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Type of Document Dissertation Author Cha, Jooyoung URN etd-04192007-123301 Title Penicillin-Binding Proteins in Pathogens: Characterizations of Catalytic Properties in Antibiotic Resistance Degree Doctor of Philosophy Department Chemistry and Biochemistry Advisory Committee
Advisor Name Title Brian M. Baker Committee Member Patricia L. Clark Committee Member Shahriar Mobashery Committee Member Thomas L. Nowak Committee Member Keywords
- Regulatory protein BlaR1 and MecR1
- lysine carboxylation
- beta-lactamase
- Staphylococcus aureus
- Penicillin-binding proteins
- Tp47
Date of Defense 2007-04-17 Availability restricted Abstract Treponema pallidum is the causative agent of syphilis and is exquisitely sensitiveto penicillins and other ��-lactam antibiotics. The Tp47, the novel 47 kDa membrane-bound
lipoprotein is abundant penicillin-binding protein (PBP) in T. pallidum.
Interestingly its X-ray structure reveals that it is distinct from any known PBPs or ��-
lactamases. Our studies further revealed that Tp47 hydrolyzes the ��-lactam bonds of
penicillins. The reaction is unique and represents a bona fide novel mechanistic strategy
in turnover of these antibiotics. The activity of Tp47 as a ��-lactamase is held back by a
significant inhibition of the activity by the products of turnover, accounting for the
sensitivity of T. pallidum to penicillins. The penicillin-binding protein activity and the ��-
lactamase reaction take place at different active sites in this protein, with the latter
reaction proceeding at a rate of over 2000-fold faster than that of the former.
Staphylococci have evolved to produce both ��-lactamases and PBP 2a as
resistance mechanism. The expression of these two proteins to have resistant phenotype
is regulated by BlaR1 and MecR1. BlaR1 is the sensor-transducer protein that binds with
��-lactam antibiotics on the cell surface (C-terminus) and transduces the signal into the
cytoplasm. The C-terminal domain of BlaR1 (BlaRS) is related to class D ��-lactamases. The carboxylation on active site lysine is important for the activation of serine as a
nucleophile. BlaRS has slow deacylation step, which exhibits the characteristic of PBPs.
BlaRS differs from class D ��-lactamases by exhibiting barrierless N��-decarboxylation in
the event of acylation, which traps acyl-enzyme complex. The recarboxylation is
prevented by interaction between the side chain of decarboxylated, free lysine and
asparagine. The significant conformational changes of BlaRS are observed by circular
dichroism and FT-IR upon interaction with ��-lactams, which may indicate critical
involvement in signal transduction. The C-terminal surface domain of MecR1 (MecRS)
has also been extensively studied. This protein also possesses N��-carboxylated lysine in
the active site. Unlike its homolog BlaRS, the kinetics of acylation of MecRS is
substantially defective, which may explain the reason why mec regulating system is
diminishing from clinical strains that exhibit high resistance level against drugs.
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