Computing and Library Services - delivering an inspiring information environment

The inactivation of bacterial DD-peptidase by β-sultams

Llinas, Antonio, Ahmed, Naveed, Cordaro, Massimiliano, Laws, Andrew P., Frere, Jean-Marie, Delmarcelle, Michael, Silvaggi, Nicholas R., Kelly, Judith A. and Page, Michael I. (2005) The inactivation of bacterial DD-peptidase by β-sultams. Biochemistry, 44 (21). pp. 7738-7746. ISSN 1520-4995

Metadata only available from this repository.


N-Acyl--sultams are time-dependent, irreversible active site-directed inhibitors of Streptomyces R61 DD-peptidase. The rate of inactivation is first order with respect to -sultam concentration, and the second-order rate constants show a dependence on pH similar to that for the hydrolysis of a substrate. Inactivation is due to the formation of a stable 1:1 enzyme-inhibitor complex as a result of the active site serine being sulfonylated by the -sultam as shown by ESI-MS analysis and by X-ray crystallography. A striking feature of the sulfonyl enzyme is that the inhibitor is not bound to the oxyanion hole but interacts extensively with the "roof" of the active site where the Arg 285 is located

Item Type: Article
Additional Information: Copyright © 2005 American Chemical Society
Subjects: Q Science > Q Science (General)
Q Science > QD Chemistry
Schools: School of Applied Sciences
School of Applied Sciences > Biomolecular Sciences Research Centre
Related URLs:

1. Spratt, B. G. (1975) Distinct penicillin binding proteins involved
in the division elongation and shape of Escherichia coli K12, Proc.
Natl. Acad. Sci. U.S.A. 72, 2999-3003.
2. Koch, A. L. (2000) Penicillin binding proteins, â-lactams, and
lactamases: offensives attacks, and defensive countermeasures,
Crit. ReV. Microbiol. 26, 205-220.
3. Ghuysen, J. M. (1991) Serine beta-lactamases and penicillinbinding
proteins, Annu. ReV. Microbiol. 45, 37-67.
4. Goffin, C., and Ghuysen, J. M. (2002) Biochemistry and comparative
genomics of SxxK superfamily acyltransferases offer a clue
to the mycobacterial paradox: presence of penicillin-susceptible
target proteins versus lack of efficiency of penicillin as therapeutic
agent, Microbiol. Mol. Biol. ReV. 66, 702-738.
5. Tipper, D. J., and Strominger, J. L. (1965) Mechanism of action
of penicillins: a proposal based on their structural similarity to
acyl-D-alanyl-alanine, Proc. Natl. Acad. Sci. U.S.A. 54, 1133-
6. Kelly, J. A., Dideberg, O., Charlier, P., Wery, J.-P., Libert, M.,
Moews, P. C., Knox J. R., Duez, C., Fraipont, C., Joris, B., Dusart,
J., Fre`re, J.-M., and Ghuysen, J.-M. (1986) On the origin of
bacterial resistance to penicillin: comparison of a â-lactamase
and a penicillin target, Science 231, 1429-1431.
7. Massova, I., and Mobashery, S. (1999) Structural and mechanistic
aspects of evolution of beta-lactamases and penicillin-binding
proteins, Curr. Pharm. Des. 5, 929-937.
8. Anderson, J. W., and Pratt, R. F. (2000) Dipeptide binding to the
extended active site of the Streptomyces R61 D-alanyl-D-alanine
peptidase: The path to a specific substrate, Biochemistry 39,
9. Fre`re, J.-M., and Joris, B. (1985) Penicillin-sensitive enzymes in
peptidoglycan biosynthesis, CRC Crit. ReV. Microbiol. 11, 299-
10. Kelly, J. A., Knox, J. R., Moews, P. C., Hite, G. J., Bartolone, J.
B., Zhao, H., Joris B., Fre`re, J.-M., and Ghuysen, J.-M. (1985)
2.8Å Structure of penicillin-sensitive D-alanylcarboxypeptidasetranspeptidase
from Streptomyces R61 and complexes with
â-lactams, J. Biol. Chem. 260, 6449-6458.
11. Jamin, M., Adam, M., Damblon, C., Christiaens, L., and Fre`re,
J.-M. (1991) Accumulation of acyl-enzyme in DD-peptidasecatalysed
reactions with analogues of peptide substrates, Biochem.
J. 280, 499-506.
12. Kiener, P. A., and Waley, S. G. (1978) Reversible inhibitors of
penicillinases, Biochem. J. 169, 197-204; Beesley, T., Gascoyne,
N., Knott-Hunziker, V., Petursson, S., Waley, S. G., Jaurin, B.,
and Grunstrom, T. (1983) The inhibition of class C beta-lactamases
by boronic acids, Biochem. J. 209, 229-233; Crompton, I. E.,
Cuthbert, B. K., Lowe, G., and Waley, S. G. (1988) Beta-lactamase
inhibitors. The inhibition of serine beta-lactamases by specific
boronic acids, Biochem. J. 251, 453-459; Ness, S., Martin, R.,
Kindler, A. M., Paetzel, M., Gold, M., Jensen, S. E., Jones, J. B.,
and Strynadka, N. C. (2000) Structure-based design guides the
improved efficacy of deacylation transition state analogue inhibitors
of TEM-1 beta-Lactamase, Biochemistry 39, 5312-5321;
Powers, R. A., Blazquez, J., Weston, G. S., Morosini, M. I.,
Baquero, F., and Shoichet, B. K. (1999) The complexed structure
and antimicrobial activity of a non-beta-lactam inhibitor of AmpC
beta-lactamase, Protein Sci. 8, 2330-2337.
13. Pratt, R. F. (1989) Inhibition of a class C â-lactamase by a specific
phosphonate monoester, Science 246, 917-919; Rahil, J., and
Pratt, R. F. (1991) Phosphonate monoester inhibitors of class A
beta-lactamases, Biochem. J. 275, 793-795; Rahil, J., and Pratt,
R. F. (1992) Mechanism of inhibition of the class C beta-lactamase
of Enterobacter cloacae P99 by phosphonate monoesters, Biochemistry
31, 5869-5878; Rahil, J., and Pratt, R. F. (1993)
Structure-activity relationships in the inhibition of serine betalactamases
by phosphonic acid derivatives, Biochem. J. 296, 389-
393; Lobkovsky, E., Billings, E. M., Moews, P. C., Rahil, J., Pratt,
R. F., and Knox, J. R. (1994) Crystallographic structure of a
phosphonate derivative of the Enterobacter cloacae P99 cephalosporinase:
mechanistic interpretation of a beta-lactamase transition-
state analog, Biochemistry 33, 6762-6772; Rahil, J., and
Pratt, R. F. (1994) Characterization of covalently bound enzyme
inhibitors as transition-state analogs by protein stability measurements:
phosphonate monoester inhibitors of a beta-lactamase,
Biochemistry 33, 116-125; Kaur, K., Lan, M. J., and Pratt, R. F.
(2001) Mechanism of inhibition of the class C beta-lactamase of
Enterobacter cloacae P99 by cyclic acyl phosph(on)ates: rescue
by return, J. Am. Chem. Soc. 123, 10436-10443; Kaur, K.,
Adediran, S. A., Lan, M. J.,and Pratt, R. F. (2003) Inhibition of
beta-lactamases by monocyclic acyl phosph(on)ates, Biochemistry
42, 1529-1536; Nagarajan, R., and Pratt, R. F. (2004) Thermodynamic
evaluation of a covalently bonded transition state
analogue inhibitor: inhibition of â-lactamases by phosphonates,
Biochemistry 43, 9664-9673.
14. Baxter, N. J., Laws, A. P., Rigoreau, L., and Page, M. I. (1996)
The hydrolytic reactivity of â-sultams, J. Chem. Soc., Perkin
Trans. 2, 2245-2246.
15. Beardsell, M., Hinchliffe, P. S., Wood, J. M., Wilmouth, R. C.,
Schofield, C. J., and Page, M. I. (2001) â-SultamssA novel class
of serine protease inhibitors, Chem. Commun., 497-498.
16. Hinchliffe, P. S., Wood, J. M., Davis, A. M., Austin, R. P., Beckett,
R. P., and Page, M. I. (2003) Structure-reactivity relationships
in the inactivation of elastase by â-sultams, Org. Biomol. Chem.
1, 67-80.
17. Lee, W., McDonough, M. A., Kotra, L., Li, Z. H., Silvaggi, N.
R., Takeda, Y., Kelly, J. A., and Mobashery, S. (2001) A 1.2Å
snapshot of the final step of bacterial cell wall biosynthesis, Proc.
Natl. Acad. Sci. U.S.A. 98, 1427-1431.
18. Silvaggi, N. R., Anderson, J. W., Brinsmade, S. R., Pratt, R. F.,
and Kelly, J. A. (2003) Crystal structure of phosphonate-inhibited
D-Ala-D-Ala peptidase reveals an analog of a tetrahedral transition
state, Biochemistry 42, 1199-1208.
19. Silvaggi, N. R., Kaur, K., Adediran, S. A., Pratt, R. F., and Kelly
J. A. (2004) Toward better antibiotics: crystallographic studies
of a novel class of DD-peptidase/beta-lactamase inhibitors, Biochemistry
43, 7046-7053.
20. Kuzin, A. P., Liu, H., Kelly, J. A., and Knox, J. R. (1995) Binding
of cephalothin and cefotaxime to D-ala-D-ala peptidase reveals a
functional basis of a natural mutation in a low-affinity penicillinbinding
protein and in extended-spectrum â-lactamases, Biochemistry
34, 9532-9540.
21. Fre`re, J.-M., Leyh-Bouille, M., Ghuysen, J.-M., Nieto, M., and
Perkins, H. R. (1976) Extracellular DD-carboxypeptidasestranspeptidases
from Streptomyces, Methods Enzymol. 45, 610-
22. Champseix, A., Chanet-Ray, J., Ettienne, A., Le Berre, A., Masson,
J., Napierale, C., and Vessiere, R. (1985) Synthe`ses de â-sultames
(thiaze´tidines-1,2 dioxyde-1,1), Bull. Soc. Chim. Fr. 3, 463-472.
23. Dean, J. A., Ed. (1973) Lange’s Handbook of Chemistry, 11 ed.,
pp 5-7, McGraw-Hill, New York.
24. Kelly, J. A., Knox, J. R., Zhao, H., Fre`re, J.-M., and Ghuysen,
J.-M. (1989) Crystallographic mapping of â-lactams bound to a
D-alanyl-D-alanine peptidase target enzyme, J. Mol. Biol. 209,
25. Otwinowski, Z., and Minor, W. (1997) Processing of X-ray
diffraction data collected in oscillation mode, Methods Enzymol.
276, 307-326.
26. Bru¨nger, A. T., Adams, P. D., Clove, G. M., Delano, W. L., Gros,
P., Grosse-Kunstleve, R. W., Jiang, J.-S., Kuszewski, J., Nilges,
M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and
Warren, G. L. (1998) Crystallography and NMR system: A new
software suite for macromolecular structure determination, Acta
Crystallogr. D54, 905-921.
27. Sheldrick, G. M., and Schneider, T. R. (1997) SHELXL: highresolution
refinement, Methods Enzymol. 277, 319-343.
28. McRee, D. E. (1999) XtalView/XfitsA versatile program for
manipulating atomic coordinates and electron density, J. Struct.
Biol. 125, 156-165.
29. Moews, P. C., Knox, J. R., Dideberg, O., Charlier, P., and Fre`re,
J.-M. (1990) The structure of the â-lactamase of Bacillus
licheniformis 749/C at 2.0Å resolution, Proteins: Struct., Funct.,
Genet. 7, 156-171.
30. Massova, I., and Mobashery, S. (1998) Kinship and diversification
of bacterial penicillin-binding proteins and â-lactamases, Antimicrob.
Agents Chemother. 42, 1-17.
31. Varetto, L., Fre`re, J.-M., Nguyen-Disteche, M., Ghuysen, J.-M.,
and Houssier, C. (1987) The pH dependence of the active-site
serine DD-peptidase of Streptomyces R61, Eur. J. Biochem. 162,
32. Fisher, J. F., Meroueh, S. O., and Mobashery, S. (2005) Bacterial
resistance to â-lactam antibiotics: compelling opportunism,
compelling opportunity, Chem. ReV. 105, 395-424.
33. McDonough, M. A., Anderson, J. W., Silvaggi, N. R., Pratt, R.
F., Knox, J. R., and Kelly, J. A. (2002) Structures of two kinetic
intermediates reveal species specificity of penicillin binding
proteins, J. Mol. Biol. 322, 111-122.
Inactivation of Peptidase by â-Sultams Biochemistry, Vol. 44, No. 21, 2005 7745 34. Fahrney, D. E., and Gold, A. M. (1963) Sulfonyl fluorides as
inhibitors of esterases. I. Rates of reaction with acetylcholinesterase,
R-chymotrypsin and trypsin, J. Am. Chem. Soc. 85, 997-
1000; Turini, P., Kurooka, S., Steer, M., Corbascio, A. M., and
Singer, T. P. (1967) The action of phenylmethysulfonyl fluoride
on human acetylcholinesterase, chymotrypsin and trypsin, J.
Pharm. Exp. Ther. 167, 98-104; Lawrence, J. B. (1972) Urea
denaturation of active-site spin-labeled R-chymotrypsin, Biochemistry
11, 2921-2924; Lawrence, J. B., and Shan, S. W. (1974)
Spin-labeled sulfonyl fluorides as active site probes of protease
structure. I. Comparison of the active site environments in
R-chymotrypsin and trypsin, J. Biol. Chem. 249, 1668-1677;
Hideaki, T., Hiroyasu, N., and Lawrence, J. B. (1984) Synthesis
and evaluation of 19-F labelled sulfonyl fluorides as probes of
protease structure: R-chymotrypsin, Biochem. J. 96, 349-355.
35. Gold, A. M., and Fahrney, D. (1964) Sulfonyl fluorides as
inhibitors of esterases. II. Formation and Reactions of phenylmethanesulfonyl
alpha-chymotrypsin, Biochemistry 3, 783-791.
36. Page, M. I., and Williams, A. (1997) in Organic and Bio-organic
Mechanisms, pp 52-80, Longmans, Harlow.
37. Baxter, N. J., Laws, A., Rigoreau, L. J. M., and Page, M. I. (2000)
Reactivity and mechanism in the hydrolysis of â-sultams, J. Am.
Chem. Soc. 122, 3375-3385.
38. Kraulis, P. J. (1991) J. Appl. Crystallogr. 24, 946-950; Fenn, T.
D., Ringe, D., and Petsko, G. A. (2003) J. Appl. Crystallogr. 36,

Depositing User: Sara Taylor
Date Deposited: 08 Feb 2008 10:57
Last Modified: 20 Oct 2008 08:46


Downloads per month over past year

Repository Staff Only: item control page

View Item View Item

University of Huddersfield, Queensgate, Huddersfield, HD1 3DH Copyright and Disclaimer All rights reserved ©