Construction of a homodimeric dihydrofolate reductase-thymidylate synthase bifunctional enzyme.
A gene encoding a bifunctional homodimeric dihydrofolate reductase-thymidylate synthase (DHFR-TS) was constructed by destroying the stop codon of Escherichia coli dihydrofolate reductase (DHFR) and joining the coding sequences of the monofunctional enzymes by a five amino acid linker. The protein was designed to mimic features of active site proximity and electrostatics in the protozoan DHFR-TSs which are believed to be important in channeling of the DHFR substrate, H2folate, to TS. The genetically engineered catalytically active homodimeric bifunctional DHFR-TS was expressed, purified and characterized. The component activities of the purified bifunctional enzyme had kinetic properties similar to those of the monofunctional TS and DHFR, but unlike the authentic bifunctional enzymes from protozoa this enzyme did not kinetically channel dihydrofolate from DHFR to TS.
Trujillo
M
M
Department of Biochemistry and Biophysics, University of California, San Francisco 94143-0448, USA.
Duncan
R
R
Santi
D V
DV
D000595:Amino Acid Sequence; D002384:Catalysis; D018388:Codon, Terminator; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology; D005492:Folic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000096:biosynthesis* / Q000235:genetics; D009249:NADP / Q000378:metabolism; D013762:Tetrahydrofolate Dehydrogenase / Q000096:biosynthesis* / Q000235:genetics; D013940:Thymidylate Synthase / Q000096:biosynthesis* / Q000235:genetics
D018388:Codon, Terminator; D009097:Multienzyme Complexes; C056930:thymidylate synthase-dihydrofolate reductase; C010920:dihydrofolate; D009249:NADP; D005492:Folic Acid; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase