title: Combinatorial mutagenesis to restrict amino acid usage in an enzyme to a reduced set.
abstract: We developed an effective strategy to restrict the amino acid usage in a relatively large protein to a reduced set with conservation of its in vivo function. The 213-residue Escherichia coli orotate phosphoribosyltransferase was subjected to 22 cycles of segment-wise combinatorial mutagenesis followed by 6 cycles of site-directed random mutagenesis, both coupled with a growth-related phenotype selection. The enzyme eventually tolerated 73 amino acid substitutions: In the final variant, 9 amino acid types (A, D, G, L, P, R, T, V, and Y) occupied 188 positions (88%), and none of 7 amino acid types (C, H, I, M, N, Q, and W) appeared. Therefore, the catalytic function associated with a relatively large protein may be achieved with a subset of the 20 amino acid. The converged sequence also implies simpler constituents for proteins in the early stage of evolution.
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000596:Amino Acids / Q000737:chemistry; D004798:Enzymes / Q000737:chemistry* / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D019143:Evolution, Molecular; D005798:Genes, Bacterial; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009962:Orotate Phosphoribosyltransferase / Q000737:chemistry / Q000235:genetics; D010641:Phenotype; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid
chemical_list: D000596:Amino Acids; D004798:Enzymes; D009962:Orotate Phosphoribosyltransferase