Permissible amino acid substitutions within the putative nucleoside binding site of herpes simplex virus type 1 encoded thymidine kinase established by random sequence mutagenesis [corrected].
We determined the essentiality of all amino acid replacements within an 11-codon sequence in the putative nucleoside-binding site of thymidine kinase encoded by herpes simplex virus type 1. This involved partial randomization of 11 codons in the gene to create a degenerate library, followed by genetic complementation using a tk- Escherichia coli strain and selection of unnatural active enzymes. We produced and tested 53,000 variants; of which 190 were found to be biologically active. Sequence analyses of functional variants revealed a high degree of flexibility in accommodating different types of amino acid substitutions in this region. However, no replacement was tolerated at proline-173, whereas tyrosine-172 could be replaced by only phenylalanine. To further define permissible substitutions at specified positions, we constructed a library with randomization at only four test codons. We produced and tested 600,000 variants; of which only 5 were active. Again proline-173 was conserved, and only tyrosine and phenylalanine were found at position 172. The identification of these conserved amino acids should provide important insights into the understanding of the structural basis of catalysis by this enzyme.
Munir
K M
KM
Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, University of Washington, Seattle 98195.
French
D C
DC
Dube
D K
DK
Loeb
L A
LA
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D003062:Codon; D005816:Genetic Complementation Test; D008969:Molecular Sequence Data; D016296:Mutagenesis; D009705:Nucleosides / Q000378:metabolism*; D010957:Plasmids; D018139:Simplexvirus / Q000201:enzymology* / Q000378:metabolism; D013937:Thymidine Kinase / Q000235:genetics*
D000596:Amino Acids; D003062:Codon; D009705:Nucleosides; D013937:Thymidine Kinase