Zinc Metallo--Lactamase from Bacteroides fragilis: A Quantum
Chemical Study on Model Systems of the Active Site
Natalia Dı ´az, Dimas Sua ´ rez,
²
and Kenneth M. Merz, Jr.*
Contribution from the Departamento de Quı ´mica Fı ´sica y Analı ´tica, UniVersidad de OViedo,
Julia ´ n ClaVerı ´a 33006, OViedo. Spain, and 152 DaVey Laboratory, Department of Chemistry,
The PennsylVania State UniVersity, UniVersity Park, PennsylVania 16802-6300
ReceiVed December 21, 1999
Abstract: Quantum chemical optimizations of the small model systems ([Zn(NH
3
)
3
(H
2
O)]
2+
,
[Zn(NH
3
)
3
(OH)]
+
,
[Zn(NH
3
)(SH) (HCOO)(OH)]
-1
(H
2
O) and [Zn(NH
3
)(SH)(HCOO)(H
2
O)] (H
2
O)) were performed at different
levels of quantum mechanical theory (HF/6-31G*, B3LYP/6-31G*, and MP2/6-31G*) to characterize the Zn-
ligand bonds for the Zn1 and Zn2 binding sites of metallo--lactamases. The nature of the zinc coordination
environment was further studied by considering larger mononuclear complexes at the B3LYP/6-31G*//HF/
6-31G* level of theory ([Zn(Me-Im)
3
(H
2
O)]
2+
,
[Zn(Me-Im)(SCH
3
)(CH
3
COO)(H
2
O)](H
2
O), etc.). The structure
and properties of a series of binuclear model compounds showing an hydroxy-mediated Zn1‚‚‚Zn2 interaction
were also analyzed at the same level of theory. One of the binuclear models with a global charge of +2,
reproduces the main structural features of the Bacteroides fragilis active site as determined by X-ray
crystallography. The proposed -lactamase model has a monoprotonated state characterized by a strong H-bond
interaction between a zinc-shared water molecule and a Zn2-bound Asp carboxylic group. The theoretical
results are discussed in the context of experimental kinetic and structural data on the B. fragilis active site,
resulting in insights into the nature of the zinc-ligand interactions, the location of the mechanistically relevant
water molecules, and the actual protonation state of the active site. By combining the present results with
previous theoretical and experimental work, mechanistic details for the mode of action of zinc -lactamases
are discussed.
Introduction
-lactam antibiotics account for 50% of the world’s total
antibiotic market.
1
The various families of -lactam antibiotics
differ in their spectrum of antibacterial activity and in their
susceptibility to -lactamase enzymes. -lactamases, which
constitute the most common and growing form of antibacterial
resistance,
2,3
catalyze the hydrolysis of -lactams to give ring-
opened -amino acids which are no longer effective as inhibitors
against their targets: bacterial membrane-bound transpeptidases
enzymes.
The mechanistic division of -lactamases is into serine
proteases (classes A, C, and D; according to their amino acid
sequence homology) and zinc enzymes (class B).
3
For the serine
proteases, the catalytic mechanism involves the formation of
an acyl enzyme intermediate generated by the nucleophilic attack
on the -lactam of the hydroxyl group of the essential serine
residue.
3
Fortunately, through the screening of natural chemical
resources (i.e., plants) as well as through molecular studies on
serine -lactamases effective inhibitors have been discovered
(e.g., cefoxitin, clavulanic acid, penicillanic acid sulfones, etc.).
4
These and other mechanism-based inhibitors selectively prevent
substrate binding between -lactamases and -lactams while
not interfering with cellular metabolism. On the other hand, the
metallo--lactamases
5
(class B) require Zn(II) ions for their
ability to efficiently hydrolyze nearly all -lactams including
the versatile broad-spectrum antibacterial carbapenem deriva-
tives (see Scheme 1).
The first metallo--lactamase to be identified was found in
the relatively innocuous bacteria Bacillus cereus in the 1960s.
Since this time most mechanistic and structural information of
Zn--lactamases has been derived from the B. cereus enzyme.
6-10
This enzyme is remarkably adaptable and is able to function
with either one or two zinc ions, which are liganded by active-
²
On leave from Departamento de Quı ´mica Fı ´sica y Analı ´tica, Univer-
sidad de Oviedo, Spain.
(1) The Chemistry of -Lactams. Page, M. I., Ed.; Blackie
Academic&Professional: London, 1992.
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228.
(4) Pratt, R. F. -lactamase: Inhibition. In ref 1, pp 229-265.
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Chem. Biol. 1999, 3, 614-622.
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J. M.; Carfi, A.; Dideberg, O.; Wouters, J.; Hemmingsen, L.; Bauer, R.
Biochemistry 1999, 38, 16500-16506.
(7) Paul-Soto, R.; Bauer, R.; Fre `re, J. M.; Galleni, M.; Meyer-Klaucke,
W.; Nolting, H.; Rossolini, G. M.; de Seny, D.; Herna ´ndez-Valladares, M.;
Zeppezauer, M.; Adolph, H. W. J. Biol. Chem. 1999, 274, 13242-13249.
(8) Fabiane, S. M.; Sohi, M. K.; Wan, T.; Payne, D. J.; Bateson, J. H.;
Mitchell, T.; Sutton, B. J. Biochemistry 1998, 37, 12404-12411.
(9) Carfi, A.; Due ´e, E.; Galleni, M.; Fre `re, J. M.; Dideberg, O. Acta
Crystallogr. D 1998, 54, 313-323.
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1998, 331, 703-711.
Scheme 1
4197 J. Am. Chem. Soc. 2000, 122, 4197-4208
10.1021/ja994462s CCC: $19.00 © 2000 American Chemical Society
Published on Web 04/18/2000