Catalytic Mechanism and Evolutionary traits of zinc b-lactamases:Does it take two to tango?

Catalytic Mechanism and Evolutionary traits of zinc b-lactamases:
Does it take two to tango?

Speaker: Alejandro J. Vila

Affiliation: Instituto de Biologia Molecular y Celular de Rosario (IBR), Argentina

Host: Ricardo Louro

Abstract

blactamases represent the prevalent resistance mechanism to b-lactam antibiotics. In the last decade, the dissemination of genes coding for metallo-b-lactamases (MBL´s) has become an emergent clinical problem. MBL´s are zinc-dependent enzymes. The exponential growth of MBL sequences being characterized has revealed an initially unforeseen structural diversity, that gives rise to the presence of mono- and dinuclear metal sites. MBL´s have been recently subdivided into classes B1, B2 and B3, each of them displaying different zinc ligands and coordination geometries (1).
We have studied the structural features of MBL´s from different subclasses with the aim of finding common structural and catalytic features. By means of mutagenesis, functional and structural studies, we conclude that a Zn site, previously regarded as non essential for catalysis, plays a major role in substrate binding and catalysis (2-5).
Non-steady state kinetic studies, aided by time-resolved electronic, EPR and Resonance Raman spectroscopy have allowed us to trap a key intermediate in β-lactam hydrolysis, and to assess the role of each metal binding site in the mechanism and stabilization of this intermediate (6,7).
Finally, directed evolution was used as an evolutionary engineering tool to explore the effect of challenging MBLs towards different antibiotics. BcII (the MBL from B.cereus) has been considered as a precursor of more efficient MBL’s present in pathogenic bacteria. In vitro evolution experiments on BcII by DNA shuffling with a cephalosporin substrate resulted in a expanded substrate spectrum of this enzyme, without sacrificing its stability nor the hydrolytic efficiency towards classical substrates of BcII (8). The mutations that give rise to these effects parallel others naturally found in MBL´s from pathogenic bacteria, and are related to the second-shell ligands of the zinc ions, expected to play a supramolecular control of reactivity. These results suggest that evolution of the chemical landscape can be predicted by means of this approach.

CV:

Education
1. Analytical Chemist, UCA, 1984.
2.  Licenciate in Chemistry (with Honors), UCA, 1986.
3. Ph. D., University of Rosario (UNR), 1990 (Summa cum laudæ).

Present Positions
1. Associate Professor, Biophysics Section, Department of Biological Chemistry, UNR, 1999-.
2. Principal Researcher, CONICET (National Research Council - Argentina), 2006.
3. International Research Scholar, Howard Hughes Medical Institute (2002-2006 and 2007-2011).
4. Head of the Biomolecular NMR Facility, Rosario.

Research Group Composition
Two postdocs, seven PhD students, two undergraduates.

Teaching Experience and Previous Positions
1. Teaching Assistant,  Inorganic Section, Dept.Physical Chemistry, UNR, 1986-1991.
2. Assistant Professor, Biophysics Section, Dept.Biological Chemistry, FCByF, UNR, 1993-1999.
3. Visiting Researcher, Beckman Institute, California Institute of Technology, 1996.
4. Visiting Professor, University of Valencia (Spain), 1998.
5. Visiting Professor, SISSA (Trieste, Italy), 2000.

Honors and Awards
• Eduardo de Robertis Award for Young Researchers, Federal Secretary of Science and Technology (Argentina, 1994).
• Ernesto E. Galloni Award in Experimental Physical Chemistry, National Academy of Sciences (Argentina, 1995). This Award is given every year to an outstanding scientist under the age of 35 in the field of chemistry, physics or mathematics.
• Ranwell Caputto Award in Chemistry, National Academy of Sciences (Argentina, 2000). This Award is given yearly to an outstanding researcher in Chemistry under 40 years.
• Rafael Labriola Award, Argentinean Chemical Society, 2004. This Award is given every 3 years to an outstanding researcher in Chemistry under 45 years.
• International Research Scholar, Howard Hughes Medical Institute, USA, Selected for two periods (2002-2006 and 2007-2011).
• José Gómez Ibáñez Lecturer, Department of Chemistry, Wesleyan University, 2008.

Involvement in Scientific Journals (member of boards)
Member of Editorial Advisory Boards: Journal of Biological Inorganic Chemistry, 1998-2001, European Journal of Inorganic Chemistry, (2000-2005); Journal of Inorganic Biochemistry, 2003-, Accounts of Chemical Research, 2005-.

Actual Grant Support from HHMI (USA), NIH (USA) and ANPCyT (Argentina).

Professional Activities and Services to the Community (selected)
• Chairman of the XXXIII Meeting of the Argentinean Biophysical Society, 2006.
• Council Member, Institute of Molecular and Cell Biology, IBR.
• Member of the International Organizing Committee, First Latin American Protein Society Meeting, Angra dos Reis, Brasil, 2004.
• Council Member, Argentinean Biophysical Society, 1997-present.
• Ad hoc member of study sections, CONICET.
• External reviewer, NSF.
• Reviewer of articles: Proc.Natl.Acad.Sci.USA, Journal of the American Chemical Society, Angewandte Chemie, Chemical Society Reviews, Journal of Proteome Research, Biochemistry, Biochemical Journal, Protein Science, Analytical Biochemistry, PROTEINS: Structure, Function, and Bioinformatics, FEBS Letters, Inorganic Chemistry, Biochimica et Biophysica Acta, Inorganica Chimica Acta, European Journal of Inorganic Chemistry, Journal of Inorganic Biochemistry, Journal of Biological Inorganic Chemistry, Bioinorganic Chemistry and Applications.

Scientific Publications in international peer-reviewed journals (since 2001)
Total number of citations (SCI): more than 1300 (December 2007). h-index: 21.
30. I Bertini, S.Ciurli, A. Dikiy, C. O. Fernández, C. Luchinat, N. Safarov, S. Shumilin and A.J.Vila “The first solution structure of an oxidized paramagnetic copper(II) protein: The case of plastocyanin from the cyanobacterium Synechocystis sp. PCC 6803”, J. Am. Chem. Soc , 123, 2405-2413 (2001).
31. C. O. Fernández, J. A. Cricco, C. E. Slutter, J.H.Richards, H.B.Gray and A.J.Vila, “Weak Axial Ligand Modulation of the Electronic Structure of CuA Sites: A Paramagnetic 1H NMR Study of Met160Gln CuA”, J.Am.Chem.Soc., 123, 11678-11685 (2001).
32. Bertini, D.A. Bryant, S.Ciurli, A. Dikiy, C. O. Fernández, C. Luchinat, N. Safarov, A.J.Vila and J. Zhao “Backbone dynamics of plastocyanin in both oxidation states. Solution structure of the reduced form and comparison with the oxidized state”, J. Biol.Chem., 276, 47217-47226 (2001).
33. A.J.Vila and C.O.Fernández “Copper in Electron-Transfer Proteins”, in “Handbook of Metalloproteins” (A. Sigel, H. Sigel & I. Bertini, editores), Marcel Dekker, New York, pp 813-856 (2001).
34. R.M. Rasia and A.J.Vila, “Exploring the role and the binding affinity of a second zinc equivalent in B.cereus metallo-–lactamase”, Biochemistry, 41, 1853-1860 (2002).
35. G. L. Estiú, R.M.Rasia, J.A.Cricco, A.J.Vila and M. E. Zerner “Is there a bridging ligand in metal-substituted -lactamases?: A spectroscopic and theoretical answer”, Int.J.Quantum Chem., 88, 118-132 (2002).
36. M. Dal Peraro, A.J.Vila, and P. Carloni “Structural Determinants and H-Bond Network of Mononuclear Zinc--Lactamase Active Site”, J.Biol.Inorg.Chem., 7, 704-712 (2002).
37. A.Donaire, B.Jiménez, C.O.Fernández, R.Pierattelli, T.Niizeki, J.M.Moratal, J.F.Hall, T.Kohzuma, S.S.Hasnain, and A.J.Vila “Metal-ligand interplay in blue copper proteins studied by 1H NMR spectroscopy: Cu(II)-pseudoazurin and rusticyanin”, J. Am. Chem. Soc., 124, 13698-13708 (2002).
38. L. Banci, R. Pierattelli and A.J.Vila “NMR of Copper Proteins”, Adv. Protein Chem. (E. Gralla & J.S.Valentine, editores), 60, 397-449 (2002).
39. A.R.Díaz; M.C.Mansilla, A.J.Vila and D. de Mendoza “Membrane topology of the acyl-lipid desaturase from Bacillus subtilis”, J.Biol.Chem., 277, 48099-48016 (2002).
40. C.O.Fernández, T. Niizeki, T. Kohzuma and A.J.Vila “Metal-ligand interactions in perturbed blue copper sites: A Paramagnetic 1H NMR Study of Co(II)-pseudoazurin”, J.Biol.Inorg.Chem., 8, 75-82 (2003).
41. R.M. Rasia, M.Ceolín and A.J.Vila, “Grafting a new metal ligand in the cocatalytic site of B.cereus metallo--lactamase: structural flexibility without loss of activity”, Protein Science, 12, 1538-1546 (2003).
47. M.Dal Peraro, A.J.Vila, and P. Carloni “Binuclear Zn(II)--lactamase from Bacteroides fragilis: an ab initio study”, Inorg.Chem., 42, 4245-4257 (2003).
48. C.O.Fernández and A.J.Vila “Paramagnetic NMR of Electron Transfer Copper Proteins”, in “Paramagnetic Resonance of Metallobiomolecules” (J.Telser, editor), ACS Symposium Series, American Chemical Society, pp. 287-303 (2003).
49. R.M. Rasia and Alejandro J. Vila. "Mechanistic study of the hydrolysis of nitrocefin mediated by B.cereus metallo--lactamase" Arkivoc, X, 507-516 (2003). Issue dedicated to Prof. E.A.Rúveda.
50. M.Dal Peraro, A.J.Vila, and P. Carloni “Substrate Binding to Mononuclear Metallo--lactamase from Bacillus cereus”, Proteins, 54, 412-423 (2004).
51. R.M. Rasia and A.J.Vila, “Structural determinants of substrate binding to Bacillus cereus metallo--lactamase”, J. Biol.Chem., 279, 26046–26051 (2004).
52. M.Dal Peraro, L.I.Llarrull, U.Rothlisberger, A.J.Vila and P.Carloni “Water-assisted reaction mechanism of monozinc -lactamases”, J. Am.Chem.Soc., 126, 12661-12668 (2004).
53. A.M.Davies, R.M.Rasia, A.J.Vila, B.J. Sutton and S.M.Fabiane “Effect of pH on the active site of an Arg121Cys mutant of the metallo-β-lactamase from Bacillus cereus: Implications for the enzyme mechanism”, 44, 4841-4849 (2005).
54. P.E.Tomatis, R.M.Rasia, L.Segovia and A.J.Vila “Mimicking natural evolution in metallo-β-lactamases  through second-shell ligand mutations”, Proc.Natl.Acad.Sci.USA, 102, 13761-13766 (2005). Highlighted in the cover. 
55. P.Marchiaro, M.A.Mussi, V.Ballerini, F.Pasterán, A.M.Viale, A.J.Vila, and A.S. Limansky “Sensitive EDTA-Based Microbiological Assays for the Detection of Metallo-β-lactamases in Non-Fermentative Gram-Negative Bacteria”, J. Clin.Microb., 43, 5648-52 (2005).
56. G.Battistuzzi, M.Bellei, A.Leonardi, R.Pierattelli, A.De Candia, A.J.Vila and M.Sola  “Reduction Thermodynamics of the T1 Cu-site in plant and fungal laccases”, J. Biol.Inorg.Chem., 10, 867-73 (2005).
57. G. E. Schujman, M.Guerin, A.Buschiazzo, F.Schaeffer, L.I.Llarrull, A.J.Vila, P.M.Alzari and D.de Mendoza “Structural basis of lipid biosynthesis regulation in Gram-positive bacteria”, EMBO J., 25, 4074-83 (2006).
58. M. W. Crowder, J. Spencer and A.J.Vila, “Metallo--lactamases: Novel Weaponry for Antibiotic Resistance in Bacteria”, Acc. Chem.Res, 39, 721-728 (2006).
59. M. Dal Peraro, A.J.Vila, P.Carloni and M. L.Klein “Role of zinc content on the catalytic efficiency of B1 metallo-β-lactamases”, J.Am.Chem.Soc., 129, 2808-2816 (2007).
60. L.I.Llarrull, S.M.Fabiane, J.M.Kowalski, B.Bennett, B.J.Sutton and A.J.Vila “Asp120 locates Zn2 for optimal Metallo--Lactamase Activity”, J. Biol.Chem., 282, 18276-18285 (2007). Highlighted in the cover. 
61. J.Morán-Barrio, J.M.González, M.N. Lisa, A.L.Costello, M.Dal Peraro, P.Carloni, B. Bennett, D.L.Tierney, A.S. Limansky, A.M.Viale and A.J.Vila “The Metallo--lactamase GOB is a mono-Zn(II) enzyme with a novel active site”, J.Biol.Chem., 282, 18286-93 (2007).
62. A.A.Poeylaut–Palena, P.E.Tomatis, A.I.Karsisiotis, C.Damblon, E.G.Mata and A.J.Vila “A Minimalistic Approach to Identify Substrate Binding Features in B1 Metallo–β–Lactamases”, Bioorg.Med.Chem.Lett., 17, 5171–5174 (2007).
63. F.Simona, A. Magistrato, M.Vera, G. Garau, A.J. Vila and P.Carloni “Protonation state and substrate binding to B2 metallo--lactamase CphA from Aeromonas hydrophila”, Proteins, 69, 595-605 (2007).
64. V.A.Campos-Bermudez, N.R.Leite, R.Krog, A.J.Costa-Filho, F.C.Soncini, G.Oliva and A.J.Vila “Biochemical and Structural Characterization of Salmonella typhimurium glyoxalase II: New insights in metal ion selectivity”, Biochemistry, 46, 11069-11079 (2007).
65. G.N.Ledesma, D.H.Murgida, H.K.Ly, H.Wackerbarth, J.Ulstrup, A.J.Costa-Filho and A.J.Vila “The Met axial ligand determines the redox potential in CuA Sites”, J.Am.Chem.Soc., 129, 11884-11885 (2007).
66. J.M.González, F.J.Medrano, A.L.Costello, D.L.Tierney and A.J.Vila “The Zn2 Position in Metallo-β-Lactamases is Critical for Activity: A study on Chimeric Metal Sites on a Conserved Protein Scaffold”, J.Mol.Biol., 373, 1141–1156 (2007).
67. L.I.Llarrull, M.F.Tioni, J.Kowalski, B.Bennett and A.J.Vila “Evidence for a dinuclear active site in the metallo-beta-lactamase BcII with substoichiometric Co(II): a new mechanism for Co(II) uptake”, J.Biol.Chem., 282, 30586-95 (2007).
68. P.Marchiaro, V.Ballerini, T.Spalding, G.Cera, M.A.Mussi, J.Morán-Barrio, A.J.Vila, A.M. Viale and A.S.Limansky “A convenient microbiological assay employing cell-free extracts for the rapid characterization of Gram-negative carbapenemase producers”, J.Antimicrob.Chemother., in press.
69. P.Marchiaro, P.E.Tomatis, M.A.Mussi, F.Pasteran, A.M.Viale, A.S.Limansky and A.J.Vila “Biochemical characterization of metallo--lactamase VIM-11 from a Pseudomonas aeruginosa clinical strain”, Antimicrob. Agents Chemother., in press.
70. L.I.Llarrull, M.F.Tioni and A.J.Vila “Metal Content and Localization during Turnover in B.cereus Metallo--Lactamase”, submitted.
71. M.F.Tioni, L.I.Llarrull, A.A.Poeylaut-Palena, M.A.Martí, M.Saggu, G.R.Periyannan, E.G.Mata, B.Bennett, D.H.Murgida and A.J.Vila “Trapping and Characterization of a Reaction Intermediate in Imipenem Hydrolysis by B. cereus Metallo--lactamase”, submitted.
72. P.E.Tomatis, S.M.Fabiane, F.Simona, P.Carloni, B.J.Sutton and A.J.Vila “Adaptive protein evolution grants organismal fitness by improving catalysis and flexibility”, submitted.

 
Invited Conferences
He has been invited to give seminars in the following institutions: Department of Chemistry and Chemical Enginering, California Institute of Technology (Pasadena, USA); BIOMAC Research School, Leiden Institute of Chemistry (University of Leiden, Holanda); Departamento de Química Inorgánica, Universitat de València (España); International Centre for Theoretical Physics (Trieste, Italia); International School for Advanced Studies-SISSA (Trieste, Italia); Departamento de Bioquímica, Università di Roma “Tor Vergata”; Centro de Estudios Atomicos - Sección de Bioenergética (Saclay, Francia); Instituto Pasteur (París, Francia); Center of Magnetic Resonance & Department of Chemistry (Universidad de Firenze, Italia); Department of Chemistry y Department of Biochemistry and Microbiology (University of Sydney, Australia); Department of Chemistry and Biochemistry (University of California, Los Angeles, USA); Instituto de Biotecnología (Universidad Autónoma de México, Cuernavaca); Instituto de Química (Universidad Autónoma de México); Max Planck Institute for Biophysical Chemistry (Goettingen, Alemania); Department of Biochemistry, University of Frankfurt); Department of Chemistry (University of California, San Diego, USA); Department of Chemistry (University of Rochester, USA); Department of Biophysics (Medical School, Johns Hopkins University, USA); Department of Chemistry and Biochemistry (University of Illinois at Urbana-Champaign, USA), Department of Chemistry (Northwestern University, USA), Max Planck Institute (Muelheim), Deparment of Chemistry (University of Miami at Ohio).
He has been invited speaker to many international meetings, including: XXXII International Conference on Coordination Chemistry, III Iberoamerican Congress of Biophysics, 4th European Conference on Bio-inorganic Chemistry, XXXIII International Conference on Coordination Chemistry, IX International Conference on Biological Inorganic Chemistry, XIX International Conference on Magnetic Resonance in Biological Systems, International Workshop on Spectroscopy for Biology, X International Conference on Biological Inorganic Chemistry, IX Workshop on Metallo-b-Lactamases, International Biophysics Congress, Buenos Aires, 2002, International Colloquia on NMR in Biology, Gordon Research Conference “Metals in Biology”, 7th International Symposium on Applied Bioinorganic Chemistry, Worskhop on Nuclear Magnetic Resonance in Biology, 2nd International Workshop on Spectroscopy for Biology, 36th International Conference on Coordination Chemistry, 1st Latin American Protein Society Meeting, 1st Iberoamerican NMR Meeting, 9th Workshop on beta-lactamases, XII International Conference on Biological Inorganic Chemistry, Conference for Drug Development for the Third World, 9th European Conference on Bio-inorganic Chemistry, 46th Annual Interscience Conference on Antimicrobial Agents and Chemotherapy, Symposium “The Biological Chemistry of Macromolecules”; International Symposium on Molecular Recognition Phenomena in Biopolymers; 6th International Conference of Biological Physics; V International Conference on Peroxynitrite and Reactive Nitrogen Species.