CONSTRUCTION OF A HOMODIMERIC DIHYDROFOLATE REDUCTASE-THYMIDYLATE SYNTHASE BIFUNCTIONAL ENZYME

A gene encoding a bifunctional homodimeric dihydrofolate reductase-thy midylate synthase (DHFR-TS) was constructed by destroying the stop cod on of Escherichia coli dihydrofolate reductase (DHFR) and joining the coding sequences of the monofunctional enzymes by a five amino acid li nker. The protein was designed to mimic features of active site proxim ity and electrostatics in the protozoan DHFR-TSs which are believed to be important in channeling of the DHFR substrate, H(2)folate, to TS. The genetically engineered catalytically active homodimeric bifunction al DHFR-TS was expressed, purified and characterized, The component ac tivities of the purified bifunctional enzyme had kinetic properties si milar to those of the monofunctional TS and DHFR, but unlike the authe ntic bifunctional enzymes from protozoa this enzyme did not kineticall y channel dihydrofolate from DHFR to TS.