Abstract

The mutant 554 of TEM-2 β-lactamase was selected for a decrease in the resistance to carbenicillin of an Escherichia coli K12 carrier. The amino acid sequence of the mutant β-lactamase was determined by manual Edman degradation analysis of proteolytic peptides. A single substitution Val for Ala was localized at position 237. The mutant exhibited only 2% of the catalytic efficiency of the wild-type enzyme towards carbenicillin and ticarcillin, whereas it retained 30–60% of the hydrolytic activity towards other penicillin and cephalosporin substrates. Carfecillin, the phenyl ester of the side-chain carboxyl group of carbenicillin, was hydrolysed as a good substrate. This suggests that the behaviour of the mutant enzyme towards carbenicillin may result from ionic rather than steric constraints. A molecular model of the Val-237 TEM-2 mutant suggests possible electrostatic interaction between Glu-171 and the carboxylic group of the side chain of carbenicillin.

References

1

Ambler
R.P.
(
1980
)
The structure of β-lactamase
Phil. Trans. R. Soc. London
,
B289
,
321
331
.

2

Dibeberg
O.
Charlier
P.
Wéry
J.-P.
Dehottay
P.
Dusart
J.
Erpicum
T.
Frère
J.-M.
Ghuysen
J.-M.
(
1987
)
The crystal structure of the β-lactamase of Streptomyces albus G at 0.3 nm resolution
Biochem. J.
,
245
,
911
913
.

3

Herzberg
O.
(
1991
)
Refined crystal structure of β-lactamase from Staphylococcus aureus PCl at 2.0 Å
J. Mol. Biol.
,
217
,
701
719
.

4

Knox
J.R.
Moews
P.C.
(
1991
)
β-Lactamase of Bacillus licheniformis 749/C. Refinement at 2 Å resolution and analysis of hydratation
J. Mol. Biol.
,
220
,
435
455
.

5

Jelsch
C.
Mourey
L.
Masson
J.-M.
Samama
J.-P.
(
1993
)
Crystal structure of Escherichia coli TEM-1 β-lactamase at 1.8 Å
Proteins Struct. Funct. Genet.
,
16
,
364
383
.

6

Bush
K.
(
1989
)
Classification of 3-lactamases: Groups 1, 2a, 2b and 2b′
Antimicrob. Agents Chemother.
,
33
,
264
270
.

7

Bush
K.
(
1989
)
Classification of β-lactamases: Groups 2c, 2d, 2e, 3, and 4
Antimicrob. Agents Chemother
,
33
,
271
276
.

8

Matagne
A.
Misselyn-Bauduin
A.-M.
Joris
B.
Erpicum
T.
Granier
B.
Frère
J.-M.
(
1990
)
The diversity of the catalytic properties of class A β-lactamases
Biochem. J.
,
265
,
131
146
.

9

Dalbadie-McFarland
G.
Riggs
A.
Richards
J.H.
(
1984
)
Directed mutagenesis as a technique to study protein function: application to β-lactamase
Biochem. Soc. Transac.
,
12
,
226
288
.

10

Sigal
S.I.
DeGrado
W.F.
Thomas
B.J.
Pettewey
S.R.
Jr.
(
1984
)
Purification and properties of thiol β-lactamase
J. Biol. Chem.
,
259
,
5327
5332
.

11

Schulz
S.
Richards
J.H.
(
1986
)
Site-saturation of β-lactamase. Production and characterization of mutant β-lactamase with all possible amino acid substitutions at residue 71
,
83
, In
Proc. Natl. Acad. Sci. USA
, pp
1588
1592
.

12

Toth
M.J.
Murgola
E.J.
Schimmel
P.
(
1988
)
Evidence for a unique first position codon-anticodon mismatch in vivo
J. Mol. Biol.
,
201
,
451
454
.

13

Healey
W.J.
Labgold
M.R.
Richards
J.H.
(
1989
)
Substrate specificities in class A β-lactamases: preference for penams vs. cephams. The role of residue 237
Proteins Struct. Funct. Genet.
,
6
,
275
283
.

14

Lenfant
F.
Labia
R.
Masson
J.-M.
(
1990
)
Probing the active site of β-lactamase RTEM-1 by informational suppression
Biochimie
,
72
,
495
503
.

15

Adachi
H.
Ohta
T.
Matsuzawa
H.
(
1991
)
Site-directed mutants, at position 166, of RTEM-1 β-lactamase that form a stable acyl enzyme intermediary with penicillin
J. Biol. Chem.
,
266
,
3186
3191
.

16

Delaire
M.
Lenfant
F.
Labia
R.
Masson
J.-M.
(
1991
)
Site-directed mutagenesis on TEM-1 β-lactamase: role of Glu 166 in catalysis and substrate binding
Prot. Eng.
,
4
,
805
810
.

17

Lenfant
F.
Labia
R.
Masson
J.-M.
(
1991
)
Replacement of lysine 234 affects transition state stabilization in the active site of β-lactamase TEM-1
J. Biol. Chem.
,
266
,
17187
17194
.

18

Sowek
J.A.
Singer
S.B.
Ohringer
S.
Malley
M.F.
Dougherty
T.J.
Gougoutas
J.Z.
Bush
K.
(
1991
)
Substitution of lysine at position 104 or 240 of TEM-1pTZ18Rβ-lactamase enhances the effect of serine-164 substitution on hydrolysis or affinity for cephalosporins and the monobactam aztreonam
Biochemistry
,
30
,
3179
3188
.

19

Delaire
M.
Labia
R.
Samama
J.-P.
Masson
J.-M.
(
1992
)
Site-directed mutagenesis at the active site of Escherichia coli TEM-1 β-lactamase. Suicide inhibitor-resistant mutant reveal the role of arginine 244 and methionine 69 in catalysis
J. Biol. Chem.
,
267
,
20600
20606
.

20

Zafaralla
G.
Manavathu
E.K.
Lerner
S.A.
Mobashery
S.
(
1992
)
Elucidation of the role of arginine-244 in the turnover process of class A β-lactamases
Biochemistry
,
31
,
3847
3852
.

21

Lenfant
F.
Labia
R.
Maveyraud
L.
Samama
J.-P.
Masson
J.-M.
(
1993
)
Site-directed mutagenesis of β-lactamase TEM- 1. Investigating the potential role of specific residues on the activity of Pseudomonas-specific enzymes
Eur. J. Biochem.
,
217
,
3939
3946
.

22

Novick
R.P.
(
1963
)
Analysis by transduction of mutations affecting penicillinase formation in Staphylococcus aureus
J. Gen. Microbiol.
,
33
,
121
136
.

23

Hall
A.
Knowles
J.R.
(
1976
)
Directed selective pressure on a β-lactamase to analyse molecular changes involved in development of enzyme function
Nature
,
264
,
803
804
.

24

Barthélémy
M.
Péduzzi
J.
Verchère-Béaur
C.
Brive-le-Bougenec
C.
Labia
R.
(
1983
)
Single substitution in amino acid composition between TEM-2 β-lactamase and a variant with altered kinetic constants
FEMS Microbiol. Lett.
,
17
,
151
155
.

25

Philippon
A.
Labia
R.
Jacoby
G.
(
1989
)
Extended-spectrum β-lactamases
Antimicrob. Agents Chemother.
,
33
,
1131
1136
.

26

Jacoby
G.A.
Medeiros
A.A.
(
1991
)
More extended-spectrum β-lactamases
Antimicron. Agents Chemother.
,
35
,
1697
1704
.

27

Labia
R.
Andrillon
J.
Le Goffic
F.
(
1973
)
Computerized microacidimetric determination of β-lactamase Michaelis-Menten constants
FEBS Lett.
,
33
,
42
44
.

28

Barthéléme
M.
Péduzzi
J.
Labia
R.
(
1988
)
Complete amino acid sequence of p453-plasmid-mediated PIT-2 β-lactamase (SHV-1)
Biochem. J.
,
251
,
73
79
.

29

Barthélémy
M.
Péduzzi
J.
Bernard
H.
Tancrède
C.
Labia
R.
(
1991
)
Close amino acid sequence relationship between the new plasmid-mediated extended-spectrum β-lactamase MEN-1 and chromosomally encoded enzymes of Klebsiella oxytoca
Biochim. Biophys. Acta
,
1122
,
15
22
.

30

Knott-Hunziker
V.
Waley
S.G.
Orlek
B.S.
Sammes
P.G.
(
1979
)
Penicillinase active sites: labelling of serine-44 in β-lactamase I by 6β-bromopenicillanic acid
FEBS Lett.
,
99
,
59
61
.

31

Fisher
J.
Belasco
J.G.
Charnas
R.L.
Khosla
S.
Knowles
J.R.
(
1980
)
β-Lactamase proceeds via an acyl-enzyme intermediaire. Interaction of Escherichia coli R-TEM enzyme with cefoxitin
Biochemistry
,
19
,
2895
2901
.

32

Knott-Hunzinker
V.
Redhead
K.
Petursson
S.
Waley
S.G.
(
1980
)
β-Lactamase action: isolation of an active-site serine peptide from the Pseudomonas enzyme and a penicillin
FEBS Lett.
,
121
,
8
10
.

33

Ambler
R.P.
Coulson
A.F.W.
Frère
J.-M.
Ghuysen
J.-M.
Joris
B.
Forsman
M.
Levesque
R.C.
Tiraby
G.
Waley
S.G.
(
1991
)
A standard numbering scheme for the class A β-lactamase
Biochem. J.
,
276
,
269
272
.

34

Joris
B.
Ghuysen
J.-M.
Dive
G.
Renard
A.
Dideberg
O.
Charlier
P.
Frère
J.-M.
Kelly
J.A.
Boyington
J.C.
Moews
P.C.
Knox
J.R.
(
1988
)
The active-site-serine penicillin-recognizing enzymes as members of the Streptomyces R61 DD-peptidase family
Biochem. J.
,
250
,
313
324
.

35

Jacob
F.
Joris
B.
Lepage
S.
Dusart
J.
Frère
J.-M.
(
1990
)
Role of the conserved amino acids of the ‘SDN’ loop (Ser130, Asp131 and Asn132) in a class A β-lactamase studied by site-directed mutagenesis
Biochem. J.
,
271
,
399
406
.

36

Ellerby
L.M.
Escobar
W.A.
Fink
A.L.
Mitchinson
C.
Wells
J.A.
(
1990
)
The role of lysine-234 in β-lactamase catalysis probed by site-directed mutagenesis
Biochemistry
,
29
,
5797
5806
.

37

Brannigan
J.
Matagne
A.
Jacob
F.
Damblon
C.
Joris
B.
Klein
D.
Spratt
B.G.
Frere
J.-M.
(
1991
)
The mutation Lys234His yields a class A β-lactamase with a novel pH-dependence
Biochem. J.
,
278
,
673
678
.

38

von Tigerstrom
R.G.
Boras
G.J.
(
1990
)
β-lactamase of Lysobacter enzymoqenes: induction, purification and characterization
J. Gen. Microbiol.
,
136
,
521
527
.

39

Boras
G.J.
Au
S.
Roy
K.L.
von Tigerstrom
R.G.
(
1993
)
β-Lactamase of Lysobacter enzymogenes: cloning, characterization and expression of the gene and comparison of the enzyme to other lactamases
J. Gen. Microbiol.
,
139
,
1245
1252
.

40

Reynaud
A.
Péduzzi
J.
Baron
P.
Barthélémy
M.
Labia
R.
(
1993
) In
33rd Intersci. Conf. Antimicrob. Agents Chemother. Poster No. 1516
Chromosomal cefotaxime-hydrolysing cephalosporinase from Proteus vulgaris RO104 belongs to Ambler's class A.
,

41

Lenzini
M.V.
Ishihara
H.
Dusart
J.
Ogawara
H.
Joris
B.
Van Beeumen
J.
Frère
J.-M.
Ghuysen
J.-M.
(
1988
)
Nucleotide sequence of the gene encoding the active-site serine β-lactamase from Streptomyces cacaoi
FEMS Microbiol. Lett.
,
49
,
371
376
.

42

Strynadka
N.C.J.
Adachi
H.
Jensen
S.E.
Johns
K.
Sielecki
A.
Betzel
C.
Sutoh
K.
James
M.N.G.
(
1992
)
Molecular structure of the acyl-enzyme intermediary in β-lactam hydrolysis at 1.7 Å resolution
Nature
,
359
,
700
705
.

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