The rate-limiting step in the folding of thecis-Pro167Thr mutant of TEM-1 β-lactamase is thetranstocisisomerization of a non-proline peptide bond

Vanhove, Marc; Raquet, Xavier; Palzkill, Timothy; Pain, Roger H.; Frère, Jean‐Marie

doi:10.1002/(sici)1097-0134(199605)25:1<104::aid-prot8>3.0.co;2-j

Cited by 27 publications

(5 citation statements)

References 30 publications

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“…It was also shown previously, from double-jump experiments, that the two major slow refolding phases of the wild-type enzyme were rate-limited by cis\trans isomerization around Xaa-Pro bonds [10,11]. Under the same conditions, the C77S mutant exhibited a similar behaviour, the two slow refolding phases being sensitive to double-jump experiments (results not shown).…”

Section: Unfolding and Refolding Kineticssupporting

confidence: 79%

“…These originate from proline isomerization, as indicated by the doublejump experiments. Recent analyses suggest a key role for Pro-167 in the case of the wild-type enzyme [11].…”

Section: Effect Of the Disulphide Bond On The Unfolding And Refolding Reactionsmentioning

confidence: 99%

“…rate-limited by cis\trans isomerization of two or more Xaa-Pro peptide bonds, one of which involves Pro-167 [11]. Interestingly, the disulphide bond of RTEM β-lactamase is not essential for enzymic activity.…”

Section: Introductionmentioning

confidence: 99%

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Kinetic and thermodynamic consequences of the removal of theCys-77–Cys-123 disulphide bond for the folding of TEM-1 β-lactamase

Vanhove

Guillaume

Ledent

et al. 1997

Biochemical Journal

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Class A beta-lactamases of the TEM family contain a single disulphide bond which connects cysteine residues 77 and 123. To clarify the possible role of the disulphide bond in the stability and folding kinetics of the TEM-1 beta-lactamase, this bond was removed by introducing a Cys-77-->Ser mutation, and the enzymically active mutant protein was studied by reversible guanidine hydrochloride-induced denaturation. The unfolding and refolding rates were monitored using tryptophan fluorescence. At low guanidine hydrochloride concentrations, the refolding of the wild-type and mutant enzymes followed biphasic time courses. The characteristics of the two phases were not significantly affected by the mutation. Double-jump experiments, in which the protein was unfolded in a high concentration of guanidine hydrochloride for a short time period and then refolded by diluting out the denaturant, indicated that, for both the wild-type and mutant enzymes, the two refolding phases could be ascribed to proline isomerization reactions. Equilibrium unfolding experiments monitored by fluorescence spectroscopy and far-UV CD indicated a three-state mechanism (N<-->H<--U). Both the folded mutant protein (N) and, to a lesser extent the thermodynamically stable intermediate, H. were destabilized relative to the fully unfolded state, U. Removal of the disulphide bond resulted in a decrease of 14.2 kJ/mol (3.4 kcal/mol) in the global free energy of stabilization. Similarly, the mutation also induced a drastic increase in the rate of thermal inactivation.

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Section: Unfolding and Refolding Kineticssupporting

confidence: 79%

“…These originate from proline isomerization, as indicated by the doublejump experiments. Recent analyses suggest a key role for Pro-167 in the case of the wild-type enzyme [11].…”

Section: Effect Of the Disulphide Bond On The Unfolding And Refolding Reactionsmentioning

confidence: 99%

See 1 more Smart Citation

Kinetic and thermodynamic consequences of the removal of theCys-77–Cys-123 disulphide bond for the folding of TEM-1 β-lactamase

Vanhove

Guillaume

Ledent

et al. 1997

Biochemical Journal

Self Cite

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“…Usually, in β-lactamases, the proline is found in the cis configuration. Studies on TEM-1 β-lactamase have already revealed the importance of the proline cis configuration in the protein folding and correct orientation of residues involved in the catalytic reaction ( Vanhove et al, 1996 ; Vanhove et al, 1995 ). Mutations at this proline, near the binding site prevents the enzyme from functioning properly ( Vanhove et al, 1996 ).…”

Section: Resultsmentioning

confidence: 99%

Gating interactions steer loop conformational changes in the active site of the L1 metallo-β-lactamase

Zhao

Shen

Chen

et al. 2023

eLife

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β-Lactam antibiotics are the most important and widely used antibacterial agents across the world. However, the widespread dissemination of β-lactamases among pathogenic bacteria limits the efficacy of β-lactam antibiotics. This has created a major public health crisis. The use of β-lactamase inhibitors has proven useful in restoring the activity of β-lactam antibiotics, yet, effective clinically approved inhibitors against class B metallo-β-lactamases are not available. L1, a class B3 enzyme expressed by Stenotrophomonas maltophilia, is a significant contributor to the β-lactam resistance displayed by this opportunistic pathogen. Structurally, L1 is a tetramer with two elongated loops, α3-β7 and β12-α5, present around the active site of each monomer. Residues in these two loops influence substrate/inhibitor binding. To study how the conformational changes of the elongated loops affect the active site in each monomer, enhanced sampling molecular dynamics simulations were performed, Markov State Models were built, and convolutional variational autoencoder-based deep learning was applied. The key identified residues (D150a, H151, P225, Y227, and R236) were mutated and the activity of the generated L1 variants was evaluated in cell-based experiments. The results demonstrate that there are extremely significant gating interactions between α3-β7 and β12-α5 loops. Taken together, the gating interactions with the conformational changes of the key residues play an important role in the structural remodeling of the active site. These observations offer insights into the potential for novel drug development exploiting these gating interactions.

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“…Such configurations are defined by the ω torsion angle of proline (cis: close to 0°, trans: nearly 180°). Usually, in βlactamases, the proline is found in the cis configuration.39 Studies on TEM-1 β-lactamase have already revealed the importance of the proline cis configuration in the protein folding and correct orientation of residues involved in the catalytic reaction (Vanhove et al, 1996(Vanhove et al, , 1995. Mutations at this proline, near the binding site prevents the enzyme from functioning properly (Vanhove et al, 1996).39 However, in L1 MBL, P225 is found in trans configuration.…”

Section: The Conformational Dynamics Of the α3-β7 And β12-α5 Loopsmentioning

confidence: 99%

Gating interactions steer loop conformational changes in the active site of the L1 metallo-β-lactamase

Zhao

Shen

Chen

et al. 2022

Preprint

View full text Add to dashboard Cite

β-lactam antibiotics are the most important and widely used antibacterial agents across the world. However, the widespread dissemination of β-lactamases among pathogenic bacteria limits the efficacy of β-lactam antibiotics. This has created a major public health crisis. The use of β-lactamase inhibitors has proven useful in restoring the activity of β-lactam antibiotics, yet, effective clinically inhibitors against class B metallo-β-lactamases (MBLs) are not available. L1, a class B3 enzyme expressed by Stenotrophomonas maltophilia, is a significant contributor to the β-lactam resistance displayed by this opportunistic pathogen. Structurally, L1 is a tetramer with two elongated loops, α3-β7 and β12-α5, present around the active site of each monomer. Residues in these two loops influence substrate/inhibitor binding. To study how the conformational changes of these elongated loops affect the active site in each monomer, enhanced sampling molecular dynamics (MD) simulations were performed, Markov State Model (MSM) were built, and convolutional variational autoencoder (CVAE)-based deep learning was applied. The key identified residues (D150a, H151, P225, Y227, R236) were mutated and the activity of the generated L1 variants was evaluated in cell-based experiments. The results demonstrate that there are extremely significant gating interactions between α3-β7 and β12-α5 loops. Taken together, the gating interactions with the conformational changes of the key residues play an important role the structural remodeling of the active site. These observations offer insights into the potential for novel drug development exploiting these gating interactions.

show abstract

The rate-limiting step in the folding of thecis-Pro167Thr mutant of TEM-1 β-lactamase is thetranstocisisomerization of a non-proline peptide bond

Cited by 27 publications

References 30 publications

Kinetic and thermodynamic consequences of the removal of theCys-77–Cys-123 disulphide bond for the folding of TEM-1 β-lactamase

Kinetic and thermodynamic consequences of the removal of theCys-77–Cys-123 disulphide bond for the folding of TEM-1 β-lactamase

Gating interactions steer loop conformational changes in the active site of the L1 metallo-β-lactamase

Gating interactions steer loop conformational changes in the active site of the L1 metallo-β-lactamase

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