Table 1.

Sequence data of functional variants obtained by additional mutagenesis experiments

Wild-type residue	Candidate amino acids in additional mutagenesis	Amino acids observed in functional variants*	Simp-2
M197	A, D, L, M, R, T, V, Y†	M(9), Y(3)	Y
N41	D, N, R, T, Y‡	N(9), R(2), T(1)	R
Q117	D, Q, R, T, Y§	D(2), Q(4), R(2), T(4)	T
Q168	D, Q, R, T, Y§	D(2), Q(6), R(2), T(3)	T
Q141	D, Q, R, T, Y§	D(1), R(5), Q(3), T(2), Y(1)	R
Q5	D, Q, R, T, Y§	Q(8), T(4)	T

^*An average of 12 functional variants were analyzed. These variants were obtained by in vivo complementation of the pyrE-defective E. coli following the site-directed random mutagenesis at the indicated positions. The frequency of an amino acid is given in parentheses next to the amino acid. 

^† The codon was randomized by using four mutagenic primers that contained RCG, CKG, KAC, and RTG, respectively (R = A + G; K = G + T). 

^‡ The codon was randomized by using two mutagenic primers that contained DAT and ASA, respectively (D = A + G + T; S = C + G). 

^§ Each of these codons was randomized by using three mutagenic primers that contained KAT, CRA, and ACT, respectively (K = G + T; R = A + G).