PMID:12361984

Citation	Akanuma, S, Kigawa, T and Yokoyama, S (2002) Combinatorial mutagenesis to restrict amino acid usage in an enzyme to a reduced set. Proc. Natl. Acad. Sci. U.S.A. 99:13549-53
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.
Links	PubMed PMC129711 Online version:10.1073/pnas.222243999
Keywords	Amino Acid Sequence; Amino Acid Substitution; Amino Acids/chemistry; Enzymes/chemistry; Enzymes/genetics; Escherichia coli/enzymology; Escherichia coli/genetics; Evolution, Molecular; Genes, Bacterial; Models, Molecular; Molecular Sequence Data; Mutagenesis; Mutagenesis, Site-Directed; Orotate Phosphoribosyltransferase/chemistry; Orotate Phosphoribosyltransferase/genetics; Phenotype; Protein Conformation; Sequence Homology, Amino Acid
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