M. Suzuki et al., RANDOM MUTAGENESIS OF THERMUS-AQUATICUS DNA-POLYMERASE-I - CONCORDANCE OF IMMUTABLE SITES IN-VIVO WITH THE CRYSTAL-STRUCTURE, Proceedings of the National Academy of Sciences of the United Statesof America, 93(18), 1996, pp. 9670-9675
Expression of Thermus aquaticus (Tag) DNA polymerase I (pol I) in Esch
erichia coli complements the growth defect caused by a temperature-sen
sitive mutation in the host pol I. We replaced the nucleotide sequence
encoding amino acids 659-671 of the O-helix of Tag DNA pol I, corresp
onding to the substrate binding site, with an oligonucleotide containi
ng random nucleotides. Functional Tag pol I mutants were selected base
d on colony formation at the nonpermissive temperature, By using a lib
rary with 9% random substitutions at each of 39 positions, we identifi
ed 61 active Taq pol I mutants, each of which contained from one to fo
ur amino acid substitutions, Some amino acids, such as alanine-661 and
threonine-664, were tolerant of several or even many diverse replacem
ents, In contrast, no replacements or only conservative replacements m
ere identified at arginine-659, lysine-663, and tyrosine-671. By using
a library with totally random nucleotides at five different codons (a
rginine-659, arginine-660, lysine-663, phenylalanine-667, and glycine-
668), we confirmed that arginine-659 and lysine-663 were immutable, an
d observed that only tyrosine substituted for phenylalanine-667. The t
wo immutable residues and the two residues that tolerate only highly c
onservative replacements lie on the side of O-helix facing the incomin
g deoxynucleoside triphosphate, as determined by x-ray analysis. Thus,
we offer a new approach to assess concordance of the active conformat
ion of an enzyme, as interpreted from the crystal structure, with the
active conformation inferred from in vivo function.