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Combinatorial Mutagenesis to Restrict Amino Acid Usage in an Enzyme to a Reduced Set
Satoshi Akanuma, Takanori Kigawa and Shigeyuki Yokoyama
Proceedings of the National Academy of Sciences of the United States of America
Vol. 99, No. 21 (Oct. 15, 2002), pp. 13549-13553
Published by: National Academy of Sciences
Stable URL: http://www.jstor.org/stable/3073444
Page Count: 5
You can always find the topics here!Topics: Amino acids, Enzymes, Codons, Mutagenesis, Plasmids, Mathematical functions, Polymerase chain reaction, Amino acid substitution, Phenotypes, DNA
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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.
Proceedings of the National Academy of Sciences of the United States of America © 2002 National Academy of Sciences