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

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