We devised strict evolutionary criteria for analyzing DHFR amino acid sequences from 233 species ranging from human to bacteria and found three evolutionarily important sequence divergent sites, defined herein as phylogenetically coherent events (PCEs). Next we experimentally probed the catalytic consequences of the different PCEs as they were introduced into wild type ecoli DHFR. Empirical valence bond molecular dynamics simulations were able to reproduce the kinetic data of various PCEs and provided further comparisons among ecoli DHFR, human DHFR, and the ecoli DHFR variants with PCE components artificially added. One of the identified PCEs drastically altered the native ecoli DHFR's binding affinity to its cofactor NADPH, product NADP+,and TMP to more closely resemble its human counterpart. Guided by these results, we then were able to engineer a human-like ecoli DHFR variant by introducing the three PCEs into native ecoli DHFR through mutagenesis. The binding constant between ecoli DHFR and trimethoprim is greatly weakened because of the the PCE mutation as seen by the Isothermal titration calorimetry (ITC) experiment.(Table 1) ITC experiments were all done using Micro Cal Auto-itC200(GE) at the Automated Biological Calorimetry Facility.
|TMP Kd,M||NADPH Kd, M||NADP+ Kd, M||Kp=Kd(NADH)/Kd(NADP+)|
|WT ecoli DHFR||6 x 10-9||1.75 x 10-7||2.3 x 10-5||.0076|
|G ecoli DHFR||(1.65 +/- 0.2) x 10-6||(9.2 +/- 0.8) x 10-7||(1.1 +/- 0.2) x 10-6||0.88|
|NG ecoli DHFR||(1.2 +/- 0.5) x 10-6||(2.6 +/- 0.4) x 10-6||(4.0 +/- 0.3) x 10-6||0.67|
|NLG ecoli DHFR||(5.0 +/- 0.7) x 10-6||(3.0 +/-0.1) x 10-6||(7.0 +/- 1.0) x 10-6||0.45|
|WT human||10-6||2.2 x 10-5||1.9 x 10-6||11.6|
(Table 1) Dissociation constants (Kd: reciprocal of the binding constants) of binary DHFR complexes of E:TMP, E:NADPH. and E:NADP+ in aqueous medium at pH 7.0 and 298 K.