Production of hetero-dimeric dihydrofolate reductase-thymidylate synthase bifunctional enzyme.
As a result of the expression of a single open reading frame composed of the coding sequence for a cysteine-free mutant (Cys85-->Ala, Cys152-->Ser) of Escherichia coli dihydrofolate reductase (DHFR; 18K monomeric protein), that for the E. coli thymidylate synthase (TS; dimeric protein with a 30K promoter), and a spacer sequence (coding 7 amino acids) with a Shine-Dargarno sequence, an active hetero-dimeric bifunctional enzyme with 50K DHFR-TS and 30K TS polypeptides was stably produced in the transformed E. coli cell in addition to an overproduction of the TS dimeric enzyme. The highly purified hetero-dimeric enzyme has similar Vmax and Km values in both DHFR and TS activities to those of the natural counterparts, monomeric DHFR and dimeric TS. Although the hetero-dimeric enzyme did not show an apparent channeling transfer of dihydrofolate (the intermediate substrate) between the spatially discrete DHFR and TS active sites, the coupling efficiency of the TS and DHFR reactions in the artificial enzyme was better than that in the separated enzymes, as shown by a decrease in the intermediate concentration at the steady state in the coupled reaction.
Joint Research Center for Atom Technology (JRCAT), National Institute for Advanced Interdisciplinary Research, Ibaraki.
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Iwakura
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Kokubu
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics*; D011993:Recombinant Fusion Proteins; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics*; D013940:Thymidylate Synthase / Q000235:genetics*
D009097:Multienzyme Complexes; D011993:Recombinant Fusion Proteins; D013762:Tetrahydrofolate Dehydrogenase; D013940:Thymidylate Synthase