Characterization of a PSE-4 Mutant with Different Properties in Relation to Penicillanic Acid Sulfones: Importance of Residues 216 to 218 in Class A β-Lactamases

Sabbagh, Yves; Thériault, Esther; Sanschagrin, François; Voyer, Normand; Palzkill, Timothy; Levesque, Roger C.

doi:10.1128/aac.42.9.2319

Cited by 9 publications

(3 citation statements)

References 31 publications

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“…Escherichia coli transformed with pMonTEM1 and pMonPSE4 were selected on LB‐agar plates containing a range of ampicillin concentrations (5 to 5000 μg/mL). Cells containing tem‐1 and pse‐4 grew on plates containing up to ∼1000 and 3000 μg/mL of ampicillin, respectively, similar to that previously reported (Sabbagh et al 1998). The minimum inhibitory concentration (MIC) of ampicillin for the strain of E. coli used was <5 μg/mL.…”

Section: Resultssupporting

confidence: 85%

“…This process was repeated using the ligated products until the full‐length genes were assembled. After assembly, pse‐4 and tem‐1 were amplified by PCR and cloned into the vector pMon•1A2; this bacterial vector contains a constitutive PSE‐4 promoter and kanamycin selectable marker (Sabbagh et al 1998). Escherichia coli transformed with pMonTEM1 and pMonPSE4 were selected on LB‐agar plates containing a range of ampicillin concentrations (5 to 5000 μg/mL).…”

Section: Resultsmentioning

confidence: 99%

“…Lactamases were cloned into the vector pMon•1A2, which was created by cloning the gene encoding the heme domain of cytochrome P450 1A2 into pMon711 (Sabbagh et al 1998). This vector was used for all selections.…”

Section: Methodsmentioning

confidence: 99%

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Library analysis of SCHEMA‐guided protein recombination

et al. 2003

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The computational algorithm SCHEMA was developed to estimate the disruption caused when amino acid residues that interact in the three-dimensional structure of a protein are inherited from different parents upon recombination. To evaluate how well SCHEMA predicts disruption, we have shuffled the distantly-related ␤-lactamases PSE-4 and TEM-1 at 13 sites to create a library of 2 14 (16,384) chimeras and examined which ones retain lactamase function. Sequencing the genes from ampicillin-selected clones revealed that the percentage of functional clones decreased exponentially with increasing calculated disruption (E ‫ס‬ the number of residue-residue contacts that are broken upon recombination). We also found that chimeras with low E have a higher probability of maintaining lactamase function than chimeras with the same effective level of mutation but chosen at random from the library. Thus, the simple distance metric used by SCHEMA to identify interactions and compute E allows one to predict which chimera sequences are most likely to retain their function. This approach can be used to evaluate crossover sites for recombination and to create highly mosaic, folded chimeras.Keywords: Chimera; lactamase; PSE-4; recombination; schema; TEM-1; directed evolution Numerous protein traits can be improved or altered when recombination is coupled with a screening or selection strategy (for review, see Minshull and Stemmer 1999; Arnold 2001a,b). During laboratory evolution, as in nature, recombination promotes the rapid accumulation of beneficial mutations from multiple parents onto a single offspring (Stemmer 1994b;Moore et al. 1997;Crameri et al. 1998). It also explores a part of sequence space that is particularly rich in folded and functional proteins. Recombination plays a key role in natural evolution of proteins through the swapping of well-defined structural domains . Where a domain structure is not obvious, however, how recombination contributes to the evolution and diversification of protein sequence and function is less well understood.Recently we developed an algorithm, called SCHEMA, for predicting which fragments of homologous proteins can be recombined without disturbing the integrity of the structure (Voigt et al. 2002). Based on the 3D structures of the parent proteins, the algorithm identifies pairs of amino acids that are interacting, defined as those residues within a cutoff distance of 4.5 Å, and determines the net number of interactions broken when a chimeric protein inherits portions of its sequence from different parents (defined as E). Because calculating E for all possible crossover combinations is computationally intractable, it is difficult to identify which crossover locations are optimal with respect to their ability to yield folded chimeras. One version of SCHEMA circumvents this computational difficulty by finding compact, con-

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Section: Resultssupporting

confidence: 85%

Section: Resultsmentioning

confidence: 99%

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Library analysis of SCHEMA‐guided protein recombination

et al. 2003

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Experimental Evolution of an Essential Bacillus Gene in an E. coli Host

Larios‐Sanz¹,

Travisano

2009

Horizontal Gene Transfer

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The acquisition of foreign genes by HGT potentially greatly speeds up adaptation by allowing faster evolution of beneficial traits. The evolutionary integration of novel genes into host gene expression and physiology is critical for adaptation by HGT, but remains largely unknown. We are exploring the evolutionary consequences of gene acquisition in populations of Escherichia coli in real time. A plasmid bearing the genes necessary for sucrose catabolism was constructed and introduced into a single E. coli genotype. Wild-type E. coli is generally incapable of utilizing sucrose, but E. coli transformants were able to grow on sucrose as a sole carbon and energy source, albeit poorly. Twelve replicate populations were initiated and propagated in sucrose minimal media for 300 generations. Over this time, we observed large fitness improvements in the selected environment. These results demonstrate the potential for HGT to substantially increase microbial niche breadth.

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NMR Dynamics of PSE-4 β-Lactamase: An Interplay of ps-ns Order and μs-ms Motions in the Active Site

Morin

Gagné

2009

Biophysical Journal

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The backbone dynamics for the 29.5 kDa class A beta-lactamase PSE-4 is presented. This solution NMR study was performed using multiple field (15)N spin relaxation and amide exchange data in the EX2 regime. Analysis was carried out with the relax program and includes the Lipari-Szabo model-free approach. Showing similarity to the homologous enzyme TEM-1, PSE-4 is very rigid on the ps-ns timescale, although slower mus-ms motions are present for several residues; this is especially true near the active site. However, significant dynamics differences exist between the two homologs for several important residues. Moreover, our data support the presence of a motion of the Omega loop first detected using molecular dynamics simulations on TEM-1. Thus, class A beta-lactamases appear to be a class of highly ordered proteins on the ps-ns timescale despite their efficient catalytic activity and high plasticity toward several different beta-lactam antibiotics. Most importantly, catalytically relevant mus-ms motions are present in the active site, suggesting an important role in catalysis.

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Characterization of a PSE-4 Mutant with Different Properties in Relation to Penicillanic Acid Sulfones: Importance of Residues 216 to 218 in Class A β-Lactamases

Cited by 9 publications

References 31 publications

Library analysis of SCHEMA‐guided protein recombination

Library analysis of SCHEMA‐guided protein recombination

Experimental Evolution of an Essential Bacillus Gene in an E. coli Host

NMR Dynamics of PSE-4 β-Lactamase: An Interplay of ps-ns Order and μs-ms Motions in the Active Site

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