Transcript Secondary Drug Resistance Mutation of TEM-1

Secondary Drug Resistance Mutation
of TEM-1 -lactamase
Amino acid sequence determinants of -lactamase
structure and activity
J. Mol. Biol. 258, 688-703 (1996)
A natural polymorphism in -lactamase is a global
suppressor
Proc. Natl. Acad. Sci. USA 94, 8801-8806 (1997)
A secondary drug resistance mutation of TEM-1 lactamase that suppresses misfolding and aggregation
Proc. Natl. Acad. Sci. USA 98, 283-288 (2001)
授課教授：楊孝德老師
指導教授：呂平江老師
報告學生：徐芝琪
Background
-lactam antibiotics inhibit the cross-linking
transpeptidase and interfere with the synthesis of the
bacterial cell wall.
TEM-1 -lactamase provides the major mechanism
of plasmid-mediated -lactam resistance.
Clavulanic acid is the natural inhibitor of -lactamase.
Natural variants of TEM-1 have more efficient at
catalyzing -lactam hydrolysis or more resistant to
inhibitors.
Previous study in 1996
In a previous study, they performed saturation
mutagenesis in which each of the 263 codons of
the gene for TEM-1 -lactamase were randomized
by oligonucleotide-directed mutagenesis.
The essential residues are largely located in the
active site pocket or at buried positions in the
protein.
One of the critical residues that is located at a
buried position in the enzyme is Leu76. However,
buried residues are often tolerant of substitutions
of other hydrophobic residues.
Previous study in 1997
Introducing the gene for the L76N -lactamase
into a mutS mismatch repair deficient strain.
Then the plasmid DNA was isolated and used to
transform E. coli XL1-Blue.
The transformants were grown on agar plates
containing 500 ug/ml ampicillin.
The DNA sequence of six revertants were
determined:
One mutant reverted codon 76 to Leu.
One mutant mutated codon 76 to Ile.
Four mutants converted codon 182 from Met to Thr.
Naturally occurring
extended-spectrum and inhibitor-resistant
-lactamases
‘‘Secondary’’ mutations
compensate for defects?
Among the 90 TEM-1 natural isolates found to date,
substitutions at residues that directly affect substrate
binding and catalysis (R164, G238, and E104) or
inhibitor binding (M69 and R244) are repeatedly found.
M182T does not have a direct role in catalysis or
substrate/inhibitor binding and is never found alone.
In the 3D structure, Met182 is far away from the sites of
the coupled primary mutation so that any suppression
mechanism must operate at a distance.
Minimal inhibitory concentration of
ampicillin and specific activities
•M182T causes a slight (20–30%) increase in sensitivity to ampicillin and
comparable decrease in the -lactamase activity.
•However, in combination with L76N, the M182T suppressor restores ampicillin
resistance to near wild-type values and increases the -lactamase activity to 40% of
the wild type.
Kinetic parameters of -lactamase
catalysis of ampicillin hydrolysis
•The catalytic efficiency of L76N is 30% of wild type, but M182T by itself
has no effect on kcat or Km values.
•When M182T is combined with L76N, it causes a slight increase in
activity against ampicillin, resulting in a double mutant with 53% the
catalytic efficiency of the wild type.
Expression Levels of Mutant and Wild-Type -lactamase
206%
10715%
+70%
 When combined with L76N,
the suppressor M182T
largely restores the amount
of -lactamase.
 If the low expression of
L76N is due to increased
proteolysis, expressing it in
SB646 strain should
enhance its expression
levels.
 But the majority (67%) of
L76N is in a catalytically
inactive.
 -lactamase express to
the correctly folded,
soluble, and
enzymatically active form
in M182T/L76N mutant.
Equilibrium denaturation of TEM-1 -lactamase
 CD spectroscopy:
The final protein concen-tration
was 15 mM.
For the wild-type protein, the first
transition (NI) has a mid-point
at 0.8 M and the second (IU) at
2.3 M.
 Fluorescence spectroscopy:
The final protein concen-tration
was 0.6 mM (excited at 290 nm,
and emission was monitored at
340 nm).
Fluorescence reveals only one
transition corresponds to the first
transition observed by CD.
Thermodynamic parameters for the
equilibrium transitions
The native protein is destabilized only in the double
Aggregation properties of wildtype and mutant -lactamases
Because the effects of these mutations cannot be
attributed to changes in stability or intrinsic catalytic
activity, the remaining possibility is an effect on
protein folding and/or aggregation.
The periplasmic extracts were denatured in GdnHCl
and then diluted to allow refolding.
These results suggest that the L76N mutation
promotes the accumulation of misfolded and
aggregated -lactamase.
Conclusion
Because the majority of the double mutant
(L76N/M182T) is soluble and active within
the periplasm, the M182T mutation
suppresses this enhanced aggregation
and misfolding caused by the L76N
mutation.
M182T may destroy an aggregation-prone
site created by L76N or alter the folding
mechanism to kinetically disfavor
Synthesis of the bacterial cell wall
Back
-lactamase catalyzes
the hydrolysis of -lactam ring
Most clinically important -lactamases employ a
mechanism involving a nucleophilic serine
residue and proceeding via a hydrolytically labile
acyl-enzyme intermediate.
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Inhibition of serine -lactamase by
clavulanic acid
Back
It inhibits serine -lactamases through a complex
mechanism involving sequential acylation,
decarboxylation and loss of a four carbon
fragment to give stable acyl-enzyme complexes.
Oligonucleotide-directed mutagenesis
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Structure
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