Deep sequencing of systematic combinatorial libraries reveals β-lactamase sequence constraints at high resolution.


Abstract

In this study, combinatorial libraries were used in conjunction with ultrahigh-throughput sequencing to comprehensively determine the impact of each of the 19 possible amino acid substitutions at each residue position in the TEM-1 β-lactamase enzyme. The libraries were introduced into Escherichiacoli, and mutants were selected for ampicillin resistance. The selected colonies were pooled and subjected to ultrahigh-throughput sequencing to reveal the sequence preferences at each position. The depth of sequencing provided a clear, statistically significant picture of what amino acids are favored for ampicillin hydrolysis for all 263 positions of the enzyme in one experiment. Although the enzyme is generally tolerant of amino acid substitutions, several surface positions far from the active site are sensitive to substitutions suggesting a role for these residues in enzyme stability, solubility, or catalysis. In addition, information on the frequency of substitutions was used to identify mutations that increase enzyme thermodynamic stability. Finally, a comparison of sequence requirements based on the mutagenesis results versus those inferred from sequence conservation in an alignment of 156 class A β-lactamases reveals significant differences in that several residues in TEM-1 do not tolerate substitutions and yet extensive variation is observed in the alignment and vice versa. An analysis of the TEM-1 and other class A structures suggests that residues that vary in the alignment may nevertheless make unique, but important, interactions within individual enzymes.

Submission Details

ID: d5RV5da23

Submitter: Shu-Ching Ou

Submission Date: July 25, 2018, 4:54 p.m.

Version: 1

Publication Details
Deng Z;Huang W;Bakkalbasi E;Brown NG;Adamski CJ;Rice K;Muzny D;Gibbs RA;Palzkill T,J Mol Biol (2012) Deep sequencing of systematic combinatorial libraries reveals β-lactamase sequence constraints at high resolution. PMID:23017428
Additional Information

Sequence Assay Result Units