title: 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.
authors.affiliation: Department of Pharmacology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
authors.forename: Zhifeng
authors.initials: Z
authors.lastname: Deng
authors.forename: Wanzhi
authors.initials: W
authors.lastname: Huang
authors.forename: Erol
authors.initials: E
authors.lastname: Bakkalbasi
authors.forename: Nicholas G
authors.initials: NG
authors.lastname: Brown
authors.forename: Carolyn J
authors.initials: CJ
authors.lastname: Adamski
authors.forename: Kacie
authors.initials: K
authors.lastname: Rice
authors.forename: Donna
authors.initials: D
authors.lastname: Muzny
authors.forename: Richard A
authors.initials: RA
authors.lastname: Gibbs
authors.forename: Timothy
authors.initials: T
authors.lastname: Palzkill
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000667:Ampicillin / Q000378:metabolism* / Q000494:pharmacology; D000668:Ampicillin Resistance; D001483:Base Sequence; D004252:DNA Mutational Analysis; D004795:Enzyme Stability; D004926:Escherichia coli / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing; D006868:Hydrolysis; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry / Q000378:metabolism; D059645:Mutation Rate; D012641:Selection, Genetic; D012995:Solubility; D013816:Thermodynamics; D001618:beta-Lactamases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
chemical_list: D050505:Mutant Proteins; D000667:Ampicillin; D001618:beta-Lactamases; C034413:beta-lactamase TEM-1