Startseite Comparative studies of Aspergillus fumigatus 2-methylcitrate synthase and human citrate synthase
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Comparative studies of Aspergillus fumigatus 2-methylcitrate synthase and human citrate synthase

  • Caleb R. Schlachter , Vincent Klapper , Taylor Radford und Maksymilian Chruszcz ORCID logo EMAIL logo
Veröffentlicht/Copyright: 29. Mai 2019
Biological Chemistry
Aus der Zeitschrift Biological Chemistry Band 400 Heft 12

Abstract

Aspergillus fumigatus is a ubiquitous fungus that is not only a problem in agriculture, but also in healthcare. Aspergillus fumigatus drug resistance is becoming more prominent which is mainly attributed to the widespread use of fungicides in agriculture. The fungi-specific 2-methylcitrate cycle is responsible for detoxifying propionyl-CoA, a toxic metabolite produced as the fungus breaks down proteins and amino acids. The enzyme responsible for this detoxification is 2-methylcitrate synthase (mcsA) and is a potential candidate for the design of new anti-fungals. However, mcsA is very similar in structure to human citrate synthase (hCS) and catalyzes the same reaction. Therefore, both enzymes were studied in parallel to provide foundations for design of mcsA-specific inhibitors. The first crystal structures of citrate synthase from humans and 2-methylcitrate synthase from A. fumigatus are reported. The determined structures capture various conformational states of the enzymes and several inhibitors were identified and characterized. Despite a significant homology, mcsA and hCS display pronounced differences in substrate specificity and cooperativity. Considering that the active sites of the enzymes are almost identical, the differences in reactions catalyzed by enzymes are caused by residues that are in the vicinity of the active site and influence conformational changes of the enzymes.

Keywords: Aspergillus; coenzyme A; cooperativity; human citrate synthase; 2-methylcitrate synthase; structure

Acknowledgments:

  1. We would also like to thank Dr. Lesa Offermann and Dr. Thomas Makris for help with analyzing kinetic data. Structural results shown in this report are derived from work performed at Argonne National Laboratory, Structural Biology Center (19BM/ID) at the APS. Argonne is operated by U. Chicago Argonne, LLC, for the U.S. Department of Energy, Office of Biological and Environmental Research under contract DE-AC02-06CH11357. Use of LS-CAT (21ID) is supported by the Michigan Economic Development Corporation and the Michigan Technology Tri-Corridor (Grant 085P100081). Data were also collected at Southeast Regional Collaborative Access Team (SER-CAT) (22BM/ID) at the APS, Argonne National Laboratory. Use of the APS was supported by the U.S. Department of Energy, Office of Science, Office of Basic Energy Sciences, under Contract No. DE-AC02-06CH11357 and W-31-109-Eng-38. This work is partially supported by an ASPIRE III and SPARC grants from the Office of the Vice President of Research at the University of South Carolina.

References

Alter, G.M., Casazza, J.P., Zhi, W., Nemeth, P., Srere, P.A., and Evans, C.T. (1990). Mutation of essential catalytic residues in pig citrate synthase. Biochemistry 29, 7557–7563.10.1021/bi00485a003Suche in Google Scholar

Ashkenazy, H., Erez, E., Martz, E., Pupko, T., and Ben-Tal, N. (2010). ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids. Nucleic Acids Res. 38, W529–W533.10.1093/nar/gkq399Suche in Google Scholar

Askew, D.S. (2008). Aspergillus fumigatus: virulence genes in a street-smart mold. Curr. Opin. Microbiol. 11, 331–337.10.1016/j.mib.2008.05.009Suche in Google Scholar

Bayer, E., Bauer, B., and Eggerer, H. (1981). Evidence from inhibitor studies for conformational changes of citrate synthase. Eur. J. Biochem. 120, 155–160.10.1111/j.1432-1033.1981.tb05683.xSuche in Google Scholar

Binder, U. and Lass-Florl, C. (2013). New insights into invasive aspergillosis – from the pathogen to the disease. Curr. Pharm. Des. 19, 3679–3688.10.2174/13816128113199990366Suche in Google Scholar

Booth, W.T., Schlachter, C.R., Pote, S., Ussin, N., Mank, N.J., Klapper, V., Offermann, L.R., Tang, C., Hurlburt, B.K., and Chruszcz, M. (2018). Impact of an N-terminal polyhistidine tag on protein thermal stability. ACS Omega 3, 760–768.10.1021/acsomega.7b01598Suche in Google Scholar

Bradford, M.M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248–254.10.1016/0003-2697(76)90527-3Suche in Google Scholar

Brock, M., Fischer, R., Linder, D., and Buckel, W. (2000). Methylcitrate synthase from Aspergillus nidulans: implications for propionate as an antifungal agent. Mol. Microbiol. 35, 961–973.10.1046/j.1365-2958.2000.01737.xSuche in Google Scholar PubMed

Chen, V.B., Arendall 3rd, W.B., Headd, J.J., Keedy, D.A., Immormino, R.M., Kapral, G.J., Murray, L.W., Richardson, J.S., and Richardson, D.C. (2010). MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D: Biol. Crystallogr. 66, 12–21.10.1107/97809553602060000884Suche in Google Scholar

Cormier, C.Y., Mohr, S.E., Zuo, D., Hu, Y., Rolfs, A., Kramer, J., Taycher, E., Kelley, F., Fiacco, M., Turnbull, G., et al. (2010). Protein Structure Initiative Material Repository: an open shared public resource of structural genomics plasmids for the biological community. Nucleic Acids Res. 38, D743–D749.10.1093/nar/gkp999Suche in Google Scholar PubMed PubMed Central

Cormier, C.Y., Park, J.G., Fiacco, M., Steel, J., Hunter, P., Kramer, J., Singla, R., and LaBaer, J. (2011). PSI: Biology-materials repository: a biologist’s resource for protein expression plasmids. J. Struct. Funct. Genomics 12, 55–62.10.1007/s10969-011-9100-8Suche in Google Scholar

DeLano, W.L. (2002). The PyMOL Molecular Graphics System. (San Carlos: DeLano Scientific).Suche in Google Scholar

Emsley, P., Lohkamp, B., Scott, W.G., and Cowtan, K. (2010). Features and development of Coot. Acta Crystallogr. D: Biol. Crystallogr. 66, 486–501.10.1107/S0907444910007493Suche in Google Scholar

Fraczek, M.G., Bromley, M., and Bowyer, P. (2011). An improved model of the Aspergillus fumigatus CYP51A protein. Antimicrob. Agents Chemother. 55, 2483–2486.10.1128/AAC.01651-10Suche in Google Scholar

Hayward, S. and Berendsen, H.J. (1998). Systematic analysis of domain motions in proteins from conformational change: new results on citrate synthase and T4 lysozyme. Proteins 30, 144–154.10.1002/(SICI)1097-0134(19980201)30:2<144::AID-PROT4>3.0.CO;2-NSuche in Google Scholar

Hayward, S., Kitao, A., and Berendsen, H.J. (1997). Model-free methods of analyzing domain motions in proteins from simulation: a comparison of normal mode analysis and molecular dynamics simulation of lysozyme. Proteins 27, 425–437.10.1002/(SICI)1097-0134(199703)27:3<425::AID-PROT10>3.0.CO;2-NSuche in Google Scholar

Howard, S.J. and Arendrup, M.C. (2011). Acquired antifungal drug resistance in Aspergillus fumigatus: epidemiology and detection. Med. Mycol. 49(Suppl 1), S90–S95.10.3109/13693786.2010.508469Suche in Google Scholar

Ibrahim-Granet, O., Dubourdeau, M., Latge, J.-P., Ave, P., Huerre, M., Brakhage, A.A., and Brock, M. (2008). Methylcitrate synthase from Aspergillus fumigatus is essential for manifestation of invasive aspergillosis. Cell. Microbiol. 10, 134–148.10.1111/j.1462-5822.2007.01025.xSuche in Google Scholar

Johansson, C.J. and Pettersson, G. (1979). Inhibition of pig-heart citrate synthase by carboxylic acids structurally related to oxaloacetate. Eur. J. Biochem. 93, 505–513.10.1111/j.1432-1033.1979.tb12849.xSuche in Google Scholar

Karpusas, M., Branchaud, B., and Remington, S.J. (1990). Proposed mechanism for the condensation reaction of citrate synthase: 1.9-A structure of the ternary complex with oxaloacetate and carboxymethyl coenzyme A. Biochemistry 29, 2213–2219.10.1021/bi00461a002Suche in Google Scholar

Karpusas, M., Holland, D., and Remington, S.J. (1991). 1.9-A structures of ternary complexes of citrate synthase with D- and L-malate: mechanistic implications. Biochemistry 30, 6024–6031.10.1021/bi00238a028Suche in Google Scholar

Krissinel, E. and Henrick, K. (2007). Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797.10.1016/j.jmb.2007.05.022Suche in Google Scholar PubMed

Kurz, L.C., Shah, S., Frieden, C., Nakra, T., Stein, R.E., Drysdale, G.R., Evans, C.T., and Srere, P.A. (1995). Catalytic strategy of citrate synthase: subunit interactions revealed as a consequence of a single amino acid change in the oxaloacetate binding site. Biochemistry 34, 13278–13288.10.1021/bi00041a003Suche in Google Scholar PubMed

Laskowski, R.A., Watson, J.D., and Thornton, J.M. (2005). ProFunc: a server for predicting protein function from 3D structure. Nucleic Acids Res. 33, W89–W93.10.1093/nar/gki414Suche in Google Scholar PubMed PubMed Central

Latge, J.P. (1999). Aspergillus fumigatus and aspergillosis. Clin. Microbiol. Rev. 12, 310–350.10.1128/9781555815523Suche in Google Scholar

Lavinder, J.J., Hari, S.B., Sullivan, B.J., and Magliery, T.J. (2009). High-throughput thermal scanning: a general, rapid dye-binding thermal shift screen for protein engineering. J. Am. Chem. Soc. 131, 3794–3795.10.1021/ja8049063Suche in Google Scholar PubMed PubMed Central

Maerker, C., Rohde, M., Brakhage, A.A., and Brock, M. (2005). Methylcitrate synthase from Aspergillus fumigatus. Propionyl-CoA affects polyketide synthesis, growth and morphology of conidia. FEBS J. 272, 3615–3630.10.1111/j.1742-4658.2005.04784.xSuche in Google Scholar PubMed

Minor, W., Cymborowski, M., Otwinowski, Z., and Chruszcz, M. (2006). HKL-3000: the integration of data reduction and structure solution – from diffraction images to an initial model in minutes. Acta Crystallogr. D: Biol. Crystallogr. 62, 859–866.10.1107/S0907444906019949Suche in Google Scholar PubMed

Murshudov, G.N., Vagin, A.A., and Dodson, E.J. (1997). Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr. D: Biol. Crystallogr. 53, 240–255.10.1107/S0907444996012255Suche in Google Scholar PubMed

Murshudov, G.N., Vagin, A.A., Lebedev, A., Wilson, K.S., and Dodson, E.J. (1999). Efficient anisotropic refinement of macromolecular structures using FFT. Acta Crystallogr. D: Biol. Crystallogr. 55, 247–255.10.1107/S090744499801405XSuche in Google Scholar PubMed

Murshudov, G.N., Skubak, P., Lebedev, A.A., Pannu, N.S., Steiner, R.A., Nicholls, R.A., Winn, M.D., Long, F., and Vagin, A.A. (2011). REFMAC5 for the refinement of macromolecular crystal structures. Acta Crystallogr. D: Biol. Crystallogr. 67, 355–367.10.1107/S0907444911001314Suche in Google Scholar PubMed PubMed Central

Otwinowski, Z. and Minor, W. (1997). Processing of X-ray diffraction data collected in oscillation mode. Methods Enzymol. 276, 307–326.10.1016/S0076-6879(97)76066-XSuche in Google Scholar

Painter, J. and Merritt, E.A. (2006). Optimal description of a protein structure in terms of multiple groups undergoing TLS motion. Acta Crystallogr. D: Biol. Crystallogr. 62, 439–450.10.1107/S0907444906005270Suche in Google Scholar

Pantoliano, M.W., Petrella, E.C., Kwasnoski, J.D., Lobanov, V.S., Myslik, J., Graf, E., Carver, T., Asel, E., Springer, B.A., Lane, P., et al. (2001). High-density miniaturized thermal shift assays as a general strategy for drug discovery. J. Biomol. Screen. 6, 429–440.10.1177/108705710100600609Suche in Google Scholar

Paulussen, C., Hallsworth, J.E., Alvarez-Perez, S., Nierman, W.C., Hamill, P.G., Blain, D., Rediers, H., and Lievens, B. (2017). Ecology of aspergillosis: insights into the pathogenic potency of Aspergillus fumigatus and some other Aspergillus species. Microb. Biotechnol. 10, 296–322.10.1111/1751-7915.12367Suche in Google Scholar

Pettersen, E.F., Goddard, T.D., Huang, C.C., Couch, G.S., Greenblatt, D.M., Meng, E.C., and Ferrin, T.E. (2004). UCSF Chimera--a visualization system for exploratory research and analysis. J. Comput. Chem. 25, 1605–1612.10.1002/jcc.20084Suche in Google Scholar

Remington, S., Wiegand, G., and Huber, R. (1982). Crystallographic refinement and atomic models of two different forms of citrate synthase at 2.7 and 1.7 A resolution. J. Mol. Biol. 158, 111–152.10.1016/0022-2836(82)90452-1Suche in Google Scholar

Robert, X. and Gouet, P. (2014). Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 42, W320–W324.10.1093/nar/gku316Suche in Google Scholar PubMed PubMed Central

Rosenbaum, G., Alkire, R.W., Evans, G., Rotella, F.J., Lazarski, K., Zhang, R.G., Ginell, S.L., Duke, N., Naday, I., Lazarz, J., et al. (2006). The Structural Biology Center 19ID undulator beamline: facility specifications and protein crystallographic results. J. Synchrotron Radiat. 13, 30–45.10.1107/S0909049505036721Suche in Google Scholar PubMed PubMed Central

Schrodinger, L.L.C. (2015). The PyMOL Molecular Graphics System, Version 1.8 (New York, NY: Schrödinger LLC).Suche in Google Scholar

Segal, B.H. (2009). Aspergillosis. N. Engl. J. Med. 360, 1870–1884.10.1056/NEJMra0808853Suche in Google Scholar PubMed

Seiler, C.Y., Park, J.G., Sharma, A., Hunter, P., Surapaneni, P., Sedillo, C., Field, J., Algar, R., Price, A., Steel, J., et al. (2014). DNASU plasmid and PSI: Biology-Materials repositories: resources to accelerate biological research. Nucleic Acids Res. 42, D1253–D1260.10.1093/nar/gkt1060Suche in Google Scholar PubMed PubMed Central

Smitherman, T.C., Mukherjee, A., Robinson, J.B.J., Butsch, R.W., Richards, E.G., and Srere, P.A. (1979). Human heart citrate synthase: purification, properties, kinetic and immunologic studies. J. Mol. Cell. Cardiol. 11, 149–160.10.1016/0022-2828(79)90460-7Suche in Google Scholar

Wang, A.H., Sherman, M.L., and Rich, A. (1978). A crystallographic investigation of citrate synthase from pig and chicken heart muscle. Biochem. Biophys. Res. Commun. 82, 150–156.10.1016/0006-291X(78)90589-2Suche in Google Scholar

Wiegand, G. and Remington, S.J. (1986). Citrate synthase: structure, control, and mechanism. Annu. Rev. Biophys. Biophys. Chem. 15, 97–117.10.1146/annurev.bb.15.060186.000525Suche in Google Scholar

Winn, M.D., Isupov, M.N., and Murshudov, G.N. (2001). Use of TLS parameters to model anisotropic displacements in macromolecular refinement. Acta Crystallogr. D: Biol. Crystallogr. 57, 122–133.10.1107/S0907444900014736Suche in Google Scholar

Winn, M.D., Murshudov, G.N., and Papiz, M.Z. (2003). Macromolecular TLS refinement in REFMAC at moderate resolutions. Methods Enzymol. 374, 300–321.10.1016/S0076-6879(03)74014-2Suche in Google Scholar

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.W., McCoy, A., et al. (2011). Overview of the CCP4 suite and current developments. Acta Crystallogr. D: Biol. Crystallogr. 67, 235–242.10.1107/S0907444910045749Suche in Google Scholar PubMed PubMed Central

Zoldak, G., Sut’ak, R., Antalik, M., Sprinzl, M., and Sedlak, E. (2003). Role of conformational flexibility for enzymatic activity in NADH oxidase from Thermus thermophilus. Eur. J. Biochem. 270, 4887–4897.10.1046/j.1432-1033.2003.03889.xSuche in Google Scholar PubMed

Supplementary Material

The online version of this article offers supplementary material (https://doi.org/10.1515/hsz-2019-0106).

Received: 2019-01-09
Accepted: 2019-05-15
Published Online: 2019-05-29
Published in Print: 2019-12-18

©2019 Walter de Gruyter GmbH, Berlin/Boston

Artikel in diesem Heft

  1. Frontmatter
  2. Review
  3. Recent advances in the development of legumain-selective chemical probes and peptide prodrugs
  4. Genes and Nucleic Acids
  5. Research Articles/Short Communications
  6. Tauroursodeoxycholate protects from glycochenodeoxycholate-induced gene expression changes in perfused rat liver
  7. Protein Structure and Function
  8. Comparative studies of Aspergillus fumigatus 2-methylcitrate synthase and human citrate synthase
  9. Assessment of the denaturation of collagen protein concentrates using different techniques
  10. Membranes, Lipids, Glycobiology
  11. Red blood cells participate in reverse cholesterol transport by mediating cholesterol efflux of high-density lipoprotein and apolipoprotein A-I from THP-1 macrophages
  12. Molecular Medicine
  13. A transgenic zebrafish model of hepatocyte function in human Z α1-antitrypsin deficiency
  14. Cell Biology and Signaling
  15. Geranylgeranyl diphosphate synthase deficiency aggravates lung fibrosis in mice by modulating TGF-β1/BMP-4 signaling
  16. Proteolysis
  17. The lysosomal aminopeptidase tripeptidyl peptidase 1 displays increased activity in malignant pancreatic cysts
https://doi.org/10.1515/hsz-2019-0106
Schlagwörter für diesen Artikel
Aspergillus; coenzyme A; cooperativity; human citrate synthase; 2-methylcitrate synthase; structure

Artikel in diesem Heft

  1. Frontmatter
  2. Review
  3. Recent advances in the development of legumain-selective chemical probes and peptide prodrugs
  4. Genes and Nucleic Acids
  5. Research Articles/Short Communications
  6. Tauroursodeoxycholate protects from glycochenodeoxycholate-induced gene expression changes in perfused rat liver
  7. Protein Structure and Function
  8. Comparative studies of Aspergillus fumigatus 2-methylcitrate synthase and human citrate synthase
  9. Assessment of the denaturation of collagen protein concentrates using different techniques
  10. Membranes, Lipids, Glycobiology
  11. Red blood cells participate in reverse cholesterol transport by mediating cholesterol efflux of high-density lipoprotein and apolipoprotein A-I from THP-1 macrophages
  12. Molecular Medicine
  13. A transgenic zebrafish model of hepatocyte function in human Z α1-antitrypsin deficiency
  14. Cell Biology and Signaling
  15. Geranylgeranyl diphosphate synthase deficiency aggravates lung fibrosis in mice by modulating TGF-β1/BMP-4 signaling
  16. Proteolysis
  17. The lysosomal aminopeptidase tripeptidyl peptidase 1 displays increased activity in malignant pancreatic cysts
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