Amino Acids

, Volume 42, Issue 6, pp 2283–2291 | Cite as

δ1-Pyrroline-5-carboxylate reductase as a new target for therapeutics: inhibition of the enzyme from Streptococcus pyogenes and effects in vivo

  • Giuseppe Forlani
  • Davide Petrollino
  • Massimo Fusetti
  • Letizia Romanini
  • Bogusław Nocek
  • Andrzej Joachimiak
  • Łukasz Berlicki
  • Paweł Kafarski
Original Article


Compounds able to interfere with amino acid biosynthesis have the potential to inhibit cell growth. In both prokaryotic and eukaryotic microorganisms, unless an ornithine cyclodeaminase is present, the activity of δ1-pyrroline-5-carboxylate (P5C) reductase is mandatory to proline production, and the enzyme inhibition should result in amino acid starvation, blocking in turn protein synthesis. The ability of some substituted derivatives of aminomethylenebisphosphonic acid and its analogues to interfere with the activity of the enzyme from the human pathogen Streptococcus pyogenes was investigated. Several compounds were able to suppress activity in the micromolar range of concentrations, with a mechanism of uncompetitive type with respect to the substrate P5C and non-competitive with respect to the electron donor NAD(P)H. The actual occurrence of enzyme inhibition in vivo was supported by the effects of the most active derivatives upon bacterial growth and free amino acid content.


Amino acid metabolism Antibiotics P5C reductase Proline Streptococcus sp 



Concentration causing 50% inhibition of enzyme activity


Concentration causing 50% inhibition of growth rate


Ornithine cyclodeaminase


δ1-Pyrroline-5-carboxylic acid



This work was supported in part by a grant from the University of Ferrara within the frame of the projects FAR2009-2010 Approcci biotecnologici per un incremento della sostenibilità della produzione agro-zootecnica. Davide Petrollino gratefully acknowledges an applied research fellowship from Spinner Consortium, Emilia Romagna Region (prot. N. 626/09).

Supplementary material

726_2011_970_MOESM1_ESM.pdf (232 kb)
Supplementary material 1 (PDF 231 kb)


  1. Aral B, Kamoun P (1997) The proline biosynthesis in living organisms. Amino Acids 13:189–217CrossRefGoogle Scholar
  2. Bradford MM (1976) A rapid and sensitive method for the quantization of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  3. Cascante M, Boros LG, Comin-Anduix B, de Atauri P, Centelles JJ, Lee PW (2002) Metabolic control analysis in drug discovery and disease. Nat Biotechnol 20:243–249PubMedCrossRefGoogle Scholar
  4. Chen LF, Chopra T, Kaye KS (2009) Pathogens resistant to antibacterial agents. Infect Dis Clin North Am 23:817–845PubMedCrossRefGoogle Scholar
  5. Culham DE, Dalgado C, Gyles CL, Mamelak D, MacLellan S, Wood JM (1998) Osmoregulatory transporter ProP influences colonization of the urinary tract by Escherichia coli. Microbiology 144:91–102PubMedCrossRefGoogle Scholar
  6. Cunin R, Glansdorff N, Piérard A, Stalon V (1986) Biosynthesis and metabolism of arginine in bacteria. Microbiol Rev 50:314–352PubMedGoogle Scholar
  7. Empadinhas N, da Costa MS (2008) Osmoadaptation mechanisms in prokaryotes: distribution of compatible solutes. Int Microbiol 11:151–161PubMedGoogle Scholar
  8. Engel LS (2010) The dilemma of multidrug-resistant Gram-negative bacteria. Am J Med Sci 340:232–237PubMedCrossRefGoogle Scholar
  9. Ferretti JJ, McShan WM, Ajdic D, Savic DJ, Savic G, Lyon K, Primeaux C, Sezate S, Suvorov AN, Kenton S, Lai HS, Lin SP, Qian Y, Jia HG, Najar FZ, Ren Q, Zhu H, Song L, White J, Yuan X, Clifton SW, Roe BA, McLaughlin R (2001) Complete genome sequence of an M1 strain of Streptococcus pyogenes. Proc Natl Acad Sci USA 98:4658–4663PubMedCrossRefGoogle Scholar
  10. Finney DJ (1971) Probit analysis, 3rd edn. Cambridge University Press, CambridgeGoogle Scholar
  11. Fischbach MA, Walsh CT (2009) Antibiotics for emerging pathogens. Science 325:1089–1093PubMedCrossRefGoogle Scholar
  12. Forlani G, Giberti S, Berlicki Ł, Petrollino D, Kafarski P (2007) Plant P5C reductase as a new target for aminomethylenebisphosphonates. J Agric Food Chem 55:4340–4347PubMedCrossRefGoogle Scholar
  13. Forlani G, Occhipinti A, Berlicki Ł, Dziędzioła G, Wieczorek A, Kafarski P (2008a) Tailoring the structure of aminophosphonates to target plant P5C reductase. J Agric Food Chem 56:3193–3199PubMedCrossRefGoogle Scholar
  14. Forlani G, Pavan M, Gramek M, Kafarski P, Lipok J (2008b) Biochemical bases for a widespread tolerance of cyanobacteria to the phosphonate herbicide glyphosate. Plant Cell Physiol 49:443–456PubMedCrossRefGoogle Scholar
  15. Forlani G, Prearo V, Wieczorek D, Kafarski P, Lipok J (2011) Polyphosphonate degradation by Spirulina spp: a cyanobacterial biofilter for the removal of anticorrosive polyphosphonates from wastewater. Enzyme Microb Tech 48:299–305CrossRefGoogle Scholar
  16. Ge M, Pan XM (2009) The contribution of proline residues to protein stability is associated with isomerization equilibrium in both unfolded and folded states. Extremophiles 13:481–489PubMedCrossRefGoogle Scholar
  17. Goodman JL, Wang S, Alam S, Ruzicka FJ, Frey PA, Wedekind JE (2004) Ornithine cyclodeaminase: Structure, mechanism of action, and implications for the mu-crystallin family. Biochemistry 43:13883–13891PubMedCrossRefGoogle Scholar
  18. Guo R-T, Cao R, Liang P-H, Ko T-P, Chang T-S, Hudock MP, Jeng W-Y, Chen CK-M, Zhang Y, Song Y, Kuo C-J, Yin F, Oldfield E, Wang AH-J (2007) Bisphosphonates target multiple sites in both cis- and trans-prenyltransferases. Proc Natl Acad Sci USA 104:10022–10027PubMedCrossRefGoogle Scholar
  19. Haydel SE (2010) Extensively drug-resistant tuberculosis: a sign of the times and an impetus for antimicrobial discovery. Pharmaceuticals (Basel) 3:2268–2290CrossRefGoogle Scholar
  20. Hall MJ, Kern AC, Middleton RF, Worthington HEC (1984) Roche Susceptibility Test (RST) medium, a defined formulation for susceptibility testing. II. Manufacture, use and stability. J Microbiol Methods 2:215–220CrossRefGoogle Scholar
  21. Höper D, Völker U, Hecker M (2005) Comprehensive characterization of the contribution of individual SigB-dependent general stress genes to stress resistance of Bacillus subtilis. J Bacteriol 187:2810–2826PubMedCrossRefGoogle Scholar
  22. Harth G, Horwitz MA (2003) Inhibition of Mycobacterium tuberculosis glutamine synthetase as a novel antibiotic strategy against tuberculosis: demonstration of efficacy in vivo. Infect Immun 71:456–464PubMedCrossRefGoogle Scholar
  23. Hutton CA, Perugini MA, Gerrard JA (2007) Inhibition of lysine biosynthesis: an evolving antibiotic strategy. Mol Biosyst 3:458–465PubMedCrossRefGoogle Scholar
  24. Lehmann S, Funck D, Szabados L, Rentsch D (2010) Proline metabolism and transport in plant development. Amino Acids 39:949–962PubMedCrossRefGoogle Scholar
  25. Liu JS, Cheng WC, Wang HJ, Chen YC, Wang WC (2008) Structure-based inhibitor discovery of Helicobacter pylori dehydroquinate synthase. Biochem Biophys Res Commun 373:1–7PubMedCrossRefGoogle Scholar
  26. Mazzucotelli E, Tartari A, Cattivelli L, Forlani G (2006) GABA metabolism during cold acclimation and freezing in barley and wheat. J Exp Bot 57:3755–3766PubMedCrossRefGoogle Scholar
  27. Nakajima K, Inatsu S, Mizote T, Nagata Y, Aoyama K, Fukuda Y, Nagata K (2008) Possible involvement of put A gene in Helicobacter pylori colonization in the stomach and motility. Biomed Res 29:9–18PubMedCrossRefGoogle Scholar
  28. Nathan C (2004) Antibiotics at the crossroads. Nature 431:899–902PubMedCrossRefGoogle Scholar
  29. Nocek B, Chang C, Li H, Lezondra L, Holzle D, Collart F, Joachimiak A (2005) Crystal structures of δ1-pyrroline-5-carboxylate reductase from human pathogens Neisseria meningitides and Streptococcus pyogenes. J Mol Biol 354:91–106PubMedCrossRefGoogle Scholar
  30. Pathania R, Brown ED (2008) Small and lethal: searching for new antibacterial compounds with novel modes of action. Biochem Cell Biol 86:111–115PubMedCrossRefGoogle Scholar
  31. Sakoulas G, Moellering RC Jr (2008) Increasing antibiotic resistance among methicillin-resistant Staphylococcus aureus strains. Clin Infect Dis 46:S360–S367PubMedCrossRefGoogle Scholar
  32. Silber AM, Colli W, Ulrich H, Alves MJ, Pereira CA (2005) Amino acid metabolic routes in Trypanosoma cruzi: possible therapeutic targets against Chagas’ disease. Curr Drug Targets Infect Disord 5:53–64PubMedCrossRefGoogle Scholar
  33. Snedecor GW, Cochran WG (1898) Statistical methods, 8th edn. The Iowa State University Press, AmesGoogle Scholar
  34. Tan S, Evans R, Singh B (2006) Herbicidal inhibitors of amino acid biosynthesis and herbicide-tolerant crops. Amino Acids 30:195–204PubMedCrossRefGoogle Scholar
  35. Takagi H (2008) Proline as a stress protectant in yeast: physiological functions, metabolic regulations, and biotechnological applications. Appl Microbiol Biotechnol 81:211–223PubMedCrossRefGoogle Scholar
  36. Williams I, Frank L (1975) Improved chemical synthesis and enzymatic assay of δ1-pyrroline-5-carboxylic acid. Anal Biochem 64:85–97PubMedCrossRefGoogle Scholar
  37. Ziebart KT, Dixon SM, Avila B, El-Badri MH, Guggenheim KG, Kurth MJ, Toney MD (2010) Targeting multiple chorismate-utilizing enzymes with a single inhibitor: validation of a three-stage design. J Med Chem 53:3718–3729PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Giuseppe Forlani
    • 1
  • Davide Petrollino
    • 1
  • Massimo Fusetti
    • 1
  • Letizia Romanini
    • 1
  • Bogusław Nocek
    • 2
  • Andrzej Joachimiak
    • 2
  • Łukasz Berlicki
    • 3
  • Paweł Kafarski
    • 3
  1. 1.Department of Biology and EvolutionUniversity of FerraraFerraraItaly
  2. 2.Biosciences Division, Argonne National LaboratoryMidwest Center for Structural GenomicsArgonneUSA
  3. 3.Department of Bioorganic ChemistryFaculty of Chemistry, Wrocław University of TechnologyWrocławPoland

Personalised recommendations