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Rex F. Pratt, Ph.D.
Professor of Biochemistry
and Beach Professor of Chemistry
(860)685-2629
rpratt@wesleyan.edu
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Biochemistry: Enzyme mechanisms; bio-organic chemistry; beta-lactamases
and beta-lactam antibiotics; enzyme inhibitor design.
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 catalyzing reactions of beta-lactam antibiotics,
the penicillins and cephalosporins. One important class of these enzymes,
the beta-lactamases, catalyzes the hydrolysis and thus destruction of beta-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
this groups of enzymes and the relationships between their active sites.
Our approach to problems of mechanisms of this type is through the design,
synthesis, and study of the mode of interaction of enzymes substrates and
inhibitors, through protein chemical modification studies, and through
use of the methods of enzyme kinetics. The rich chemistry of beta-lactams
permits the design of very subtle active site probes.
With respect to the second part of the problem referred to above, we
have been trying to assess the contribution that the conformational mobility
of beta-lactamases contributes to their catalysis. We have also been looking
at the binding of peptides to the peptide antibiotic vancomycin, as a model
for substrate binding processes. The basic thrust of our efforts is best
put as a question. What can we learn about the functioning of a biological
system from a detailed knowledge of a chemistry involved?
Selected Publications
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K. Graves-Woodward and R.F. Pratt, "Reaction of Soluble Penicillin-Binding
Protein 2a of Methicillin-Resistant Staphylococcus aureus with ß-Lactams
and Acyclic Substrates: Kinetics in Homogeneous Solution", Biochem.J.
332,
755 (1998).
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R.F. Pratt and N. Li, "Inhibition of Serine ß-Lactamases by Acyl
Phosph(on)ates: A New Source of Inert Acyl [and Phosph(on)yl]-Enzymes", J.
Amer. Chem. Soc., 120, 4264 (1998).
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R.F. Pratt and N.J. Hammar, "Salicyloyl Cyclic Phosphate, A "Penicillin-Like"
Inhibitor of ß-Lactamases", J. Amer. Chem. Soc. 120,
3004 (1998).
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L. Maveyraud, R.F. Pratt, and J.-P. Samama, "Crystal Structure of an Acylation
Transition-State Analog of the TEM-1 ß-Lactamase. Mechanistic Implications
for the Class A ß-Lactamases", Biochemistry 37,
2622 (1998).
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S.A. Adediran and R.F. Pratt, "ß-Secondary and Solvent Deuterium
Kinetic Isotope Effects on Catalysis by the StreptomycesR61 DD-Peptidase:Comparisons
with a Structurally Similar Class C ß-Lactamase", Biochemistry38,
1469 (1999).
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D. Cabaret, S.A. Adediran, M.J. Garcia-Gonzalez, R.F.Pratt and M. Wakselman,
"Synthesis and Reactivity with ß-Lactamases of "Penicillin-like"
Cyclic Depsipeptides", J. Org. Chem. 64, 713 (1999).
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S.A. Adediran, D. Cabaret, R.F. Pratt, and M. Wakselman, "A "Cephalosporin-like"
Cyclic Depsipeptide: Synthesis and Reaction with
ß-Lactam-Recognizing
Enzymes", Bioorg. Med. Chem. Letts. 9, 341 (1999).
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N.J. Bernstein and R.F. Pratt, "On the Importance of a Methyl Group in
ß-Lactamase Evolution: Free Energy Profiles and Molecular Modeling",
Biochemistry, 38,
10499 (1999).
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K. Graves-Woodward and R.F. Pratt, "Interactions of Soluble Penicillin-Binding
Protein 2a of Methicillin-Resistant Staphylococcus aureus with
Moenomycin" Biochemistry, 38, 10533 (1999).
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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).
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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).
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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).
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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).
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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).
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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).
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R.F. Pratt, "Functional Evolution of the ß-Lactamase Active Site",
J.
Chem. Soc. Perkin Trans. II, 851 (2002).
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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", Biochemistry, 41, 4329 (2002).
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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).
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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).
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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" Biochemistry, 42,
1199 (2003).
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K. Kaur, S.A. Adediran, M.J.K. Lan and R.F. Pratt, "Inhibition of
ß-Lactamases
by Monocyclic Acyl Phosph(on)ates" Biochemistry 42, 1429
(2003).
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D. Cabaret, S.A. Adediran, R.F. Pratt and M. Wakselman, "New Substrates
for ß-Lactam-Recognizing Enzymes: Aryl Malonamates" Biochemistry,
42, 6719 (2003).
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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).
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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).
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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).
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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).
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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).
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H.R. 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).
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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).
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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).
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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).
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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).
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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).
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S.K. Perumal and R.F. Pratt, "Ketophosph(on)ates - A New Lead to Inhibitors of
ß-Lactamases" Faseb Journal, 19, A862-A862 (2005).
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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).
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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).
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Ish 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" Biochemistry, 44,
9971-9979 (2005).
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Ish 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).
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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).
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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).
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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).
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S.A. Adediran, I. Kumar, and R.F. Pratt, "Deacylation Transition States of a
Bacterial DD-Peptidase" Biochemistry, 45, 13074-13082 (2006).
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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).
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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).
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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. DPI 10.1021/ja072370u (2007).
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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, Vol. 2 No. 9, 620-624 (2007).
Education
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B.S. 1965 University of Melbourne, Australia
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Ph.D. 1969 University of Melbourne, Australia
[Chemistry][Wesleyan]
Last updated: August 10, 2007 (rncb)