Random mutagenesis of Thermus aquaticus DNA polymerase I: concordance of immutable sites in vivo with the crystal structure.
Expression of Thermus aquaticus (Taq) DNA polymerase I (pol I) in Escherichia, coli complements the growth defect caused by a temperature-sensitive mutation in the host pol I. We replaced the nucleotide sequence encoding amino acids 659-671 of the O-helix of Taq DNA pol I, corresponding to the substrate binding site, with an oligonucleotide containing random nucleotides. Functional Taq pol I mutants were selected based on colony formation at the nonpermissive temperature. By using a library with 9% random substitutions at each of 39 positions, we identified 61 active Taq pol I mutants, each of which contained from one to four amino acid substitutions. Some amino acids, such as alanine-661 and threonine-664, were tolerant of several or even many diverse replacements. In contrast, no replacements or only conservative replacements were identified at arginine-659, lysine-663, and tyrosine-671. By using a library with totally random nucleotides at five different codons (arginine-659, arginine-660, lysine-663, phenylalanine-667, and glycine-668), we confirmed that arginine-659 and lysine-663 were immutable, and observed that only tyrosine substituted for phenylalanine-667. The two immutable residues and the two residues that tolerate only highly conservative replacements lie on the side of O-helix facing the incoming deoxynucleoside triphosphate, as determined by x-ray analysis. Thus, we offer a new approach to assess concordance of the active conformation of an enzyme, as interpreted from the crystal structure, with the active conformation inferred from in vivo function.
Suzuki
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Joseph Gottstein Memorial Cancer Research Laboratory, Department of Pathology, Seattle, WA, USA.
Baskin
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D
Hood
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Loeb
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LA
D000595:Amino Acid Sequence; D001120:Arginine; D003062:Codon; D018360:Crystallography, X-Ray; D004256:DNA Polymerase I / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli; D005998:Glycine; D008239:Lysine; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D010957:Plasmids / Q000378:metabolism; D011487:Protein Conformation; D013824:Thermus / Q000201:enzymology*
D003062:Codon; D010649:Phenylalanine; D001120:Arginine; D004256:DNA Polymerase I; D008239:Lysine; D005998:Glycine