Search:
Computing and Library Services - delivering an inspiring information environment

Mutational analysis of the two zinc binding sites of the Bacillus cereus 569/H/9 metallo-β-lactamase

Seny, Dominique de, Prosperi-Meys, Christelle, Bebrone, Carine, Rossolini, Gian Maria, Page, Michael I., Noel, Phillipe, Frere, Jean-Marie and Galleni, Moreno (2002) Mutational analysis of the two zinc binding sites of the Bacillus cereus 569/H/9 metallo-β-lactamase. Biochemical Journal, 363. pp. 687-696. ISSN 0264-6021

Metadata only available from this repository.

Abstract

The metallo-b-lactamase BcII from Bacillus cereus 569/H/9 possesses a binuclear zinc centre. The mono-zinc form of the enzyme displays an appreciably high activity, although full efficiency is observed for the di-zinc enzyme. In an attempt to assign the involvement of the different zinc ligands in the catalytic properties of BcII, individual substitutions of selected amino acids were generated. With the exception of His116Ser (H116S), C221A and C221S, the mono- and di-zinc forms of all the other mutants were poorly active. The activity of H116S decreases by a factor of 10 when compared with the wild type. The catalytic efficiency of C221A and C221S was zinc-dependent. The mono-zinc forms of these mutants exhibited a low activity, whereas the catalytic efficiency of their respective di-zinc forms was comparable with that of the wild type. Surprisingly, the zinc contents of the mutants and the wild-type BcII were similar. These data suggest that the affinity of the b-lactamase for the metal was not affected by the substitution of the ligand. The pH-dependence of the H196S catalytic efficiency indicates that the zinc ions participate in the hydrolysis of the b-lactam ring by acting as a Lewis acid. The zinc ions activate the catalytic water molecule, but also polarize the carbonyl bond of the b-lactam ring and stabilize the development of a negative charge on the carbonyl oxygen of the tetrahedral reaction intermediate. Our studies also demonstrate that Asn233 is not directly involved in the interaction with the substrates.

Item Type: Article
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:
References:

1 Galleni, M., Lamotte-Brasseur, J., Rossolini, G. M., Spencer, J., Dideberg, O. and
Fre' re, J.-M. (2001) Antimicrob. Agents Chemother. 45, 660±663
2 Hussain, M., Carlino, A., Madonna, M. J. and Lampen, J. O. (1985) Cloning and
sequencing of the metallothioprotein beta-lactamase II gene of Bacillus cereus 569/H
in Escherichia coli. J. Bacteriol. 164, 223±229
3 Rasmussen, B. A., Gluzman, Y. and Tally, F. P. (1990) Cloning and sequencing of the
class B beta-lactamase gene (ccrA) from Bacteroides fragilis TAL3636. Antimicrob.
Agents Chemother. 34, 1590±1592
4 Yang, Y., Rasmussen, B. A. and Bush, K. (1992) Biochemical characterization of the
metallo-beta-lactamase CcrA from Bacteroides fragilis TAL3636. Antimicrob. Agents
Chemother. 36, 1155±1157
5 Laraki, N., Franceschini, N., Rossolini, G. M., Santucci, P., Meunier, C., de Pauw, E.,
Amicosante, G., Fre' re, J. M. and Galleni, M. (1999) Biochemical characterization of
the Pseudomonas aeruginosa 101/1477 metallo-beta-lactamase IMP-1 produced by
Escherichia coli. Antimicrob. Agents Chemother. 43, 902±906
6 Laraki, N., Galleni, M., Thamm, I., Riccio, M. L., Amicosante, G., Fre' re, J. M. and
Rossolini, G. M. (1999) Structure of In31, a blaIMP-containing Pseudomonas
aeruginosa integron phyletically related to In5, which carries an unusual array of
gene cassettes. Antimicrob. Agents Chemother. 43, 890±901
7 Senda, K., Arakawa, Y., Nakashima, K., Ito, H., Ichiyama, S., Shimokata, K., Kato, N.
and Ohta, M. (1996) Multifocal outbreaks of metallo-beta-lactamase-producing
Pseudomonas aeruginosa resistant to broad-spectrum beta-lactams, including
carbapenems. Antimicrob. Agents Chemother. 40, 349±353
8 Senda, K., Arakawa, Y., Ichiyama, S., Nakashima, K., Ito, H., Ohsuka, S., Shimokata,
K., Kato, N. and Ohta, M. (1996) PCR detection of metallo-beta-lactamase gene
(blaIMP) in Gram-negative rods resistant to broad-spectrum beta-lactams. J. Clin.
Microbiol. 34, 2909±2913
9 Car®, A., Due! e, E., Galleni, M., Fre' re, J.-M. and Dideberg, O. (1998) 1.85 AI
resolution structure of the zinc (II) beta-lactamase from Bacillus cereus. Acta
Crystallogr. Ser. D 54, 313±323
10 Concha, N. O., Rasmussen, B. A., Bush, K. and Herzberg, O. (1996) Crystal structure
of the wide-spectrum binuclear zinc beta-lactamase from Bacteroides fragilis. Structure
4, 823±836
11 Concha, N. O., Janson, C. A., Rowling, P., Pearson, S., Cheever, C. A., Clarke, B. P.,
Lewis, C., Galleni, M., Fre' re, J. M., Payne, D. J., Bateson, J. H. and Abdel-Meguid,
S. S. (2000) Crystal structure of the IMP-1 metallo beta-lactamase from Pseudomonas
aeruginosa and its complex with a mercaptocarboxylate inhibitor : binding
determinants of a potent, broad-spectrum inhibitor. Biochemistry 39, 4288±4298
12 Car®, A., Pares, S., Due! e, E., Galleni, M., Duez, C., Fre' re, J. M. and Dideberg, O.
(1995) The 3-D structure of a zinc metallo-beta-lactamase from Bacillus cereus
reveals a new type of protein fold. EMBO J. 14, 4914±4921
13 Paul-Soto, R., Bauer, R., Fre' re, J. M., Galleni, M., Meyer-Klaucke, W., Nolting, H.,
Rossolini, G. M., de Seny, D., Hernandez-Valladares, M., Zeppezauer, M. and Adolph,
H. W. (1999) Mono- and binuclear Zn2+-beta-lactamase. Role of the conserved
cysteine in the catalytic mechanism. J. Biol. Chem. 274, 13242±13249
14 Baldwin, G. S., Galdes, A., Hill, H. A. O., Smith, B. E., Waley, S. G. and Abraham,
E. P. (1978) Histidine residues of zinc ligands in beta-lactamase II. Biochem. J. 175,
441±447
15 Crowder, M. W., Wang, Z., Franklin, S. L., Zovinka, E. P. and Benkovic, S. J. (1996)
Characterization of the metal-binding sites of the beta-lactamase from Bacteroides
fragilis. Biochemistry 35, 12126±12132
16 Bicknell, R. and Waley, S. G. (1985) Cryoenzymology of Bacillus cereus betalactamase
II. Biochemistry 24, 6876±6887
17 Bicknell, R., Schaffer, A., Waley, S. G. and Auld, D. S. (1986) Changes in the
coordination geometry of the active-site metal during catalysis of benzylpenicillin
hydrolysis by Bacillus cereus beta-lactamase II. Biochemistry 25, 7208±721518 Bounaga, S., Laws, A. P., Galleni, M. and Page, M. I. (1998) The mechanism of
catalysis and the inhibition of the Bacillus cereus zinc-dependent beta-lactamase.
Biochem. J. 331, 703±711
19 Wang, Z., Fast, W. and Benkovic, S. J. (1999) On the mechanism of the metallo-betalactamase
from Bacteroides fragilis. Biochemistry 38, 10013±10023
20 Murphy, B. P. and Pratt, R. F. (1989) Evidence for an oxyanion hole in serine betalactamases
and DD-peptidases. Biochem. J. 258, 765±768
21 Sutton, B. J., Artymiuk, P. J., Cordero-Borboa, A. E., Little, C., Phillips, D. C. and
Waley, S. G. (1987) An X-ray-crystallographic study of beta-lactamase II from
Bacillus cereus at 0.35 nm resolution. Biochem. J. 248, 181±188
22 Lim, H. M., Iyer, R. K. and Pe' ne, J. J. (1991) Site-directed mutagenesis of
dicarboxylic acids near the active site of Bacillus cereus 5/B/6 beta-lactamase II.
Biochem. J. 276, 401±404
23 Lim, H. M. and Pe' ne, J. J. (1989) Mutations affecting the catalytic activity of Bacillus
cereus 5/B/6 beta-lactamase II. J. Biol. Chem. 264, 11682±11687
24 De Meester, F., Boris, B., Reckinger, G., Bellefroid-Bourguignon, C., Fre' re, J. M. and
Waley, S. G. (1987) Automated analysis of enzyme inactivation phenomena.
Application to beta-lactamases and DD-peptidases. Biochem. Pharmacol. 36,
2393±2403
25 Yang, Y., Keeney, D., Tang, X., Can®eld, N. and Rasmussen, B. A. (1999) Kinetic
properties and metal content of the metallo-beta-lactamase CcrA harboring selective
amino acid substitutions. J. Biol. Chem. 274, 15706±15711
Received 8 August 2001/8 January 2002 ; accepted 6 February 2002
26 Haruta, S., Yamaguchi, H., Yamamoto, E. T., Eriguchi, Y., Nukaga, M., O'Hara, K. and
Sawai, T. (2000) Functional analysis of the active site of a metallo-beta-lactamase
proliferating in Japan. Antimicrob. Agents Chemother. 44, 2304±2309
27 Chantalat, L., Due! e, E., Galleni, M., Fre' re, J.-M. and Dideberg, O. (2000) Structural
effects of the active site mutation cysteine to serine in Bacillus cereus zinc-betalactamase.
Protein Sci. 9, 1402±1406
28 Yanchak, M. P., Taylor, R. A. and Crowder, M. W. (2000) Mutational analysis of
metallo-beta-lactamase CcrA from Bacteroides fragilis. Biochemistry 39, 11330±11339
29 Kiefer, L. L. and Fierke, C. A. (1994) Functional characterization of human carbonic
anhydrase II variants with altered zinc binding sites. Biochemistry 33, 15233±15240
30 Chevrier, B., D'Orchymont, H., Schalk, C., Tarnus, C. and Moras, D. (1996) The
structure of the Aeromonas proteolytica aminopeptidase complexed with a
hydroxamate inhibitor. Involvement in catalysis of Glu151 and two zinc ions
of the cocatalytic unit. Eur. J. Biochem. 237, 393±398
31 Zang, T. M., Hollman, D. A., Crawford, P. A., Crowder, M. W. and Makaroff, C. A.
(2001) Arabidopsis glyoxalase II contains a zinc/iron binuclear metal center that is
essential for substrate binding and catalysis. J. Biol. Chem. 276, 4788±4795
32 de Seny, D., Heinz, U., Wommer, S., Kiefer, M., Meyer-Klauck, W., Galleni, M.,
Fre' re, J. M., Bauer, R. and Adolph, H. W. (2001) Metal ion binding and coordination
geometry for wild type and mutants of metallo-b-lactamase from Bacillus cereus
569/H/9 (BcII) : a combined thermodynamic, kinetic, and spectroscopic approach.

Depositing User: Sara Taylor
Date Deposited: 11 Feb 2008 14:24
Last Modified: 20 Oct 2008 10:27
URI: http://eprints.hud.ac.uk/id/eprint/523

Item control for Repository Staff only:

View Item

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