) was determined. The study report included three crystal structures of AbFolD in a binary complex with NADP (PDB code 4B4U), a ternary complex with NADP and an inhibitor (PDB code 4B4V), and a ternary complex with NADH and another inhibitor named LY354899 (PDB code 4B4W) [11]. The inhibitors used in this study mimicked the intermediate reaction products of this enzyme. Complex structures with two different inhibitors allowed for a more detailed elucidation of the enzyme reaction mechanism of AbFolD [12]. The crystal structure of unliganded FolD from Pseudomonas aeruginosa (PaFolD) was also determined in 2012. The loop region (residues 231–243) of the PaFolD structure shows a different conformation than that of the EcFolD and AbFolD structures [12]. Recently, the complex crystal structure of EcFolD-carolacton was determined (PDB code 5O22) [10]. The authors identified the key residues involved in carolacton binding and resistance in E. coli. They also suggested the possibility of using carolacton as an anticancer drug because it inhibits the human methylenetetrahydrofolate dehydrogenase/cyclohydrolase enzyme hsMTHFD2, which is overexpressed in cancer cells [10]. Other studies have also shown that FolD is a potential drug target [13,14].
Our research group is interested in the identification and development of effective antibacterial drugs against oral pathogens. Porphyromonas gingivalis is an anaerobic, non-motile, Gram-negative bacterium that causes chronic periodontitis [15]. Chronic infection of this pathogen has also been linked to rheumatoid arthritis, heart disease, diabetes, Alzheimer’s disease, and other systemic diseases [16,17,18,19,20]. Various antibiotics have been used to prevent infection of P. gingivalis. However, since the late 20th century, there have been several cases of acquired resistance to some antibiotics, including amoxicillin, metronidazole, macrolides, tetracyclines, and fluoroquinolones [21,22,23,24]. Considering that the resistance against antibiotics that are still available can be acquired through the mutation of the target site, it is essential to develop novel target bioactive molecules [21]. In this study, the FolD-encoding gene of P. gingivalis was identified and cloned as a target protein.
To obtain structural information, we expressed, purified, and crystallized the PgFolD protein from P. gingivalis. The crystal structure of substrate-free PgFolD was determined at a resolution of 2.05 Å. Multiple sequence alignments and comparative structural studies of PgFolD and other bacterial FolD homologs were performed to understand the differences and characteristics of the active sites of PgFolD. Considering that the species-specific treatment of growth inhibitors is crucial to preserve the beneficial oral bacteria, these new findings from the sequence and structural analysis will provide a better understanding of structure-based drug development against specific oral pathogenic bacteria such as P. gingivalis.

2. Materials and Methods

2.1. Protein Expression and Purification

The recombinant plasmid encoding PgFolD was produced by ligating synthesized PgFolD gene fragments (Bioneer, Daejeon, Republic of Korea) into the pET28a vector using NdeI and XhoI restriction enzymes. The generated plasmid was transformed into E. coli BL21 (DE3) cells for protein expression analyses. A single colony of cells was cultured in 4 L of Luria–Bertani (LB) medium with 50 μg/mL kanamycin and incubated at 150 rpm and 37 °C until the optical density reached 0.4 at 600 nm. Thereafter, 1 mM isopropyl β-D-1-thiogalactopyranoside was added to the cell culture to induce protein overexpression, and the cells were grown overnight in a shaking incubator (25 °C, 150 rpm). The cell culture was then harvested by centrifugation at 6000 rpm and 4 °C for 20 min. Harvested cell pellets were resuspended in cell lysis buffer (20 mM Tris-HCl pH 8.0, 200 mM NaCl, and 5 mM imidazole) and disrupted by ultrasonication (Vibra-Cell™, Sonics & Materials, Inc., Newtown, CT, USA). The disrupted cells were centrifugated at 16,000 rpm and 4 °C for 50 min, after which the protein-containing supernatant was separated from cell debris.
As our target protein was tagged with a poly-histidine tag, Ni-NTA agarose resin was used for purification. First, the resin was packed vertically in a column using gravity. After packing, the column was washed with distilled water and equilibrated with cell lysis buffer. The separated supernatant was loaded onto a column and bound to the Ni-NTA resin at a flow rate of 3 mL/min. Weakly bound proteins were washed with washing buffer (30 mM imidazole, 20 mM Tris-HCl (pH 8.0) and 200 mM NaCl). Poly-histidine-tagged PgFolD was eluted using an elution buffer (300 mM imidazole, 20 mM Tris-HCl pH 8.0, and 200 mM NaCl). The eluted protein was treated with 40 U of thrombin to remove the poly-histidine tag and incubated using an inverting machine at 4 °C overnight. Final purification was performed by size-exclusion chromatography using a HiLoad Superdex 200 pg column (Cytiva, Marlborough, MA, USA) and fresh buffer without imidazole (20 mM Tris-HCl pH 8.0 and 200 mM NaCl). Purified protein was concentrated to 30 mg/mL in a volume of 5 mL using an Amicon ultrafiltration filter unit with a 10 kDa cutoff value.

2.2. Crystallization and Data Collection

Purified protein was crystallized using the sitting drop vapor diffusion method in an MRC 96-well sitting drop plate (Molecular Dimensions, Rotherham, UK) at room temperature. For primary screening, 300 nL of purified protein (30 mg/mL) was mixed with 300 nL of crystallization screening solution using a ‘Mosquito’ crystallization robot (SPT Labtech, Melbourn, UK). The commercial crystallization suites, including MCSG 1T~4T (Anatrace, Maumee, OH, USA), The PGA Screen, and JCSG Plus (Molecular Dimensions, Rotherham, UK), were used for screening. For solutions in which crystals were formed, the crystallization conditions were further optimized to increase the size of the crystals using the hanging drop diffusion method. The final crystals for X-ray diffraction were obtained under the conditions of 0.1 M Na2HPO4/KH2PO4 (pH 6.2) with 35% (w/v) MPD.
A single crystal was briefly incubated in a reservoir solution at a final glycerol concentration of 20% for cryoprotection. Thereafter, the crystal was mounted on the synchrotron facility beamline 5C (BL-5C) at the Pohang Accelerator Laboratory (PAL, Pohang, Republic of Korea), and complete diffraction images were captured at 1° oscillations per frame using an Eiger 9M detector (Dectris, Baden, Switzerland). The collected data were processed using the HKL-2000 software (HKL Research Inc., Charlottesville, VA, USA) [25].

2.3. Structure Determination and Oligomerization

We used the CCP4 and Phenix suite [26] and Coot software [27] to build the PgFolD structure. The initial phasing problem was solved by the molecular replacement method [26]. A template structure from the AlphaFold database (model number AF-Q7MVE9-F1) was used [28]. Iterative refinement was performed using Refmac [29], phenix.refinement [30], and Coot [27]. The refined structure was validated using Molprobity [31,32]. The final structure was deposited in the Protein Data Bank under the accession code (PDB code PgFolD: 8WFC). The X-ray diffraction data and refinement statistics are summarized in Table 1.
Size-exclusion chromatography (SEC) was performed using a HiLoad Superdex 200 pg column (Cytiva) at a flow rate of 0.5 mL/min. We used Protein Standard Mix 15–600 kDa (Sigma-Aldrich, Burlington, MA, USA) containing Thyoglobulin bovine (670 kDa), γ-globulins from bovine blood (150 kDa), ovalbumin (44.3 kDa), ribonuclease A type I-A (13.7 kDa), and p-aminobenzoic acid (137 Da) to make a trend curve showing the relationship between the molecular weight of protein and elution volume (Column volume). PgFolD was eluted at a flow rate of 0.5 mL/min using a protein storage buffer (20 mM Tris-HCl pH 8.0 and 200 mM NaCl). After confirming that PgFolD exists as an oligomer in an aqueous environment through SEC, its dimeric structure was analyzed and visualized using PyMOL v4.6 [33].

3. Results and Discussion

3.1. Overall Structure of PgFolD

The genome of Porphyromonas gingivalis strain ATCC BAA-308/W83 was sequenced, and all gene sequence information is available [34]. The sequence UniProtKB ID Q7MVE9, corresponding to the gene encoding PgFolD, was selected for cloning, expression, and purification. Purified PgFolD was successfully crystallized using the hanging drop vapor diffusion method at 293 K. The crystal structure of the apo form of PgFolD was determined at 2.05 Å resolution using the molecular replacement method, with refinement results being 21.5% of Rfactor and 23.8% of Rfree. The final model was composed of 285 amino acids and 163 water molecules (Table 1). The overall structure of PgFolD was divided into N-terminal and C-terminal domains. The N-terminal domain has three parallel β-strands (β1-β3), surrounded by six α-helices (α1, α2, α3, α4, α5, and α6), whereas the C-terminal domain contains two separated β-sheets (β4-β7 and β8-β9) and is surrounded by six α-helices (α6, α7, α8, α9, α10, and α11) (Figure 1). The co-factor and substrate-binding sites were located between the N-terminal and C-terminal domains. One protomer of PgFolD was observed in the asymmetric unit, and the Matthew coefficient was 3.35 Å3, with a solvent content of 63.26% [35]. Analytical gel filtration results showed that PgFolD formed a stable dimer in solution. The dimer structure of PgFolD can be generated by crystallographic two-fold symmetry and is very similar to previously known dimeric structures from other bacterial FolD homologs. An accessible surface of approximately 1416 Å2 per monomer is buried upon dimerization, accounting for approximately 10.4% of the total monomer surface. The majority of the dimer interface occurs between α5 and β5 of each subunit (Figure 2).

3.2. Active Site of PgFolD

To investigate and compare the active site residues in PgFolD, the apo-PgFolD structure was superimposed onto the NADP-bound AbFolD (PDB code 4B4U) and the inhibitor (LY354899)-NADP-bound AbFolD (PDB code 4B4V) structures that have been reported previously [12]. Among the bacterial FolD structures, the AbFolD structure was selected for comparison with the PgFolD structure because both coenzyme- and inhibitor-bound AbFolD structures are available (Table 2). The amino acid sequence identity between PgFolD and AbFolD is 42.7% (Figure 3). Furthermore, we could determine the NADP-binding residues of PgFolD upon comparing the apo-PgFolD structure with the NADP-bound AbFolD structure (PDB code 4B4U) (Figure 4). The NADP-interacting residues of PgFolD and AbFolD were very similar, except for Q218 in PgFolD, corresponding to the K211 in AbFolD. When the apo-PgFolD structure was compared to the inhibitor (LY354899)-NADP-bound AbFolD (PDB code 4B4V) structure, the putative ligand-binding residues of PgFolD were identified. Through this structural comparison, we could identify different residues in the putative substrate-binding site of PgFolD compared to that in AbFolD. PgFolD has three unique residues (S55, L101, and T238) at the substrate-binding site, whereas AbFolD has M52, H98, and F231 residues at the corresponding positions (Figure 4D). The distinct arrangement of residues surrounding the inhibitor binding site suggests that the inhibitor (LY354899) is ineffective in inhibiting the activity of PgFolD. This implies the necessity of developing a customized inhibitor specifically designed for PgFolD based on the structure information. In addition, the domain-closing movement of PgFolD and AbFolD by the binding of co-factors and/or inhibitors could be observed through these structural comparison studies. The unliganded form of PgFolD contains an open conformation, and NADP binding induces domain closure. When both NADP and the inhibitor were bound to AbFolD at the same time, AbFolD had the most fully closed conformation (Figure 4E). Next, the surface charges of bacterial apo-FolD structures were investigated and compared. The NADP-binding site exhibited a similar shape and was dominated by a negatively charged electrostatic surface, whereas the surface charge distribution and shape of the substrate-binding site were considerably different (Figure 5). This suggests that it is possible to produce an inhibitor specific to P. gingivalis using the structural information of the substrate-binding site.

4. Conclusions

In the current study, we described the determination of the apo-form structure of PgFolD at a resolution of 2.05 Å. PgFolD structure has high overall structural similarity with previously known bacterial FolDs. However, multiple sequence alignment and comparative structural analysis revealed that PgFolD has unique substrate-binding residues, indicating that PgFolD has different binding modes for substrates. While it is necessary to validate the importance of these residues through mutational experiments and further structural analysis in the presence of co-factors, this information could be used to develop specific inhibitors of PgFolD against this oral pathogen.

Author Contributions

Conceptualization, Y.-S.S. and J.H.L.; methodology, S.I., H.D. and J.H.; validation, Y.-S.S. and J.H.L.; formal analysis, S.I., H.D. and J.H.; investigation, S.I., H.D. and J.H.; resources, Y.-S.S. and J.H.L.; data curation, H.D.; writing—original draft preparation, S.I., H.D. and Y.-S.S.; writing—review and editing, Y.-S.S. and J.H.L.; funding acquisition, J.H.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was a part of the project titled “Development of potential antibiotic compounds using polar organism resources (KIMST 20200610, KOPRI PM23030),” funded by the Ministry of Oceans and Fisheries, Republic of Korea.

Data Availability Statement

Not applicable.

Acknowledgments

We would like to thank the staff at the X-ray core facility of the Korea Basic Science Institute (KBSI; Ochang, Republic of Korea) and BL-5C of the Pohang Accelerator Laboratory (Pohang, Republic of Korea) for their kind assistance with data collection.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Rezk, B.M.; Haenen, G.R.M.M.; Van Der Vijgh, W.J.F.; Bast, A. Tetrahydrofolate and 5-methyltetrahydrofolate are folates with high antioxidant activity. Identification of the antioxidant pharmacophore. FEBS Lett. 2003, 555, 601–605. [Google Scholar] [CrossRef] [PubMed]
  2. Ames, T.D.; Rodionov, D.A.; Weinberg, Z.; Breaker, R.R. A eubacterial riboswitch class that senses the coenzyme tetrahydrofolate. Chem. Biol. 2010, 17, 681–685. [Google Scholar] [CrossRef] [PubMed]
  3. Kim, S.J.; Yoon, J.; Im, D.K.; Kim, Y.H.; Oh, M.K. Adaptively evolved Escherichia coli for improved ability of formate utilization as a carbon source in sugar-free conditions. Biotechnol. Biofuels 2019, 12, 207. [Google Scholar] [CrossRef]
  4. Burda, P.; Kuster, A.; Hjalmarson, O.; Suormala, T.; Bürer, C.; Lutz, S.; Roussey, G.; Christa, L.; Asin-Cayuela, J.; Kollberg, G.; et al. Characterization and review of MTHFD1 deficiency: Four new patients, cellular delineation and response to folic and folinic acid treatment. J. Inherit. Metab. Dis. 2015, 38, 863–872. [Google Scholar] [CrossRef] [PubMed]
  5. Kim, S.E. Enzymes involved in folate metabolism and its implication for cancer treatment. Nutr. Res. Pract. 2020, 14, 95–101. [Google Scholar] [CrossRef] [PubMed]
  6. Allaire, M.; Li, Y.; MacKenzie, R.E.; Cygler, M. The 3-D structure of a folate-dependent dehyhdrogenase/cyclohydrolase bifunctional enzyme at 1.5 Å resolution. Structure 1998, 6, 173–182. [Google Scholar] [CrossRef]
  7. Murta, S.M.F.; Vickers, T.J.; Scott, D.A.; Beverley, S.M. Methylene tetrahydrofolate dehydrogenase/cyclohydrolase and the synthesis of 10-CHO-THF are essential in Leishmania major. Mol. Microbiol. 2009, 71, 1386–1401. [Google Scholar] [CrossRef] [PubMed]
  8. Eadsforth, T.C.; Gardiner, M.; Maluf, F.V.; McElroy, S.; James, D.; Frearson, J.; Gray, D.; Hunter, W.N. Assessment of Pseudomonas aeruginosa N5, N10—Methylenetetrahydrofolate dehydrogenase—Cyclohydrolase as a potential antibacterial drug target. PLoS ONE 2012, 7, e35973. [Google Scholar] [CrossRef] [PubMed]
  9. De Berardinis, V.; Vallenet, D.; Castelli, V.; Besnard, M.; Pinet, A.; Cruaud, C.; Samair, S.; Lechaplais, C.; Gyapay, G.; Richez, C.; et al. A complete collection of single-gene deletion mutants of Acinetobacter baylyi ADP1. Mol. Syst. Biol. 2008, 4, 174. [Google Scholar] [CrossRef] [PubMed]
  10. Fu, C.; Sikandar, A.; Donner, J.; Zaburannyi, N.; Herrmann, J.; Reck, M.; Wagner-Döbler, I.; Koehnke, J.; Müller, R. The natural product carolacton inhibits folate-dependent C1 metabolism by targeting FolD/MTHFD. Nat. Commun. 2017, 8, 1529. [Google Scholar] [CrossRef] [PubMed]
  11. Shen, B.W.; Dyer, D.H.; Huang, J.Y.; D’Ari, L.; Rabinowitz, J.; Stoddard, B.L. The crystal structure of a bacterial, bifunctional 5, 10 methylene-tetrahydrofolate dehydrogenase/cyclohydrolase. Protein Sci. 1999, 8, 1342–1349. [Google Scholar] [CrossRef] [PubMed]
  12. Eadsforth, T.C.; Maluf, F.V.; Hunter, W.N. Acinetobacter baumannii FolD ligand complexes—Potent inhibitors of folate metabolism and a re-evaluation of the structure of LY374571. FEBS J. 2012, 279, 4350–4360. [Google Scholar] [CrossRef] [PubMed]
  13. Eadsforth, T.C.; Cameron, S.; Hunter, W.N. The crystal structure of Leishmania major N5, N10 -methylenetetrahydrofolate dehydrogenase/cyclohydrolase and assessment of a potential drug target. Mol. Biochem. Parasitol. 2012, 181, 178–185. [Google Scholar] [CrossRef] [PubMed]
  14. Bermingham, A.; Derrick, J.P. The folic acid biosynthesis pathway in bacteria: Evaluation of potential for antibacterial drug discovery. BioEssays 2002, 24, 637–648. [Google Scholar] [CrossRef]
  15. Mysak, J.; Podzimek, S.; Sommerova, P.; Lyuya-Mi, Y.; Bartova, J.; Janatova, T.; Prochazkova, J.; Duskova, J. Porphyromonas gingivalis: Major periodontopathic Pathogen Overview. J. Immunol. Res. 2014, 2014, 476068. [Google Scholar] [CrossRef]
  16. Marchesan, J.T.; Gerow, E.A.; Schaff, R.; Taut, A.D.; Shin, S.; Sugai, J.; Brand, D.; Burberry, A.; Jorns, J.; Lundy, S.K.; et al. Porphyromonas gingivalis oral infection exacerbates the development and severity of collagen-induced arthritis. Arthritis Res. Ther. 2013, 15, R186. [Google Scholar] [CrossRef] [PubMed]
  17. Aleksijević, L.H.; Aleksijević, M.; Škrlec, I.; Šram, M.; Šram, M.; Talapko, J. Porphyromonas gingivalis virulence factors and clinical significance in periodontal disease and coronary artery diseases. Pathogens 2022, 11, 1173. [Google Scholar] [CrossRef]
  18. Nishihara, R.; Sugano, N.; Takano, M.; Shimada, T.; Tanaka, H.; Oka, S.; Ito, K. The effect of Porphyromonas gingivalis infection on cytokine levels in type 2 diabetic mice. J. Periodont. Res. 2009, 44, 305–310. [Google Scholar] [CrossRef]
  19. Dominy, S.S.; Lynch, C.; Ermini, F.; Benedyk, M.; Marczyk, A.; Konradi, A.; Nguyen, M.; Haditsch, U.; Raha, D.; Griffin, C.; et al. Porphyromonas gingivalis in Alzheimer’s disease brains: Evidence for disease causation and treatment with small-molecule inhibitors. Sci. Adv. 2019, 5, eaau3333. [Google Scholar] [CrossRef]
  20. Li, X.; Kolltveit, K.M.; Tronstad, L.; Olsen, I. Systemic diseases caused by oral infection. Clin. Microbiol. Rev. 2000, 13, 547–558. [Google Scholar] [CrossRef]
  21. Kalhor, H.; Goodarzi, N.N.; Rahimi, H.; Firoozeh, F.; Badmasti, F. Identification of novel drug targets in Porphyromonas gingivalis and proposing inhibitors against acetate kinase using structure-based virtual screening. Process Biochem. 2023, 130, 310–321. [Google Scholar] [CrossRef]
  22. Jepsen, K.; Falk, W.; Brune, F.; Fimmers, R.; Jepsen, S.; Bekeredjian-Ding, I. Prevalence and antibiotic susceptibility trends of periodontal pathogens in the subgingival microbiota of German periodontis patients: A retrospective surveillance study. J. Clin. Periodontol. 2021, 48, 1216–1227. [Google Scholar] [CrossRef] [PubMed]
  23. Japoni, A.; Vazin, A.; Noushadi, S.; Kiany, F.; Japoni, S.; Alborzi, A. Antibacterial susceptibility patterns of Porphyromonas gingivalis isolated from chronic periodontitis patients. Med. Oral Patol. Oral Cir. Bucal 2011, 16, 1031–1035. [Google Scholar] [CrossRef] [PubMed]
  24. Conrads, G.; Klomp, T.; Deng, D.; Wenzler, J.S.; Braun, A.; Abdelbary, M.M. The Antimicrobial Susceptibility of Porphyromonas gingivalis: Genetic Repertoire, Global Phenotype, and Review of the Literature. Antibiotics 2021, 10, 1438. [Google Scholar] [CrossRef]
  25. Otwinowski, Z.; Minor, W. Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 1997, 276, 307–326. [Google Scholar] [CrossRef]
  26. Winn, M.D.; Ballard, C.C.; Cowtan, K.D.; Dodson, E.J.; Emsley, P.; Evans, P.R.; Keegan, R.M.; Krissinel, E.B.; Leslie, A.G.; McCoy, A.; et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 2011, 67, 235–242. [Google Scholar] [CrossRef]
  27. Emsley, P.; Cowtan, K. Coot: Model-building tools for molecular graphics. Acta Crystallogr. D Biol. Crystallogr. 2004, 60, 2126–2132. [Google Scholar] [CrossRef]
  28. Jumper, J.; Evans, R.; Pritzel, A.; Green, T.; Figurnov, M.; Ronneberger, O.; Tunyasuvunakool, K.; Bates, R.; Žídek, A.; Potapenko, A.; et al. Highly accurate protein structure prediction with AlphaFold. Nature 2021, 596, 583–589. [Google Scholar] [CrossRef]
  29. Murshudov, G.N.; Skubák, P.; Lebedev, A.A.; Pannu, N.S.; Steiner, R.A.; Nicholls, R.A.; Winn, M.D.; Long, F.; Vagin, A.A. REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D Biol. Crystallogr. 2011, 67, 355–367. [Google Scholar] [CrossRef]
  30. Afonine, P.V.; Grosse-Kunstleve, R.W.; Echols, N.; Headd, J.J.; Moriarty, N.W.; Mustyakimov, M.; Terwilliger, T.C.; Urzhumtsev, A.; Zwart, P.H.; Adams, P.D. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D 2012, 68, 352–367. [Google Scholar] [CrossRef]
  31. Chen, V.B.; Arendall, W.B., 3rd; Headd, J.J.; Keedy, D.A.; Immormino, R.M.; Kapral, G.J.; Murray, L.W.; Richardson, J.S.; Richardson, D.C. MolProbity: All-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 2010, 66, 12–21. [Google Scholar] [CrossRef] [PubMed]
  32. Williams, C.J.; Headd, J.J.; Moriarty, N.W.; Prisant, M.G.; Videau, L.L.; Deis, L.N.; Verma, V.; Keedy, D.A.; Hintze, B.J.; Chen, V.B.; et al. MolProbity: More and better reference data for improved all-atom structure validation. Protein Sci. 2018, 27, 293–315. [Google Scholar] [CrossRef] [PubMed]
  33. Schrödinger, L.; DeLano, W. PyMOL. Version 2.0 Schrödinger, LLC. 2020. Available online: http://www.pymol.org/pymol (accessed on 10 October 2023).
  34. Nelson, K.E.; Rleischmann, R.D.; DeBoy, R.T.; Paulsen, I.T.; Fouts, D.E.; Eisen, J.A.; Daugherty, S.C.; Dodson, R.J.; Durkin, A.S.; Gwinn, M.; et al. Complete genome sequence of the oral pathogenic bacterium Porphyromonas gingivalis Strain W83. J. Bacteriol. 2004, 186, 5591–5601. [Google Scholar] [CrossRef]
  35. Matthews, B.W. Solvent content of protein crystals. J. Mol. Biol. 1968, 33, 491–497. [Google Scholar] [CrossRef]
Crystals 13 01489 g001
Figure 1. Crystallization, X-ray diffraction, and structure determination of PgFolD. (A) Crystals of PgFolD in 96-well sitting drop plate. Scale bar, 250 μm. (B) X-ray diffraction results of PgFolD. The diffraction data were collected up to 2.05 Å. (C) The overall structure of PgFolD is shown as a ribbon diagram with 11 α-helices colored light blue and 9 β-stands colored gold.
Figure 1. Crystallization, X-ray diffraction, and structure determination of PgFolD. (A) Crystals of PgFolD in 96-well sitting drop plate. Scale bar, 250 μm. (B) X-ray diffraction results of PgFolD. The diffraction data were collected up to 2.05 Å. (C) The overall structure of PgFolD is shown as a ribbon diagram with 11 α-helices colored light blue and 9 β-stands colored gold.
Crystals 13 01489 g001
Crystals 13 01489 g002
Figure 2. Size-exclusion chromatography results and dimeric structure of PgFolD. (A) The black curve with five peaks represents the size-exclusion chromatography result of Protein Standard Mix, and the red curve with a single peak represents the result of PgFolD. The results show the oligomeric state of PgFolD in aqueous solution. (B) Dimeric structure of PgFolD and its secondary structure. Each molecule of PgFolD in the dimeric structure is shown in two different colors (sky blue and wheat).
Figure 2. Size-exclusion chromatography results and dimeric structure of PgFolD. (A) The black curve with five peaks represents the size-exclusion chromatography result of Protein Standard Mix, and the red curve with a single peak represents the result of PgFolD. The results show the oligomeric state of PgFolD in aqueous solution. (B) Dimeric structure of PgFolD and its secondary structure. Each molecule of PgFolD in the dimeric structure is shown in two different colors (sky blue and wheat).
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Crystals 13 01489 g003
Figure 3. Multiple sequence alignment with other structural homologs. Multiple amino acid sequence alignment was performed between PgFolD (UniProtKB code Q7MVE9) and other FolDs, namely FolD from Acinetobacter baumannii (AbFolD; UniProtKB code D0CBC8; PDB codes 4B4U, 4B4V, and 4B4W), Escherichia coli (EcFolD; UniProtKB code P24186; PDB codes 5O28, 1B0A, and 5O22), and Pseudomonas aeruginosa (PaFolD; UniProtKB code Q9I2U6; PDB code 4A5O), methylenetetrahydrofolate dehydrogenase/cyclohydrolase from Leishmania major (LmDHCH; UniProtKB code Q4Q9F9; PDB code 4A26), and bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase from Homo sapiens (HsMTHFD2; UniProtKB code P13995; PDB codes 7EHJ, 6KG2, 5TC4). When comparing PgFolD and AbFolD, three residues (S55, L101, and T238) of PgFolD that might be important for substrate binding but are different from the corresponding residues of AbFolD are marked using red circles.
Figure 3. Multiple sequence alignment with other structural homologs. Multiple amino acid sequence alignment was performed between PgFolD (UniProtKB code Q7MVE9) and other FolDs, namely FolD from Acinetobacter baumannii (AbFolD; UniProtKB code D0CBC8; PDB codes 4B4U, 4B4V, and 4B4W), Escherichia coli (EcFolD; UniProtKB code P24186; PDB codes 5O28, 1B0A, and 5O22), and Pseudomonas aeruginosa (PaFolD; UniProtKB code Q9I2U6; PDB code 4A5O), methylenetetrahydrofolate dehydrogenase/cyclohydrolase from Leishmania major (LmDHCH; UniProtKB code Q4Q9F9; PDB code 4A26), and bifunctional methylenetetrahydrofolate dehydrogenase/cyclohydrolase from Homo sapiens (HsMTHFD2; UniProtKB code P13995; PDB codes 7EHJ, 6KG2, 5TC4). When comparing PgFolD and AbFolD, three residues (S55, L101, and T238) of PgFolD that might be important for substrate binding but are different from the corresponding residues of AbFolD are marked using red circles.
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Crystals 13 01489 g004
Figure 4. Structural comparison of PgFolD and AbFolD. (A) The key residues of PgFolD involved in the substrate and NADP binding are shown using a stick model. (B) Superimposed structures of PgFolD (green) and NADP (orange)-bound AbFolD (slate blue) (PDB code 4B4U). (C,D) Superimposed structures of PgFolD (green) and inhibitor (LY354899) (orange) NADP (yellow)-bound AbFolD (red) (PDB code 4B4V). Notably, there are three residues (S55, L101, and T238) in the substrate binding site of PgFolD that are different from the substrate binding residues in AbFolD. (E) Structural comparison of apo-PgFolD (green) and two different AbFolD structures (PDB codes 4B4U and 4B4V) shows that the apo structure of PgFolD (green) has open conformation, while the binary (slate blue) and ternary (red) complexed AbFolD structures have closed conformation.
Figure 4. Structural comparison of PgFolD and AbFolD. (A) The key residues of PgFolD involved in the substrate and NADP binding are shown using a stick model. (B) Superimposed structures of PgFolD (green) and NADP (orange)-bound AbFolD (slate blue) (PDB code 4B4U). (C,D) Superimposed structures of PgFolD (green) and inhibitor (LY354899) (orange) NADP (yellow)-bound AbFolD (red) (PDB code 4B4V). Notably, there are three residues (S55, L101, and T238) in the substrate binding site of PgFolD that are different from the substrate binding residues in AbFolD. (E) Structural comparison of apo-PgFolD (green) and two different AbFolD structures (PDB codes 4B4U and 4B4V) shows that the apo structure of PgFolD (green) has open conformation, while the binary (slate blue) and ternary (red) complexed AbFolD structures have closed conformation.
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Crystals 13 01489 g005
Figure 5. Comparison of electrostatic potentials on surfaces of bacterial apo-FolD structures. Electrostatic surface representation of (A) apo-PgFolD, (B) apo-EcFolD (PDB code 5O28), and (C) apo-PaFolD (PDB code 4A5O) is shown. The NADP and substrate binding sites are indicated using green and gold dotted circles, respectively. Electrostatic surface potential was calculated with APBS software and colored using a calculated charge from red (−5 kT/e) to blue (+5 kT/e).
Figure 5. Comparison of electrostatic potentials on surfaces of bacterial apo-FolD structures. Electrostatic surface representation of (A) apo-PgFolD, (B) apo-EcFolD (PDB code 5O28), and (C) apo-PaFolD (PDB code 4A5O) is shown. The NADP and substrate binding sites are indicated using green and gold dotted circles, respectively. Electrostatic surface potential was calculated with APBS software and colored using a calculated charge from red (−5 kT/e) to blue (+5 kT/e).
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Table 1. X-ray diffraction data collection and refinement statistics.
Table 1. X-ray diffraction data collection and refinement statistics.
Data SetPgFolD
X-ray sourceBL-5C (PAL, Pohang)
Space groupP4322
Unit cell parameters (Å, °)a = 110.68, b = 110.68, c = 69.78, α = β = γ = 90
Wavelength (Å)0.9796
Resolution (Å)50–2.05 (2.09–2.05)
Total reflections723,199 (1739)
Unique reflections27,821 (1347)
Average I/σ (I) 59.67 (10.38)
Rmerge a0.100 (0.304)
Redundancy26 (26.3)
Completeness (%)99.9 (99.9)
Refinement
Resolution range (Å)35.01–2.05 (2.12–2.05)
No. of reflections of working set27,773 (2576)
No. of reflections of test set1413 (142)
No. of amino acid residues285
No. of water molecules163
Rcryst b0.215 (0.254)
Rfree c0.238 (0.288)
R.m.s. bond length (Å)0.011
R.m.s. bond angle (°)1.13
Average B value (Å2) (protein) 40.82
Average B value (Å2) (solvent)43.49
Ramachandran plot
Favored (%)94.66
Allowed (%)5.34
Outliers (%)0.00
a Rmerge = ∑|<I> − I|/∑<I>. b Rcryst = ∑||Fo| − |Fc||/∑|Fo|. c Rfree calculated with 5% of all reflections excluded from the refinement stages using high-resolution data. Values in parentheses refer to the highest-resolution shells.
Table 2. Known bacterial FolD structures.
Table 2. Known bacterial FolD structures.
Protein NamePgFolDAbFolDEcFolDPaFolD
SourcePorphyromonas gingivalisAcinetobacter baumanniiEscherichia coliPseudomonas aeruginosa
UniProtKB codeQ7MVE9D0CBC8P24186Q9I2U6
Sequence identity/similarity (%) with PgFolD 39%/57%44%/59%45%/60%
PDB code 4B4U4B4V4B4W5O28, 1B0A5O224A5O
Complexed ligandnoneNADPNADP and inhibitor LY354899NADP and an inhibitornonecarolactonnone
Domain conformationopenclosedclosedclosedopenopenopen
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Im, S.; Do, H.; Hwang, J.; Shim, Y.-S.; Lee, J.H. Crystal Structure and Sequence Analysis of N5, N10-Methylenetetrahydrofolate Dehydrogenase/Cyclohydrolase Enzyme from Porphyromonas gingivalis. Crystals 2023, 13, 1489. https://doi.org/10.3390/cryst13101489

AMA Style

Im S, Do H, Hwang J, Shim Y-S, Lee JH. Crystal Structure and Sequence Analysis of N5, N10-Methylenetetrahydrofolate Dehydrogenase/Cyclohydrolase Enzyme from Porphyromonas gingivalis. Crystals. 2023; 13(10):1489. https://doi.org/10.3390/cryst13101489

Chicago/Turabian Style

Im, Sehyeok, Hackwon Do, Jisub Hwang, Youn-Soo Shim, and Jun Hyuck Lee. 2023. "Crystal Structure and Sequence Analysis of N5, N10-Methylenetetrahydrofolate Dehydrogenase/Cyclohydrolase Enzyme from Porphyromonas gingivalis" Crystals 13, no. 10: 1489. https://doi.org/10.3390/cryst13101489

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