Skip to main content
SpringerLink
Log in

The dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase from Haemophilus influenzae contains two active-site histidine residues

  • Original Paper
  • Published:
JBIC Journal of Biological Inorganic Chemistry Aims and scope Submit manuscript

Abstract

The catalytic and structural properties of the H67A and H349A dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase (DapE) from Haemophilus influenzae were investigated. On the basis of sequence alignment with the carboxypeptidase from Pseudomonas sp. strain RS-16, both H67 and H349 were predicted to be Zn(II) ligands. The H67A DapE enzyme exhibited a decreased catalytic efficiency (180-fold) compared with wild-type (WT) DapE towards N-succinyldiaminopimelic acid. No catalytic activity was observed for H349A under the experimental conditions used. The electronic paramagnetic resonance (EPR) and electronic absorption data indicate that the Co(II) ion bound to H349A-DapE is analogous to that of WT DapE after the addition of a single Co(II) ion. The addition of 1 equiv of Co(II) to H67A DapE provides spectra that are very different from those of the first Co(II) binding site of the WT enzyme, but that are similar to those of the second binding site. The EPR and electronic absorption data, in conjunction with the kinetic data, are consistent with the assignment of H67 and H349 as active-site metal ligands for the DapE from H. influenzae. Furthermore, the data suggest that H67 is a ligand in the first metal binding site, while H349 resides in the second metal binding site. A three-dimensional homology structure of the DapE from H. influenzae was generated using the X-ray crystal structure of the DapE from Neisseria meningitidis as a template and superimposed on the structure of the aminopeptidase from Aeromonas proteolytica (AAP). This homology structure confirms the assignment of H67 and H349 as active-site ligands. The superimposition of the homology model of DapE with the dizinc(II) structure of AAP indicates that within 4.0 Å of the Zn(II) binding sites of AAP all of the amino acid residues of DapE are nearly identical.

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

Scheme 1
Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Similar content being viewed by others

Abbreviations

AAP:

Aminopeptidase from Aeromonas proteolytica

CPG2 :

Carboxypeptidase from Pseudomonas sp. strain RS-16

DapE:

dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase

EPR:

Electron paramagnetic resonance

HEPES:

4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid

ICP-AES:

Inductively coupled plasma atomic emission spectrometry

mDAP:

meso-Diaminopimelate

l,l-SDAP:

N-Succinyl-l,l-diaminopimelic acid

WT:

Wild type

References

  1. Scapin G, Blanchard JS (1998) Enzymology of bacterial lysine biosyntesis. Adv Enzymol 72:279–325

    PubMed  CAS  Google Scholar 

  2. Born TL, Blanchard JS (1999) Structure/function studies on enzymes in the diaminopimelate pathway of bacterial cell wall synthesis. Curr Opin Chem Biol 3:607–613

    Article  PubMed  CAS  Google Scholar 

  3. Born TL, Zheng R, Blanchard JS (1998) Hydrolysis of N-succinyl-L,-Ldiaminopimelic acid by the Haemophilus influenzae dapE-encoded desuccinylase: metal activation, solvent isotope effects, and kinetic mechanism. Biochemistry 37:10478–10487

    Article  PubMed  CAS  Google Scholar 

  4. Levy SB (1998) The challenge of antibiotic resistance. Sci Am 278:46–53

    Article  PubMed  CAS  Google Scholar 

  5. Karita M, Etterbeek ML, Forsyth MH, Tummuru MR, Blaser MJ (1997) Characterization of Helicobacter pylori dapE and construction of a conditionally lethal dapE mutant. Infect Immun 65:4158–4164

    PubMed  CAS  Google Scholar 

  6. Pavelka MS, Jacobs WR (1996) Biosynthesis of diaminopimelate, the precursor of lysine and a component of peptidoglycan, is an essential function of Mycobacterium smegmatis. J Bacteriol 178:6496–6507

    PubMed  CAS  Google Scholar 

  7. Velasco AM, Leguina JI, Lazcano A (2002) Molecular evolution of the lysine biosynthetic pathways. J Mol Evol 55:445–459

    Article  PubMed  CAS  Google Scholar 

  8. Bouvier J, Richaud C, Higgins W, Bögler O, Stragier P (1992) Cloning, characterization, and expression of the dapE gene of Escherichia coli. J Bacteriol 174:5265–5271

    PubMed  CAS  Google Scholar 

  9. Bienvenue DL, Gilner DM, Davis RS, Bennett B, Holz RC (2003) Substrate specificity, metal binding properties, and spectroscopic characterization of the dapE-encoded-N-succinyl-L, L-diaminopimelic acid desuccinylase from haemophilus influenzae. Biochemistry 42:10756–10763

    Article  PubMed  CAS  Google Scholar 

  10. Badger J, Sauder JM, Adams JM, Antonysamy S, Bain K, Bergseid MG, Buchanan SG, Buchanan MD, Batiyenko Y, Christopher JA, Emtage S, Eroshkina A, Feil I, Furlong EB, Gajiwala KS, Gao X, He D, Hendle J, Huber A, Hoda K, Kearins P, Kissinger C, Laubert B, Lewis HA, Lin J, Loomis K, Lorimer D, Louie G, Maletic M, Marsh CD, Miller I, Molinari J, Muller-Dieckmann HJ, Newman JM, Noland BW, Pagarigan B, Park F, Peat TS, Post KW, Radojicic S, Ramos A, Romero R, Rutter ME, Sanderson WE, Schwinn KD, Tresser J, Winhoven J, Wright TA, Wu L, Xu J, Harris TJ (2005) Structural analysis of a set of proteins resulting from a bacterial genomics project. Proteins 60:787–796

    Article  PubMed  CAS  Google Scholar 

  11. Chevrier B, Schalk C, D’Orchymont H, Rondeau J-M, Moras D, Tarnus C (1994) Crystal structure of Aeromonas proteolytica aminopeptidase: a prototypical member of the co-catalytic zinc enzyme family. Structure 2:283–291

    Article  PubMed  CAS  Google Scholar 

  12. Rowsell S, Pauptit RA, Tucker AD, Melton RG, Blow DM, Brick P (1997) Crystal structure of carboxypeptidase G2, a bacterial enzyme with applications in cancer therapy. Structure 5:337–347

    Article  PubMed  CAS  Google Scholar 

  13. Cosper NJ, Bienvenue DL, Shokes J, Gilner DM, Tsukamoto T, Scott RA, Holz RC (2004) The dapE-encoded N-succinyl-L, L-diaminopimelic acid desuccinylase from haemophilus influenzae is a dinuclear metallohydrolase. J Am Chem Soc 125:14654–14655

    Article  CAS  Google Scholar 

  14. Davis R, Bienvenue D, Swierczek SI, Gilner DM, Rajagopal L, Bennett B, Holz RC (2006) Kinetic and spectroscopic characterization of the E134A- and E134D-altered dapE-encoded N-succinyl-L, L-diaminopimelic acid desuccinylase from Haemophilus influenzae. J Biol Inorg Chem 11:206–216

    Article  PubMed  CAS  Google Scholar 

  15. Makarova KS, Grishin NV (1999) The Zn-peptidase superfamily: functional convergence after evolutionary divergence. J Mol Biol 292:11–17

    Article  PubMed  CAS  Google Scholar 

  16. Bergmann M, Stein WH (1939) Naphthalene-β-sulfonic acid as a reagent for amino acids. J Biol Chem 129:609–618

    CAS  Google Scholar 

  17. Lin Y, Myhrman R, Schrag ML, Gelb MH (1988) Bacterial N-succinyl-L-diaminopimelic acid desuccinylase. Purification, partial characterization, and substrate specificity. J Biol Chem 263:1622–1627

    PubMed  CAS  Google Scholar 

  18. D’souza VM, Bennett B, Copik AJ, Holz RC (2000) Characterization of the divalent metal binding properties of the methionyl aminopeptidase from Escherichia coli. Biochemistry 39:3817–3826

    Article  PubMed  CAS  Google Scholar 

  19. Winzor DJ, Sawyer WH (1995) Quantitative characterization of ligand binding. Wiley-Liss, New York

    Google Scholar 

  20. Eswar N, John B, Mirkovic N, Fiser A, Ilyin VA, Pieper U, Stuart AC, Marti-Renom MA, Madhusudhan MS, Yerkovich B, Sali A (2003) Tools for comparative protein structure modeling and analysis. Nucleic Acids Res 31:3375–3380

    Article  PubMed  CAS  Google Scholar 

  21. Krissinel E, Henrick K (2004) Secondary-structure matching (SSM), a new tool for fast protein structure alignment in three dimensions. Acta Crystallogr D Biol Crystallogr 60:2256–2268

    Article  PubMed  CAS  Google Scholar 

  22. Prescott JM, Wilkes SH (1976) Aeromonas aminopeptidase. Methods Enzymol 45:530–543

    Article  PubMed  CAS  Google Scholar 

  23. Fleminger G, Yaron A (1984) Soluble and immobilized clostridial aminopeptidase and aminopeptidase P as metal-requiring enzymes. Biochim Biophys Acta 789:245–256

    PubMed  CAS  Google Scholar 

  24. de Seny D, Heinz U, Wommer S, Kiefer M, Meyer-Klaucke W, Galleni M, Frere J-M, Bauer R, Adolph H-W (2001) Metal ion binding and coordination geometry for wild-type and mutants of the metallo-β-lactamase from Bacillus cereus 569/H/9 (BcII). J Biol Chem 276:45065–45078

    Article  PubMed  CAS  Google Scholar 

  25. Holz RC (2002) The aminopeptidase from aeromonas proteolytica: structure and mechanism of co-catalytic metal centers involved in peptide hydrolysis. Coord Chem Rev 232:5–26

    Article  CAS  Google Scholar 

  26. Bertini I, Luchinat C (1984) High-spin cobalt(II) as a probe for the investigation of metalloproteins. Adv Inorg Biochem 6:71–111

    PubMed  CAS  Google Scholar 

  27. Horrocks WD Jr, Ishley JN, Holmquist B, Thompson JS (1980) Structural and electronic mimics of the active site of cobalt(II)-substituted zinc metalloenzymes. J Inorg Biochem 12:131–141

    Google Scholar 

  28. Huntington KM, Bienvenue D, Wei Y, Bennett B, Holz RC, Pei D (1999) Slow-binding inhibition of the aminopeptidase from Aeromonas proteolytica by peptide thiols: synthesis and spectral characterization. Biochemistry 38:15587–15596

    Article  PubMed  CAS  Google Scholar 

  29. Crawford PA, Yang K-W, Sharma N, Bennett B, Crowder MW (2005) Spectroscopic studies on cobalt(II)-substituted metallo-β-lactamase ImiS from Aeromonas veronii bv. sobria. Biochemistry 44:5168–5176

    Article  PubMed  CAS  Google Scholar 

  30. DePaola C, Bennett B, Holz RC, Ringe D, Petsko G (1999) 1-Butaneboronic acid binding to Aeromonas proteolytica aminopeptidase: a case of arrested development. Biochemistry 38:9048–9053

    Article  CAS  Google Scholar 

  31. Holm RH, Kennepohl P, Solomon EI (1996) Structural and functional aspects of metal sites in biology. Chem Rev 96:2239–2314

    Article  PubMed  CAS  Google Scholar 

  32. Lowther WT, Matthews BW (2002) Metalloaminopeptidases: common functional themes in disparate structural surroundings. Chem Rev 102:4581–4607

    Article  PubMed  CAS  Google Scholar 

  33. Gill SC, von Hippel PH (1989) Calculation of protein extinction coefficients from amino acid sequence data. Anal Biochem 182:319–326

    Google Scholar 

Download references

Acknowledgments

This work was supported by the National Science Foundation (CHE- 0652981, RCH) and the National Institutes of Health (AI056321, RR001980 BB). The Bruker ESP-300E EPR spectrometer was purchased with funds provided by the National Science Foundation (BIR-9413530).

Author information

Authors and Affiliations

  1. Department of Chemistry, Loyola University Chicago, 1068 W Sheridan Rd, Chicago, IL, 60626, USA

    Danuta M. Gillner, David L. Bienvenue, Vincentos Zachary & Richard C. Holz

  2. Department of Chemistry, Silesian University of Technology, 44-100, Gliwice, Poland

    Danuta M. Gillner

  3. The Midwest Center for Structural Genomics, Argonne National Laboratory, Argonne, IL, 60439, USA

    Boguslaw P. Nocek & Andrzej Joachimiak

  4. The National Biomedical EPR Center, Biophysics Research Institute, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI, 53226, USA

    Brian Bennett

Authors
  1. Danuta M. Gillner
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. David L. Bienvenue
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Boguslaw P. Nocek
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Andrzej Joachimiak
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Vincentos Zachary
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Brian Bennett
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Richard C. Holz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Richard C. Holz.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gillner, D.M., Bienvenue, D.L., Nocek, B.P. et al. The dapE-encoded N-succinyl-l,l-diaminopimelic acid desuccinylase from Haemophilus influenzae contains two active-site histidine residues. J Biol Inorg Chem 14, 1–10 (2009). https://doi.org/10.1007/s00775-008-0418-z

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00775-008-0418-z

Keywords

Search

Navigation