Biochemistry: Enzyme kinetics and mechanisms; bio-organic chemistry; enzyme inhibitor design; bacterial DD-peptidases, ß-lactamases and ß-lactam antibiotics.
Biological chemistry involves chemical reactions which are relevant, directly or indirectly, to biological systems. There are, of course, many different reactions that can fall into this category and an almost equal number of ways in which they can be studied. My approach, which derives from a background of physical organic aqueous solution chemistry, is directed towards an understanding of the mechanisms of these reactions.
Since chemical reactions in living systems are usually enzyme catalyzed, some understanding of enzyme mechanisms in fundamental to biological chemistry. One can divide this problem, on paper at least, into two parts. First what is the chemistry involved, i.e., what functional groups on an enzyme interact, covalently or non-covalently, with the substrate, and how do they catalyze the reaction? Second, what is the role of the rest of the protein, i.e., how does the static and dynamic structure of the whole protein molecule contribute to its function?
With these general questions in mind we have concentrated on a particular group of bacterial enzymes, those specifically interacting with ß-lactam antibiotics, the penicillins and cephalosporins. One important class of these enzymes, the ß-lactamases, catalyzes the hydrolysis and thus destruction of ß-lactam antibiotics and is the major source of bacterial penicillin resistance, while another, the D-alanine transpeptidases, is involved in bacterial cell wall synthesis and is the site of the antibiotic action of these drugs. These enzymes are thus of practical or clinical importance as well as of fundamental interest.
Until recently very little was known about the mechanism of action of these groups of enzymes and the relationships between their active sites. The substrate specificity of the transpeptidases was also not well established. Our approach to mechanistic problems of this type is through the design and synthesis of new enzyme substrates and inhibitors, and study of their modes of interaction with the relevant enzymes by means of the methods of enzyme kinetics and physical organic chemistry. The rich chemistry of ß-lactams permits the design of very subtle active site probes. Crystal structures of enzyme/inhibitor complexes, aided by computational modeling, can lead to further inhibitor design.
- J.H. Bell, K. Curley and R.F. Pratt, “Inhibition of Serine Amidohydrolases by Complexes of Vanadate with Hydroxamic Acids”, Biochem. Biophys. Res. Commun. 274, 732 (2000).
- K. Kaur and R.F. Pratt, “Mechanism of Reaction of Acyl Phosph(on)ates with the ß-Lactamase of Enterobacter cloacae P99″, Biochemistry 40, 4610 (2001).
- S.A. Adediran, D. Cabaret, B. Drusilla, R.F. Pratt, and M. Wakselman, “The Synthesis and Evaluation of Benzofuranones as ß-Lactamase Substrates”, Bioorg. Med. Chem. 9, 1175 (2001).
- S. Kumar, S.L. Pearson, and R.F. Pratt, “Design, Synthesis, and Evaluation of alpha-Ketoheterocycles as Class C ß-Lactamase Inhibitors” Biooorg. Med. Chem.9, 2035 (2001).
- M.J. Morrison, N. Li, and R.F.Pratt, “Inverse Acyl Phosph(on)ates: Substrates or Inhibitors of ß-Lactam-Recognizing Enzymes” Bioorg. Chem. 29, 271 (2001).
- K. Kaur, M.J.K. Lan, and R.F. Pratt, “Mechanism of Inhibition of the Class C ß-Lactamase of Enterobacter cloacae by Cyclic Acyl Phosph(on)ates: Rescue by Return” J. Amer. Chem. Soc.123, 10436 (2001).
- R.F. Pratt, “Functional Evolution of the ß-Lactamase Active Site”, J. Chem. Soc. Perkin Trans. II, 851 (2002).
- J.H. Bell and R.F. Pratt, “Mechanism of Inhibition of the ß-Lactamase of Enterobacter cloacae P99 by 1:1 Complexes of Vanadate with Hydroxamic Acids”, Biochemistry41, 4329 (2002).
- J.H. Bell and R.F. Pratt, “Formation and Structure of 1:1 Complexes Between Aryl Hydroxamic Acids and Vanadate at neutral pH”, Inorg. Chem. 41, 2747 (2002).
- M.A. McDonough, J.W. Anderson, N.R. Silvaggi, R.F. Pratt, J.R. Knox and J.A. Kelly, “Structures of Two Kinetic Intermediates Reveal Species Specificity of Penicillin-binding Proteins”, J. Mol. Biol.322, 111 (2002).
- N.R. Silvaggi, J.W. Anderson, S.R. Brinsmade, R.F. Pratt and J.A. Kelly, “The Crystal Structure of Phosphonate-Inhibited D-Ala-D-Ala peptidase Reveals an Analogue of a Tetrahedral Transition State” Biochemistry42, 1199 (2003).
- K. Kaur, S.A. Adediran, M.J.K. Lan and R.F. Pratt, “Inhibition of ß-Lactamases by Monocyclic Acyl Phosph(on)ates” Biochemistry42, 1429 (2003).
- D. Cabaret, S.A. Adediran, R.F. Pratt and M. Wakselman, “New Substrates for ß-Lactam-Recognizing Enzymes: Aryl Malonamates” Biochemistry 42, 6719 (2003).
- J.W. Anderson, S.A. Adediran, P. Charlier, M. Nguyen-Disteche, J-M. Frere, R.A. Nicholas, and R.F.Pratt, “On the Substrate Specificity of Bacterial DD-Peptidases: Evidence from two Series of Peptidoglycan-mimetic Peptides” Biochem.J. 373, 949 (2003).
- M. Nukaga, S. Kumar, K. Nukaga, R. F. Pratt and J. R. Knox, Hydrolysis of Third-generation Cephalosporins by Class C ß-Lactamases: Structures of a Transition State Analog of Cefotaxime in Wild-type and Extended Spectrum Enzymes J. Biol. Chem. 279, 9344 (2004).
- S. Kumar, S. A. Adediran, M. Nukaga and R. F. Pratt, Kinetics of Turnover of Cefotaxime by the Enterobacter cloacae P99 and GCl ß-Lactamases: Two Free Enzyme Forms of the P99 ß-Lactamase Detected by a Combination of Pre- and Post-Steady State Kinetics Biochemistry 43, 2664 (2004).
- Y.M. Ahn and R. F. Pratt, Kinetic and Structural Consequences of the Leaving Group in Substrates of a Class C ß-Lactamase Bioorg. Med. Chem. 12, 1537 (2004).
- N.R. Silvaggi, K. Kaur, S.A. Adediran, R.F. Pratt, and J.A. Kelly, “Toward Better Antibiotics: Crystallographic Studies of a Novel Class of DD-Peptidase/ß-Lactamase Inhibitors” Biochemistry 43, 7046 (2004).
- H.J. Josephine, I. Kumar, and R.F. Pratt, “The Perfect Penicillin? Inhibition of a Bacterial DD-Peptidase by Peptidoglycan-Mimetic ß-Lactams” J. Amer. Chem. Soc. 126, 8122 (2004).
- R. Nagarajan and R.F. Pratt, “Thermodynamic Evaluation of a Covalently Bonded Transition State Analogue Inhibitor: Inhibition of ß-Lactamases by Phosphonates” Biochemistry 43, 9664 (2004).
- S.A. Adediran, D. Cabaret, J.-F. Lohier, M. Wakselman, and R.F. Pratt, “Benzopyranones with Retro-Amide Side Chains as (Inhibitory) ß-Lactamase Substrates” Biooorg. Med. Chem. Lett. 14, 5117 (2004).
- R. Nagarajan and R.F. Pratt, “Synthesis and Evaluation of New Substrate Analogues of the Streptomyces R61 DD-Peptidase: Dissection of a Specific Ligand” J. Org. Chem. 69, 7472 (2004).
- N.R. Silvaggi, H.R. Josephine, A.P. Kuzin, R. Nagarajan, R.F. Pratt and J.A. Kelly, “Crystal Structures of Complexes between the R61 DD-Peptidase and Peptidoglycan-mimetic ß-Lactams: A Non-Covalent Complex with a “Perfect Penicillin” J. Mol. Biol. 345, 521 (2005).
- S.A. Adediran, Z. Zhang, M. Nukaga, T. Palzkill, and R.F. Pratt, “The D-Methyl Group in ß-Lactamase Evolution: Evidence from the Y221G and GC1 Mutants of the Class C ß-Lactamase of Enterobacter cloacae P99″ Biochemistry 44, 7543 (2005).
- S.A. Adediran, Michiyoshi Nukaga, Stèphane Baurin, J.-M. Frère, and R.F. Pratt, “Inhibition of Class D ß-Lactamase by Acyl Phosphates and Phosphonates” Antimicrobial Agents and Chemotherapy 49, 4410-4412 (2005).
- S. Majumdar, S.A. Adediran, Michiyoshi Nukaga, and R. F. Pratt, “Inhibition of Class D ß-Lactamases by Diaroyl Phosphates” Biochemistry 44, 16121-16120 (2005).
- I. Kumar and R.F. Pratt, “Transpeptidation Reactions of a Specific Substrate Catalyzed by the Streptomyces R61 DD-Peptidase: Characterization of a Chromogenic Substrate and Acyl Acceptor” Biochemistry44, 9971-9979 (2005).
- I. Kumar and R.F. Pratt, “Transpeptidation Reactions of a Specific Substrate Catalyzed by the Streptomyces R61 DD-Peptidase: The Structural Basis of Acyl Acceptor Specificity” Biochemistry 44, 30 (2005).
- S.A. Adediran, J.-F, Lohier, D. Cabaret, M. Wakselman, and R.F. Pratt, “Synthesis and reactivity with ß-lactamases of a monobactam bearing a retro-amide side chain” Bioorganic & Medicinal Chemistry 16, 869-871 (2006).
- S.A. Adediran, D. Cabaret, J.A. Sammons, M. Wakselman, and R.F. Pratt, “Synthesis and ß-lactamases reactivity of a-substituted phenaceturates” Bioorganic & Medicinal Chemistry 14, 7023-7033 (2006).
- S. K. Perumal and R.F. Pratt, “Synthesis and Evaluation of Ketophosph(on)ates as ß-Lactamase Inhibitors” J. Org. Chem. 71, 4778-4785 (2006).
- S.A. Adediran, I. Kumar, and R.F. Pratt, “Deacylation Transition States of a Bacterial DD-Peptidase” Biochemistry 45, 13074-13082 (2006).
- H.J. Josephine, P. Charlier, P. Davies, R. A. Nicholas and R.F. Pratt, “Reactivity of penicillin-binging with Peptidoglycan-mimetic ß-Lactams: What’s wrong with these enzymes? Biochemistry45, 15873 (2006).
- A. Moulin, J.H. Bell, R.F. Pratt*, and D. Ringe, “Inhibition of Chymotrypsin by a Complex of Ortho-Vanadate and Benzohydroxamic Acid: Structure of the Inert Complex and its Mechanistic Interpretation” Biochemistry 46, 5982 (2007).
- E. Sauvage, C. Duez, R. Herman, F. Kerff, S. Petrella, J.W. Anderson, S.A. Adediran, R.F. Pratt, J.-M. Frere, and P. Charlier, ” Crystal Structure of the Bacillus subtilis Penicillin-Binding Protein 4a, and its Complex with the Peptidoglycan Mimetic Peptide” J. Mol. Biol.371, 528 (2007).
- P.N. Wyrembrak, K. Babaoglu, R. B. Pelto, B. K. Schoichet, and R. F. Pratt, “O-Aryloxycarbonyl Hydroxamates: New ß-Lactamase Inhibitors That Cross-Link the Active Site” J. Amer. Chem Soc. 129, 9548-9549 (2007).
- I. Kumar, H.R. Josephine, and R.F. Pratt, “Reactions of Peptidoglycan-Mimetic ß-Lactams with Penicillin-Binding Proteins in Vivo and in Membranes”, ACS Chemical Biology 2, 620-624 (2007).
- S. Perumal, S.A. Adediran, and R.F. Pratt, “ß-Ketophosphonates as ß-lactamases inhibitors: Intramolecular cooperativity between the hydrophobic subsites of class D-ß-lactamase” Bioorganic & Medicinal Chemistry 16, 6987-6994 (2008).
- E. Sauvage, A. J. Powell, H. Heilemann, H.R. Josephine, P. Charlier, C. Davies, and R.F. Pratt, “Crystal Structures of Complexes of Bacterial DD-Peptidases with Peptidoglycan-Mimetic Ligands: The Substrate Specificity Puzzle Science Direct 381, 383-393 (2008).
- R.F. Pratt, “Substrate Specificity of Bacterial DD-Peptidases (Penicillin-Binding Proteins)” Cell. Mol. Life Sci. 65, 2138 (2008).
- S.A. Adediran and R.F. Pratt, “Inhibition of Serine ß-lactamases by Vanadate – Catechol Complexes”, Biochemistry47, 9467-9474 (2008).
- E. Sauvage, A. J. Powell, J. Heilemann, H. J. Josephine, P. Charlier, C. Davies. and R. F. Pratt, “Crystal structures of complexes of bacterial DD-peptidase with peptidoglycan-mimetic ligands: The substrate specificity puzzle“ J. Mol. Biol. 381, 383 (2008).
- R. B. Pelto and R. F. Pratt, “Kinetics and Mechanism of Inhibition of a Serine ß-lactamase by O-Aryloxycarbonyl Hydroxamates.” Biochemistry 47, 12037 (2008).
- S.R. Granta, et. al. and R.F. Pratt, “Approaches to the Simultaneous Inactivation of Metallo and Serine- ß-Lactamases” Bioorg. Med. Chem. Lett.19:1618 (2009).
- S. Majumdar and R.F. Pratt, “Inhibition of class A and C ß-Lactamases by diaroyl phosphates” Biochemistry 48, 8285 (2009).
- S. Majumdar and R.F. Pratt, “Intramolecular cooperativity in the reaction of diacyl phosphates with serine ß-Lactamases” Biochemistry48, 8293 (2009).
- E. Sauvage, A. Zervosen, G. Dive, R. Herman, A. Amoroso, B. Joris, E. Fonze, R.F. Pratt, A. Luxen, P. Charlier and F. Kerff, “Structural basis of the inhibition of class A ß-Lactamases and penicillin-binding proteins by 6 ß-iodopenicillanate” J. Amer. Chem. Soc., 313, 15262 (2009).
- S.A. Adediran, D. Cabaret, J.-F. Lohier, M. Wakselman, and R. F. Pratt, “Substituted aryl malonamates as new serine ß-Lactamases substrates: Structure-activity studies”, Bioorganic & Medicinal Chemistry,18, 282-291, (2010).
- L. Dzhekieva, M. Rocaboy, F. Kerff, P. Charlier, E. Sauvage and R.F. Pratt, “Crystal Structure of a Complex between the Actinomadura R39 DD-peptidase and a Peptidoglycan-mimetic Boronate Inhibitor: Interpretation of a Transition State Analogue in Terms of Catalytic Mechanism” Biochemistry 49, 6411 (2010).
- G. Nicola, J. Tomberg, R.F. Pratt, R.A. Nicholas and C. Davies, “Crystal Structures of Covalent Complexes of ß-Lactam Antibiotics with Escherichia coli Penicillin-binding Protein 5: Towards an Understanding of Antibiotic Specificity” Biochemistry, 49, 8094 (2010).
- R.F. Pratt and M.J. McLeish, “Structural Relationships between the Active Sites of ß-Lactam-Recognizing and Amidase Signature Enzymes: Convergent Evolution?” Biochemistry, 49, 9688 (2010).
- R.B. Pelto and R.F. Pratt, “Serendipitous Discovery of a-Hydroxyalkyl Esters as ß-Lactamase Substrates” Biochemistry 49, 10496 (2010).
- S.A. Adediran, I. Kumar, R. Nagarajan, E. Sauvage and R.F. Pratt, “Kinetics of Reactions of the Actinomadura R39 DD-Peptidase with Specific Substrates” Biochemistry, 50, 376 (2011).
- A. Zervosen, R. Herman, F. Kerff, A. Herman, A. Bouillez, F. Prati, R.F. Pratt, J.-M. Frere, B. Joris, A. Luxen, P. Charlier and E. Sauvage, “Unexpected Tricovalent Binding Mode of Boronic Acids within the Active Site of a Penicillin-Binding Protein” J. Amer. Chem. Soc. 133, 10839 (2011).
- V.V. Nemmara, L. Dzhekieva, K.S. Sarkar, S.A. Adediran, C. Duez, R.A. Nicholas and R.F. Pratt, “Substrate Specificity of Low-Molecular Mass Bacterial DD-Peptidases” Biochemistry, 50, 10091-10101 (2011).
- S.A. Adediran, G. Lin, R.B. Pelto and R.F. Pratt, “Crossover inhibition as an indicator of convergent evolution of enzyme mechanisms: A ß-lactamase and a N-terminal nucleophile hydrolase” FEBS Letters, 586, 4186-4189 (2012).
- L. Dzhekieva, S.A. Adediran, R. Herman, F. Kerff, C. Duez, P. Charlier, E. Sauvage and R. F. Pratt, “Inhibition of DD-peptidases by a Specific Trifluoroketone: Crystal Structure of a Complex with the Actinomadura R39 DD-peptidase.” Biochemistry, 52, 2128-2138, 2013.
- V.V. Nemmara, S.A. Adediran, K. Dave, C. Duez, and R. F. Pratt “Dual Substrate Specificity of Bacillus subtilis PBP4a.” Biochemistry, 52, 2627-2637 (2013).
B.S. 1965 University of Melbourne, Australia
Ph.D. 1969 University of Melbourne, Australia