Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Scientific Correspondence
  • Published:

Penicillin V acylase crystal structure reveals new Ntn-hydrolase family members

Nature Structural Biology volume 6, pages 414–416 (1999)Cite this article

Two enzyme types, penicillin V acylases (PVA) and penicillin G acylases (PGA), with distinct substrate preferences, account for all the enzymic industrial production of 6-aminopenicillanic acid1,2. This β-lactam compound is then elaborated into a range of semi-synthetic penicillins. Although their industrial substrates are very similar, representative examples of the two enzyme types differ widely in molecular properties. PVA from Bacillus sphaericus is tetrameric with a monomer Mr of 35,000 while PGA from Escherichia coli is a heterodimer of Mr 90,000. Furthermore, they have no detectable sequence homology. These differences, which exist in spite of the similarity of their industrial substrates, provoked us to determine the crystal structure of PVA to establish the nature of its catalytic mechanism and to identify any biochemical and structural relationships with PGA and other Ntn (N-terminal nucleophile) hydrolases.

The PVA and PGA enzymes thus share a distinctive structural core but are otherwise unrelated in primary sequence, including the active site residue. Both PGA and PVA have approximately the same angle (+30°) between the β-strands of the two β-sheets, which are decorated by the active site residues in Ntn hydrolases. Using these β-sheets for structural alignment reveals that the catalytic regions of PVA and PGA overlap (Fig. 1 c) with a root mean square (r.m.s.) deviation of 0.46 Å for the catalytic atoms. The oxyanion hole in PVA consists of the Nδ2 of Asn 175 and the NH of Tyr 82, corresponding to the Nδ2 of Asn B241 and NH of Ala B69 in PGA. Other common interactions at the active site include those associated with the arginine (residue 228 in PVA and B263 in PGA), and the oxygens of Asp 20 and Asn 175 in PVA and Gln B23 and Asn B241 in PGA, which are critical for positioning the lone pair of the unprotonated N-terminal α-amino group. In spite of these common features, the pH profiles for optimum catalysis by PGA and PVA are quite different2. That for PGA is broad (pH 7–9) while it is sharper for PVA, centering on pH 5.5.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: a,The PVA tetramer showing the main chain as ribbons.

Accession codes

Accessions

Protein Data Bank

References

  1. Shewale, J.G. & SivaRaman, H. Process Biochem. 24 , 146–154 (1989).

    CAS  Google Scholar 

  2. Shewale, J.G. & Sudhakaran, V.K. Enzyme Microb. Technol. 20, 402–410 (1997).

    Article  CAS  Google Scholar 

  3. Duggleby, H.J. et al. Nature 373, 264–268 (1995).

    Article  CAS  Google Scholar 

  4. Brannigan, J.A. et al. Nature 378, 416–419 (1995).

    Article  CAS  Google Scholar 

  5. Olsson, A. & Uhlen, M. Gene 45, 175 –181 (1986).

    Article  CAS  Google Scholar 

  6. Artymiuk, P.J. Nature Struct. Biol. 2, 1035–1037 (1995).

    Article  CAS  Google Scholar 

  7. Lowe, J. et al. Science 268, 533–539 (1995).

    Article  CAS  Google Scholar 

  8. Groll, M. et al. Nature 386, 463–471 (1997).

    Article  CAS  Google Scholar 

  9. Oinonen, C., Tikkanen, R., Rouvinen, J. & Peltonen, L. Nature Struct. Biol. 2, 1102–1108 (1995).

    Article  CAS  Google Scholar 

  10. Smith, J.L. et al. Science 264, 1427–1433 (1994).

    CAS  Google Scholar 

  11. Tikkanen, R., Riikonen, A., Oinonen, J. & Peltonen, L. EMBO J. 15, 2954–2960 ( 1996).

    Article  CAS  Google Scholar 

  12. Guan, C. et al. J. Biol. Chem. 271, 1732– 1737 (1996).

    Article  CAS  Google Scholar 

  13. Brannigan, J.A. & Dodson, G.G. Nature Struct. Biol. 4, 334–337 ( 1997).

    Article  CAS  Google Scholar 

  14. Seemuller, E., Lupas, A. & Baumeister, W. Nature 382, 468– 470 (1996).

    Article  CAS  Google Scholar 

  15. Aronson, N.N. Glycobiology 6, 669–675 (1996).

    Article  CAS  Google Scholar 

  16. Christiaens, H., Leer, R.J., Pouwels, P.H. & Verstraete, W. Appl. Environ. Microbiol. 57, 3792– 3798 (1992).

    Google Scholar 

  17. Isupov, M.N. et al. Structure 4, 801– 810 (1996).

    Article  CAS  Google Scholar 

  18. Kim, J.H., Krahn, J.M., Tomchick, D.R., Smith, J.L. & Zalkin, H. J. Biol. Chem. 271, 15549–15557 (1996).

    Article  CAS  Google Scholar 

  19. Burton, H.S. & Abraham, E.P. Biochem. J. 50, 168–174 (1951).

    Article  CAS  Google Scholar 

  20. Pundale, A. & SivaRaman, H. Current Microbiol. 34, 144–148 (1997).

    Article  Google Scholar 

  21. Otwinowski, Z. & Minor, W. Meth. Enz. 276, 307–326 (1997).

    Article  CAS  Google Scholar 

  22. Collaborative Computational Project No. 4. Acta Crystallogr. D 50, 760–763 (1994).

  23. Otwinowski, Z. Proceedings of the CCP4 Study Weekend 80–86 (1991).

    Google Scholar 

  24. Cowtan, K. Joint CCP4 and ESF–EACBM Newsletter on protein crystallography 31, 34–38 (1994).

    Google Scholar 

  25. QUANTA96 X–ray structure analysis user's reference. (Molecular Simulations, San Diego; 1996).

  26. Lamzin, V.S. & Wilson, K.S. Acta. Crystallogr. D 49, 129–147 (1993).

    Article  CAS  Google Scholar 

  27. Murshudov, G.N., Vagin, A.A. & Dodson, E.J. Acta Crystallogr. D 53, 240 –255 (1997).

    Article  CAS  Google Scholar 

  28. Brunger, A.T. Nature 355, 472–475 ( 1992).

    Article  CAS  Google Scholar 

  29. Stehle, T., Ahmed, S.A., Claiborne, A. & Schulz, G.E. J. Mol. Biol. 221, 1325–1344 (1991).

    CAS  PubMed  Google Scholar 

  30. Navaza, J. Acta Crystallogr. A 50, 157–163 (1994).

    Article  Google Scholar 

Download references

Acknowledgements

We would like to thank G. Murshudov, A. Anston and C. SivaRaman for helpful discussions, the CLRC at Daresbury for synchrotron facilities and M. Vijayan for data collection facilities at the Indian Institute of Science in Bangalore. The British Council Higher Education Link Programme and a travel grant from Glaxo–Wellcome is acknowledged for the support that began the research. The BBSRC provided support through the Rolling Project Mode Time allocation to York. JAB and CSV are funded by the York Structural Biology Centre.

Author information

Authors and Affiliations

  1. Division of Biochemical Sciences, National Chemical Laboratory, Pune, 411008, India

    C.G. Suresh, A.V. Pundle, H. SivaRaman & K.N. Rao

  2. Chemistry Department, University of York, York, YO10 5DD, UK

    J.A. Brannigan, C.E. McVey, C.S. Verma, Z. Dauter, E.J. Dodson & G.G. Dodson

Authors
  1. C.G. Suresh
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. A.V. Pundle
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. H. SivaRaman
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. K.N. Rao
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. J.A. Brannigan
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. C.E. McVey
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. C.S. Verma
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Z. Dauter
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. E.J. Dodson
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. G.G. Dodson
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding authors

Correspondence to C.G. Suresh or J.A. Brannigan.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Suresh, C., Pundle, A., SivaRaman, H. et al. Penicillin V acylase crystal structure reveals new Ntn-hydrolase family members. Nat Struct Mol Biol 6, 414–416 (1999). https://doi.org/10.1038/8213

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1038/8213

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing