Combinatorial mutagenesis to restrict amino acid usage in an enzyme to a reduced set

Satoshi Akanuma, Takanori Kigawa, Shigeyuki Yokoyama

Research output: Contribution to journalArticle

67 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)13549-13553
Number of pages5
JournalProceedings of the National Academy of Sciences of the United States of America
Volume99
Issue number21
DOIs
Publication statusPublished - 2002 Oct 15
Externally publishedYes

ASJC Scopus subject areas

  • General

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