Skip to main content
SpringerLink
Log in

Cloning, Expression, Purification and Characterization of UMP Kinase from Staphylococcus aureus

  • Published:
The Protein Journal Aims and scope Submit manuscript

Abstract

Uridine monophosphate kinase (UMPK) an enzyme of de novo biosynthesis catalyses the formation of UDP and it is involved in cell wall and RNA biosynthesis. In the present study UMPK of Staphylococcus aureus ATCC12600 was characterized. Analysis of purified UMPK by gel filtration chromatography on Sephadex G-200 indicated a molecular weight of 150 kDa and exhibited monomeric form with molecular weight of 25 kDa in SDS-PAGE confirming homohexamer nature of UMPK in solution. The enzyme kinetics of UMPK showed Km of 2.80 ± 0.1 μM and Vmax 51.38 ± 1.39 μM of NADH/min/mg. The enzyme exhibited cooperative kinetics with ATP as substrate, as GTP decreased this cooperativity and increased affinity for ATP. The UMPK gene was amplified, sequenced (Accession number: FJ415072), cloned in pQE30 vector and overexpressed in Escherichia coli DH5α. The purified recombinant UMPK showed similar properties of native UMPK. The UMPK gene sequence showed complete homology with pyrH gene sequence of all S. aureus strains reported in the database, the 3D structure of S. aureus UMPK built from the deduced amino acid sequence was super imposed with human UMPK (PDB ID: 1TEV) to find out the structural identity using the MATRAS programme gave an RMSD value 4.24 Å indicating very low homology and extensive structural variations with human UMPK structure. Thus, UMPK may be a potential drug target in the development of antimicrobials.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Abbreviations

UMPK:

Uridine monophosphate kinase

RMSD:

Root mean square deviation

MATRAS:

MArkovian TRAnsition of Structure

PDB:

Protein Data Bank

DEAE cellulose:

Diethylaminoethyl cellulose

SDS-PAGE:

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

CMP:

Cytidine monophosphate

UMP:

Uridine monophosphate

DOPE:

Discrete Optimized Protein Energy

CMPK:

Cytidine monophosphate Kinase

UTP:

Uridine triphosphate

LDH:

Lactate dehydrogenase

PK:

Pyruvate kinase

PEP:

Phosphoenolpyruvate

References

  1. Altschul SF, Madden TL, Schäffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Nucleic Acids Res 25(17):3389–3402

    Article  CAS  Google Scholar 

  2. Arias CA, Murray BE (2009) N Engl J Med 360(5):439–443

    Article  CAS  Google Scholar 

  3. Arner ESJ, Eriksson S (1995) Pharmacol Ther 67(2):155–186

    Article  CAS  Google Scholar 

  4. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H, Shindyalov IN, Bourne PE (2000) Nucleic Acids Res 28(1):235–242

    Article  CAS  Google Scholar 

  5. Blondin C, Serina L, Wiesmuller L, Gilles A-M, Baˆrzu O (1994) Anal Biochem 220(1):219–221

    Article  CAS  Google Scholar 

  6. Bradford MM (1976) Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  7. Brown ED (2006) Nature 441:293–294

    Article  CAS  Google Scholar 

  8. Chilom C, Gazdaru D, Bucurenci N, Straut M, Geanta C, Popescu A (2007) J Optoelectron Adv Mater 9(8):2585–2588

    CAS  Google Scholar 

  9. Cui L, Ma X, Sato K, Okuma K, Tenover FC, Mamizuka EM, Gemmell CG, Kim MN, Ploy MC, Solh EN, Ferraz V, Hiramatsu K (2003) J Clin Microbiol 41(1):5–14

    Article  CAS  Google Scholar 

  10. Dalton JAR, Jackson RM (2007) Struct Bioinforma 23(15):1901–1908

    Article  CAS  Google Scholar 

  11. Danishuddin M, Chatrath R, Singh R (2011) Bioinformation 6(4):144–148

    Article  Google Scholar 

  12. Evrin C, Straut M, Slavova-Azmanova N, Bucurenci N, Onu A, Assairi L, Ionescu M, Palibroda N, Baˆrzu O, Gilles A-M (2007) J Biol Chem 282:7242–7253

    Article  CAS  Google Scholar 

  13. Gagyi C, Bucurenci N, Sîrbu O, Labesse G, Ionescu M, Ofiteru A, Assairi L, Landais S, Danchin A, Baˆrzu O, Gilles A-M (2003) Eur J Biochem 270:3196–3240

    Article  CAS  Google Scholar 

  14. Hari Prasad O, Nagarjuna V, Lakshmi Prasanna Ch, Vasu D, Yugandhar VG, Prasad UV, Srinivas Rao PVLN, Chaudhary A, Reddy OVS, Sarma PVGK (2010) J Pure Appl Microbiol 4(2):609–616

    Google Scholar 

  15. Kawabata T (2003) Nucleic Acids Res 31(13):3367–3369

    Article  CAS  Google Scholar 

  16. Kholti A, Charlier D, Gigot D, Huysveld N, Roovers M, Glansdorff N (1998) J Mol Biol 280(4):571–582

    Article  CAS  Google Scholar 

  17. Labesse G, Benkali K, Salard-Arnaud I, Gilles A-M, Munier-Lehmann H (2011) Nucleic Acids Res 39(8):3458–3472

    Article  CAS  Google Scholar 

  18. Laemmli UK (1970) Nature 227:680–685

    Article  CAS  Google Scholar 

  19. Laskowski RA, MacArthur MW, Moss DS, Thornton JM (1993) J Appl Cryst 26:283–291

    Article  CAS  Google Scholar 

  20. Lee VJ, Miller GH, Yagisawa M (1999) Curr Opin Microbiol 2:475–482

    Article  CAS  Google Scholar 

  21. Liljelund P, Lacroute F (1986) Mol Gen Genet 205(1):74–81

    Article  CAS  Google Scholar 

  22. Lowy FD (2003) J Clin Investig 111(9):1265–1273

    CAS  Google Scholar 

  23. Meier C, Carter LG, Sainsbury S, Mancini EJ, Owens RJ, Stuart DI, Esnouf RM (2008) J Mol Biol 381:1098–1105

    Article  CAS  Google Scholar 

  24. Ohta T, Hirakawa H, Morikawa K, Maruyama A, Inose Y, Yamashita A, Oshima K, Kuroda M, Hattori M, Hiramatsu K, Kuhara S, Hayashi H (2004) DNA Res 11:51–56

    Article  CAS  Google Scholar 

  25. Sambrook J, Russell DW (2001) Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory Press, New York, NY

    Google Scholar 

  26. Sandrini MP, Piskur J (2005) Trends Biochem Sci 30:225–228

    Article  CAS  Google Scholar 

  27. Serina L, Blondin C, Krin E, Sismeiro O, Danchin A, Sakamoto H, Gilles A-M, Barzu O (1995) Biochemistry 34:5066–5074

    Article  CAS  Google Scholar 

  28. Stadhouders J, Hassings F, Van Aalsten-Van Maren NO (1976) Netz Milk Diary J 30:222–229

    Google Scholar 

  29. Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG (1997) Nucleic Acids Res 25(24):4876–4882

    Article  CAS  Google Scholar 

  30. Walsh R, Martin E, Darvesh S (2010) Biochim Biophys Acta 1800:1–5

    Article  CAS  Google Scholar 

  31. Louise E-W, Martin W, Liya W, Staffan E (2007) FEBS J 24:6403–6414

    Google Scholar 

  32. Md Zeyaullah, Haque S, Nabi G, Nand KN, Ali A (2010) Afr J Biotechnol 9(25):3714–3718

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Biotechnology, Sri Venkateswara Institute of Medical Sciences, Tirupati, 517 507, Andhra Pradesh, India

    O. Hari Prasad, Y. Nanda Kumar & P. V. G. K. Sarma

  2. Department of Biochemistry, Sri Venkateswara University, Tirupati, 517 502, Andhra Pradesh, India

    O. V. S. Reddy

  3. Department of Microbiology, Sri Venkateswara Institute of Medical Sciences, Tirupati, 517 507, Andhra Pradesh, India

    Abhijit Chaudhary

Authors
  1. O. Hari Prasad
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Y. Nanda Kumar
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. O. V. S. Reddy
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Abhijit Chaudhary
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. P. V. G. K. Sarma
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to P. V. G. K. Sarma.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Hari Prasad, O., Nanda Kumar, Y., Reddy, O.V.S. et al. Cloning, Expression, Purification and Characterization of UMP Kinase from Staphylococcus aureus . Protein J 31, 345–352 (2012). https://doi.org/10.1007/s10930-012-9410-0

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s10930-012-9410-0

Keywords

Search

Navigation