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Characterization of the Dihydroorotase from Methanococcus jannaschii

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Abstract

The gene that codes for the putative dihydroorotase in the hyperthermophilic archaeon Methanococcus jannaschii was subcloned in pET-21a and expressed in Escherichia coli. A purification protocol was devised. The purity of the protein was evaluated by SDS-PAGE and the protein was confirmed by sequencing using LC–MS. The calculated molecular mass is 48104 Da. SEC-LS suggested that the protein is a monomer in solution. ICP-MS showed that there are two Zn ions per monomer. Kinetic analysis of the recombinant protein gave hyperbolic kinetics with Vmax = 12.2 µmol/min/mg and Km = 0.14 mM at 25 °C. Furthermore the activity of the protein increased with temperature consistent with the hyperthermophilic nature of the organism. A homology model was constructed using the mesophilic Bacillus anthracis protein as the template. Residues known to be critical for Zn and substrate binding were conserved. The activity of the enzyme at 85 and 90 °C was found to be relatively constant over 160 min and this correlates with the temperature of optimal growth of the organism of 85 °C. The amino acid sequences and structures of the two proteins were compared and this gave insight into some of the factors that may confer thermostability—more Lys and Ile, fewer Ala, Thr, Gln and Gly residues, and shorter N- and C-termini. Additional and better insight into the thermostabilization strategies adopted by this enzyme will be provided when its crystal structure is determined.

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Abbreviations

A. aeolicus :

Aquifex aeolicus

ATCase:

Aspartate transcarbamoylase

B. anthracis, Ba :

Bacillus anthracis

BME:

2-Mercaptoethanol

BSA:

Bovine serum albumin

CA:

Carbamoyl aspartate

CAD:

Carbamoyl phosphate synthetase/aspartate transcarbamoylase/dihydroorotase protein

CID:

Collision induced dissociation

DHO:

Dihydroorotate

DHOase:

Dihydroorotase

E. coli, Ec :

Escherichia coli

IPTG:

Isopropyl β-d-1-thiogalactopyranoside

LB:

Luria–Bertani medium

MES:

2-(N-morpholino)ethanesulfonic acid

M. jannaschii, Mj :

Methanococcus jannaschii

SRM:

Selective reaction monitoring

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

S. aureus :

Staphylococcus aureus

T. thermophilus :

Thermus thermophilus

Tris:

Tris(hydroxymethyl)aminomethane

References

  1. Holm L, Sander C (1997) An evolutionary treasure: unification of a broad set of amidohydrolases related to urease. Proteins 28:72–82

    Article  CAS  Google Scholar 

  2. Fields C, Brichta D, Shepherdson M, Farinha M, O’Donovan G (1999) Phylogenetic analysis and classification of dihydroorotases: a complex history for a complex enzyme. Paths Pyrimidines 7:49–63

    Google Scholar 

  3. Grande-García A, Lallous N, Díaz-Tejada C, Ramón-Maiques S (2014) Structure, functional characterization, and evolution of the dihydroorotase domain of human CAD. Structure 22:185–198

    Article  Google Scholar 

  4. Kim GJ, Kim HS (1998) Identification of the structural similarity in the functionally related amidohydrolases acting on the cyclic amide ring. Biochem J 330(Pt 1):295–302

    Article  CAS  Google Scholar 

  5. Thoden JB, Phillips GN Jr, Neal TM, Raushel FM, Holden HM (2001) Molecular structure of dihydroorotase: a paradigm for catalysis through the use of a binuclear metal center. Biochemistry 40:6989–6997

    Article  CAS  Google Scholar 

  6. Porter TN, Li Y, Raushel FM (2004) Mechanism of the dihydroorotase reaction. Biochemistry 43:16285–16292

    Article  CAS  Google Scholar 

  7. Rice AJ, Lei H, Santarsiero BD, Lee H, Johnson ME (2016) Ca-asp bound X-ray structure and inhibition of Bacillus anthracis dihydroorotase (DHOase). Bioorg Med Chem 24:4536–4543

    Article  CAS  Google Scholar 

  8. Edwards BF, Fernando R, Martin PD, Grimley E, Cordes M, Vaishnav A, Brunzelle JS, Evans HG, Evans DR (2013) The mononuclear metal center of type-I dihydroorotase from Aquifex aeolicus. BMC Biochem 14:1

    Article  Google Scholar 

  9. Prescott LM, Jones ME (1969) Modified methods for the determination of carbamyl aspartate. Anal Biochem 32:408–419

    Article  CAS  Google Scholar 

  10. Folta-Stogniew E, Williams KR (1999) Determination of molecular masses of proteins in solution: implementation of an HPLC size exclusion chromatography and laser light scattering service in a core laboratory. J Biomol Tech 10:51–63

    CAS  Google Scholar 

  11. Yang J, Yan R, Roy A, Xu D, Poisson J, Zhang Y (2015) The I-TASSER suite: protein structure and function prediction. Nat Methods 12:7–8

    Article  CAS  Google Scholar 

  12. Yang J, Zhang Y (2015) I-TASSER server: new development for protein structure and function predictions. Nucleic Acids Res 43:W174–W181

    Article  CAS  Google Scholar 

  13. Zhang Y (2008) I-TASSER server for protein 3D structure prediction. BMC Bioinform 9:40-2105-9-40

    Google Scholar 

  14. Roy A, Kucukural A, Zhang Y (2010) I-TASSER: a unified platform for automated protein structure and function prediction. Nat Protoc 5:725–738

    Article  CAS  Google Scholar 

  15. Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) UCSF Chimera—a visualization system for exploratory research and analysis. J Comput Chem 25:1605–1612

    Article  CAS  Google Scholar 

  16. Sali A, Blundell T (1994) Comparative protein modelling by satisfaction of spatial restraints. Protein Struct Distance Anal 64:C86

    Google Scholar 

  17. Maier JA, Martinez C, Kasavajhala K, Wickstrom L, Hauser KE, Simmerling C (2015) ff14SB: improving the accuracy of protein side chain and backbone parameters from ff99SB. J Chem Theory Comput 11:3696–3713

    Article  CAS  Google Scholar 

  18. Wang J, Wang W, Kollman PA, Case DA (2006) Automatic atom type and bond type perception in molecular mechanical calculations. J Mol Graph Model 25:247–260

    Article  Google Scholar 

  19. Laskowski RA, Rullmann JAC, MacArthur MW, Kaptein R, Thornton JM (1996) AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR 8:477–486

    Article  CAS  Google Scholar 

  20. Bult CJ, White O, Olsen GJ, Zhou L (1996) Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii. Science 273:1058

    Article  CAS  Google Scholar 

  21. Edgar RC (2004) MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 32:1792–1797

    Article  CAS  Google Scholar 

  22. Jones W, Leigh JA, Mayer F, Woese C, Wolfe R (1983) Methanococcus jannaschii sp. nov., an extremely thermophilic methanogen from a submarine hydrothermal vent. Arch Microbiol 136:254–261

    Article  CAS  Google Scholar 

  23. Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins MR, Appel RD, Bairoch A (2005) Protein identification and analysis tools on the ExPASy server. In: Walker JM (ed) The proteomics handbook. Humana Press, Totowa, pp 571–607

    Chapter  Google Scholar 

  24. Zhang P, Martin PD, Purcarea C, Vaishnav A, Brunzelle JS, Fernando R, Guy-Evans HI, Evans DR, Edwards BF (2009) Dihydroorotase from the hyperthermophile Aquifiex aeolicus is activated by stoichiometric association with aspartate transcarbamoylase and forms a one-pot reactor for pyrimidine biosynthesis. Biochemistry 48:766–778

    Article  CAS  Google Scholar 

  25. Kankanala R (2011) Characterizing the oligomeric structure and catalytic activity of the dihydroorotase and aspartate transcarbamoylase from the bacterium, Bacillus anthracis. Master’s Theses and Doctoral Dissertations. Paper 339. http://commons.emich.edu/theses/339

  26. Hack ES, Vorobyova T, Sakash JB, West JM, Macol CP, Herve G, Williams MK, Kantrowitz ER (2000) Characterization of the aspartate transcarbamoylase from Methanococcus jannaschii. J Biol Chem 275:15820–15827

    Article  CAS  Google Scholar 

  27. Martin PD, Purcarea C, Zhang P, Vaishnav A, Sadecki S, Guy-Evans HI, Evans DR, Edwards BF (2005) The crystal structure of a novel, latent dihydroorotase from Aquifex aeolicus at 1.7 Å resolution. J Mol Biol 348:535–547

    Article  CAS  Google Scholar 

  28. Washabaugh MW, Collins KD (1984) Dihydroorotase from Escherichia coli. Purification and characterization. J Biol Chem 259:3293–3298

    CAS  Google Scholar 

  29. Zimmermann BH, Evans DR (1993) Cloning, overexpression, and characterization of the functional dihydroorotase domain of the mammalian multifunctional protein CAD. Biochemistry 32:1519–1527

    Article  CAS  Google Scholar 

  30. Kelly RE, Mally MI, Evans DR (1986) The dihydroorotase domain of the multifunctional protein CAD. Subunit structure, zinc content, and kinetics. J Biol Chem 261:6073–6083

    CAS  Google Scholar 

  31. Wang C, Tsau H, Chen W, Huang C (2010) Identification and characterization of a putative dihydroorotase, KPN01074, from Klebsiella pneumoniae. Protein J 29:445–452

    Article  CAS  Google Scholar 

  32. Ho Y, Huang Y, Huang C (2013) Chemical rescue of the post-translationally carboxylated lysine mutant of allantoinase and dihydroorotase by metal ions and short-chain carboxylic acids. Amino Acids 44:1181–1191

    Article  CAS  Google Scholar 

  33. Fields PA (2001) Review: protein function at thermal extremes: balancing stability and flexibility. Comp Biochem Physiol A 129:417–431

    Article  CAS  Google Scholar 

  34. Feller G (2010) Topical review: protein stability and enzyme activity at extreme biological temperatures. J Phys Condens Matter 22:323101

    Article  Google Scholar 

  35. Elias M, Wieczorek G, Rosenne S, Tawfik DS (2014) The universality of enzymatic rate–temperature dependency. Trends Biochem Sci 39:1–7

    Article  CAS  Google Scholar 

  36. Huang DT, Kaplan J, Menz RI, Katis VL, Wake RG, Zhao F, Wolfenden R, Christopherson RI (2006) Thermodynamic analysis of catalysis by the dihydroorotases from hamster and Bacillus caldolyticus, as compared with the uncatalyzed reaction. Biochemistry 45:8275–8283

    Article  CAS  Google Scholar 

  37. Vieille C, Zeikus GJ (2001) Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability. Microbiol Mol Biol Rev 65:1–43

    Article  CAS  Google Scholar 

  38. Petsko GA (2001) Structural basis of thermostability in hyperthermophilic proteins, or ‘there’s more than one way to skin a cat’. Methods Enzymol 334:469–478

    Article  CAS  Google Scholar 

  39. Ding Y, Cai Y, Han Y, Zhao B (2012) Comparison of the structural basis for thermal stability between archaeal and bacterial proteins. Extremophiles 16:67–78

    Article  CAS  Google Scholar 

  40. Mizuguchi K, Sele M, Cubellis MV (2007) Environment specific substitution tables for thermophilic proteins. BMC Bioinform 8(Suppl 1):S15

    Article  Google Scholar 

  41. Saelensminde G, Halskau Ø, Helland R, Willassen NP, Jonassen I (2007) Structure-dependent relationships between growth temperature of prokaryotes and the amino acid frequency in their proteins. Extremophiles 11:585–596

    Article  CAS  Google Scholar 

  42. Vitali J, Colaneri MJ, Kantrowitz E (2008) Crystal structure of the catalytic trimer of Methanococcus jannaschii aspartate transcarbamoylase. Proteins 71:1324–1334

    Article  CAS  Google Scholar 

Download references

Acknowledgements

This work was supported in part by grant GM071512 (JV) from the National Institutes of Health. Molecular graphics and analyses were performed with the UCSF Chimera package. Chimera is developed by the Resource for Biocomputing, Visualization, and Informatics at the University of California, San Francisco (supported by NIGMS P41-GM103311). We thank Dr. Belinda Willard of the Lerner Research Institute in Cleveland Clinic for the LC–MS, Mr. Michael Murphy of Intertek Chemicals and Pharmaceuticals for the ICP-MS and Dr. Ewa Folta-Stogniew of the Biophysics Resource of the Keck Facility at Yale for the SEC-LS. The SEC-LS/UV/RI instrumentation was supported by NIH Award Number 1S10RR023748-01. We also thank Dr. Bin Su of Cleveland State University for use of his SPECTRAmax PLUS 384 microplate reader and Drs. Barbara Zimmermann of Los Andes University and Evan Kantrowitz of Boston College for enlightening discussions.

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Author notes

  1. Aditya K. Singh

    Present address: Department of Pharmacology and Toxicology, University of Texas Medical Branch, Galveston, TX, 77555, USA

Authors and Affiliations

  1. Department of Physics, Cleveland State University, Cleveland, OH, 44115, USA

    Jacqueline Vitali & Aditya K. Singh

  2. Department of Biology, Geology and Environmental Sciences, Cleveland State University, Cleveland, OH, 44115, USA

    Jacqueline Vitali

  3. Department of Chemistry and Physics, State University of New York at Old Westbury, Old Westbury, NY, 11568, USA

    Michael J. Colaneri

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  1. Jacqueline Vitali
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Correspondence to Jacqueline Vitali.

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Vitali, J., Singh, A.K. & Colaneri, M.J. Characterization of the Dihydroorotase from Methanococcus jannaschii . Protein J 36, 361–373 (2017). https://doi.org/10.1007/s10930-017-9729-7

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