[en] Molecular evolution has always been a subject of discussions, and researchers are interested in understanding how proteins with similar scaffolds can catalyze different reactions. In the superfamily of serine penicillin-recognizing enzymes, D-alanyl-D-alanine peptidases and β-lactamases are phylogenetically linked but feature large differences of reactivity towards their respective substrates. In particular, while β-lactamases hydrolyze penicillins very fast, leading to their inactivation, these molecules inhibit D-alanyl-D-alanine peptidases by forming stable covalent penicilloyl enzymes. In cyanobacteria, we have discovered a new family of penicillin-binding proteins (PBPs) presenting all the sequence features of class A β-lactamases but having a six-amino-acid deletion in the conserved Ω-loop and lacking the essential Glu166 known to be involved in the penicillin hydrolysis mechanism. With the aim of evolving a member of this family into a β-lactamase, PBP-A from Thermosynechococcus elongatus has been chosen because of its thermostability. Based on sequence alignments, introduction of a glutamate in position 158 of the shorter Ω-loop afforded an enzyme with a 50-fold increase in the rate of penicillin hydrolysis. The crystal structures of PBP-A in the free and penicilloylated forms at 1.9 Å resolution and of L158E mutant at 1.5 Å resolution were also solved, giving insights in the catalytic mechanism of the proteins. Since all the active-site elements of PBP-A-L158E, including an essential water molecule, are almost perfectly superimposed with those of a class A β-lactamase such as TEM-1, the question why our mutant is still 5 orders of magnitude less active as a penicillinase remains and our results emphasize how far we are from understanding the secrets of enzymes. Based on the few minor differences between the active sites of PBP-A and TEM-1,mutations were introduced in the L158E enzyme, but while activities on D-Ala-D-Ala mimicking substrates were severely impaired, further improvement in penicillinase activity was
unsuccessful.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Urbach, Carole; Université Catholique de Louvain - UCL > Institut des sciences de la vie - ISV
Evrard, Christine ; Université Catholique de Louvain - UCL > Département de Chimie > Unité de Chimie Structurale - CSTR
Pudzaitis, Vaidas; Université Catholique de Louvain - UCL > Institut des sciences de la vie - ISV
Fastrez, Jacques; Université Catholique de Louvain - UCL > Institut des sciences de la vie - ISV
Soumillion, Patrice; Université Catholique de Louvain - UCL > Institut des sciences de la vie - ISV
Declercq, Jean-Paul; Université Catholique de Louvain - UCL > Département de Chimie > Unité de Chimie Structurale - CSTR
Language :
English
Title :
Structure of PBP-A from Thermosynechococcus elongatus, a Penicillin-Binding Protein Closely Related to Class A β-Lactamases
Orengo C.A., and Thornton J.M. Protein families and their evolution-a structural perspective. Annu. Rev. Biochem. 74 (2005) 867-900
Gerlt J.A., and Babbitt P.C. Divergent evolution of enzymatic function: mechanistically diverse superfamilies and functionally distinct suprafamilies. Annu. Rev. Biochem. 70 (2001) 209-246
Joris B., Ghuysen J.M., Dive G., Renard A., Dideberg O., Charlier P., et al. The active-site-serine penicillin-recognizing enzymes as members of the Streptomyces R61 DD-peptidase family. Biochem. J. 250 (1988) 313-324
Massova I., and Mobashery S. Kinship and diversification of bacterial penicillin-binding proteins and beta-lactamases. Antimicrob. Agents Chemother. 42 (1998) 1-17
Ambler R.P., Coulson A.F., Frere J.M., Ghuysen J.M., Joris B., Forsman M., et al. A standard numbering scheme for the class A beta-lactamases. Biochem. J. 276 (1991) 269-270
Matagne A., and Frere J.M. Contribution of mutant analysis to the understanding of enzyme catalysis-the case of class-A beta-lactamases. Biochim. Biophys. Acta 1246 (1995) 109-127
Adachi H., Ohta T., and Matsuzawa H. Site-directed mutants, at position 166, of RTEM-1 beta-lactamase that form a stable acyl-enzyme intermediate with penicillin. J. Biol. Chem. 266 (1991) 3186-3191
Hermann J.C., Hensen C., Ridder L., Mulholland A.J., and Holtje H.D. Mechanisms of antibiotic resistance: QM/MM modeling of the acylation reaction of a class A beta-lactamase with benzylpenicillin. J. Am. Chem. Soc. 127 (2005) 4454-4465
Meroueh S.O., Fisher J.F., Schlegel H.B., and Mobashery S. Ab initio QM/MM study of class A beta-lactamase acylation: dual participation of Glu166 and Lys73 in a concerted base promotion of Ser70. J. Am. Chem. Soc. 127 (2005) 15397-15407
Urbach C., Fastrez J., and Soumillion P. A new family of cyanobacterial penicillin-binding proteins: a missing link in the evolution of class A beta-lactamases. J. Biol. Chem. 283 (2008) 32516-32526
Laskowski R.A., Macarthur M.W., Moss D.S., and Thornton J.M. Procheck-a program to check the stereochemical quality of protein structures. J. Appl. Crystallogr. 26 (1993) 283-291
Lovell S.C., Davis I.W., Arendall W.B., de Bakker P.I.W., Word J.M., Prisant M.G., et al. Structure validation by C alpha geometry: phi, psi and C beta deviation. Proteins 50 (2003) 437-450
Holm L., and Sander C. Mapping the protein universe. Science 273 (1996) 595-603
Tranier S., Bouthors A.T., Maveyraud L., Guillet V., Sougakoff W., and Samama J.P. The high resolution crystal structure for class A beta-lactamase PER-1 reveals the bases for its increase in breadth of activity. J. Biol. Chem. 275 (2000) 28075-28082
Minasov G., Wang X., and Shoichet B.K. An ultrahigh resolution structure of TEM-1 beta-lactamase suggests a role for Glu166 as the general base in acylation. J. Am. Chem. Soc. 124 (2002) 5333-5340
Jelsch C., Mourey L., Masson J.M., and Samama J.P. Crystal structure of Escherichia coli TEM1 beta-lactamase at 1.8 Å resolution. Proteins 16 (1993) 364-383
Ibuka A.S., Ishii Y., Galleni M., Ishiguro M., Yamaguchi K., Frere J.M., et al. Crystal structure of extended-spectrum beta-lactamase Toho-1: insights into the molecular mechanism for catalytic reaction and substrate specificity expansion. Biochemistry 42 (2003) 10634-10643
Knox J.R., and Moews P.C. Beta-lactamase of Bacillus licheniformis 749/C-refinement at 2 Å resolution and analysis of hydration. J. Mol. Biol. 220 (1991) 435-455
Herzberg O. Refined crystal-structure of beta-lactamase from Staphylococcus aureus Pc1 at 2.0-Å resolution. J. Mol. Biol. 217 (1991) 701-719
Strynadka N.C., Adachi H., Jensen S.E., Johns K., Sielecki A., Betzel C., et al. Molecular structure of the acyl-enzyme intermediate in beta-lactam hydrolysis at 1.7 Å resolution. Nature 359 (1992) 700-705
Murphy B.P., and Pratt R.F. Evidence for an oxyanion hole in serine beta-lactamases and DD-peptidases. Biochem. J. 256 (1988) 669-672
Fonze E., Vermeire M., Nguyen-Disteche M., Brasseur R., and Charlier P. The crystal structure of a penicilloyl-serine transferase of intermediate penicillin sensitivity. The DD-transpeptidase of Streptomyces K15. J. Biol. Chem. 274 (1999) 21853-21860
Zawadzke L.E., Chen C.C.H., Banerjee S., Li Z., Wasch S., Kapadia G., et al. Elimination of the hydrolytic water molecule in a class A beta-lactamase mutant: crystal structure and kinetics. Biochemistry 35 (1996) 16475-16482
Lewis E.R., Winterberg K.M., and Fink A.L. At mutation leads to altered product specificity in beta-lactamase catalysis. Proc. Natl Acad. Sci. USA 94 (1997) 443-447
Chen C.C.H., and Herzberg O. Structures of the acyl-enzyme complexes of the Staphylococcus aureus beta-lactamase mutant Glu166Asp:Asn170Gln with benzylpenicillin and cephaloridine. Biochemistry 40 (2001) 2351-2358
Escobar W.A., Miller J., and Fink A.L. Effects of site-specific mutagenesis of tyrosine-105 in a class-A beta-lactamase. Biochem. J. 303 (1994) 555-558
Doucet N., De Wals P.Y., and Pelletier J.N. Site-saturation mutagenesis of Tyr-105 reveals its importance in substrate stabilization and discrimination in TEM-1 beta-lactamase. J. Biol. Chem. 279 (2004) 46295-46303
Palzkill T., and Botstein D. Identification of amino-acid substitutions that alter the substrate-specificity of Tem-1 beta-lactamase. J. Bacteriol. 174 (1992) 5237-5243
Osuna J., Viadiu H., Fink A.L., and Soberon X. Substitution of Asp for Asn at position-132 in the active-site of Tem beta-lactamase-activity toward different substrates and effects of neighboring residues. J. Biol. Chem. 270 (1995) 775-780
Bouthors A.T., Dagoneau-Blanchard N., Naas T., Nordmann P., Jarlier V., and Sougakoff W. Role of residues 104, 164, 166, 238 and 240 in the substrate profile of PER-1 beta-lactamase hydrolysing third-generation cephalosporins. Biochem. J. 330 (1998) 1443-1449
Sabbagh Y., Theriault E., Sanschagrin F., Voyer N., Palzkill T., and Levesque R.C. Characterization of a PSE-4 mutant with different properties in relation to penicillanic acid sulfones: importance of residues 216 to 218 in class A beta-lactamases. Antimicrob. Agents Chemother. 42 (1998) 2319-2325
Pieper U., Hayakawa K., Li Z., and Herzberg O. Circularly permuted beta-lactamase from Staphylococcus aureus PC1. Biochemistry 36 (1997) 8767-8774
Osuna J., Perez-Blancas A., and Soberon X. Improving a circularly permuted TEM-1 beta-lactamase by directed evolution. Protein Eng. 15 (2002) 463-470
Guex N., and Peitsch M.C. SWISS-MODEL and the Swiss-PdbViewer: an environment for comparative protein modeling. Electrophoresis 18 (1997) 2714-2723
Zafaralla G., Manavathu E.K., Lerner S.A., and Mobashery S. Elucidation of the role of arginine-244 in the turnover processes of class A beta-lactamases. Biochemistry 31 (1992) 3847-3852
Jacob-Dubuisson F., Lamotte-Brasseur J., Dideberg O., Joris B., and Frère J.-M. Arginine 220 is a critical residue for the catalytic mechanism of the Streptomyces albus G beta-lactamase. Protein Eng. 4 (1991) 811-819
Guillaume G., Vanhove M., Lamotte-Brasseur J., Ledent P., Jamin M., Joris B., and Frere J.M. Site-directed mutagenesis of glutamate 166 in two beta-lactamases. Kinetic and molecular modeling studies. J. Biol. Chem. 272 (1997) 5438-5444
Gibson R.M., Christensen H., and Waley S.G. Site-directed mutagenesis of beta-lactamase I. Single and double mutants of Glu-166 and Lys-73. Biochem. J. 272 (1990) 613-619
Damblon C., Raquet X., Lian L.Y., Lamotte-Brasseur J., Fonze E., Charlier P., et al. The catalytic mechanism of beta-lactamases: NMR titration of an active-site lysine residue of the TEM-1 enzyme. Proc. Natl Acad. Sci. USA 93 (1996) 1747-1752
Lietz E.J., Truher H., Kahn D., Hokenson M.J., and Fink A.L. Lysine-73 is involved in the acylation and deacylation of beta-lactamase. Biochemistry 39 (2000) 4971-4981
Sauvage E., Kerff F., Fonze E., Herman R., Schoot B., Marquette J.P., et al. The 2.4-Å crystal structure of the penicillin-resistant penicillin-binding protein PBP5fm from Enterococcus faecium in complex with benzylpenicillin. Cell. Mol. Life Sci. 59 (2002) 1223-1232
Chesnel L., Zapun A., Mouz N., Dideberg O., and Vernet T. Increase of the deacylation rate of PBP2x from Streptococcus pneumoniae by single point mutations mimicking the class A beta-lactamases. Eur. J. Biochem. 269 (2002) 1678-1683
Dessen A., Mouz N., Gordon E., Hopkins J., and Dideberg O. Crystal structure of PBP2x from a highly penicillin-resistant Streptococcus pneumoniae clinical isolate: a mosaic framework containing 83 mutations. J. Biol. Chem. 276 (2001) 45106-45112
Pettersen E.F., Goddard T.D., Huang C.C., Couch G.S., Greenblatt D.M., Meng E.C., and Ferrin T.E. UCSF Chimera-a visualization system for exploratory research and analysis. J. Comput. Chem. 25 (2004) 1605-1612
Kabsch W. Automatic processing of rotation diffraction data from crystals of initially unknown symmetry and cell constants. J. Appl. Crystallogr. 26 (1993) 795-800
Read R.J. Pushing the boundaries of molecular replacement with maximum likelihood. Acta Crystallogr., Sect. D: Biol. Crystallogr. 57 (2001) 1373-1382
Cowtan K. Modified phased translation functions and their application to molecular-fragment location. Acta Crystallogr., Sect. D: Biol. Crystallogr. 54 (1998) 750-756
Collaborative Computational Project, No. 4. The CCP4 suite: programs for protein crystallography. Acta Crystallogr., Sect. D: Biol. Crystallogr. 50 (1994) 760-763
Jones T.A., Zou J.Y., Cowan S.W., and Kjeldgaard M. Improved methods for building protein models in electron-density maps and the location of errors in these models. Acta Crystallogr., Sect. A: Found. Crystallogr. 47 (1991) 110-119
Emsley P., and Cowtan K. Coot: model-building tools for molecular graphics. Acta Crystallogr., Sect. D: Biol. Crystallogr. 60 (2004) 2126-2132
Murshudov G.N., Vagin A.A., and Dodson E.J. Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr., Sect. D: Biol. Crystallogr. 53 (1997) 240-255
Lamzin V.S., and Perrakis A. Current state of automated crystallographic data analysis. Nat. Struct. Biol. 7 (2000) 978-981
Konagurthu A.S., Whisstock J.C., Stuckey P.J., and Lesk A.M. MUSTANG: a multiple structural alignment algorithm. Proteins 64 (2006) 559-574
Hall T.A. BioEdit a user-friendly biological sequence alignment editor and analysis program for Windows 95/98/NT. Nucleic Acids Symp. Ser. 41 (1999) 95-98
Silvaggi N.R., Josephine H.R., Kuzin A.P., Nagarajan R., Pratt R.F., and Kelly J.A. Crystal structures of complexes between the R61 DD-peptidase and peptidoglycan-mimetic beta-lactams: a non-covalent complex with a "perfect penicillin". J. Mol. Biol. 345 (2005) 521-533