LOOP AND SUBDOMAIN MOVEMENTS IN THE MECHANISM OF ESCHERICHIA-COLI DIHYDROFOLATE-REDUCTASE - CRYSTALLOGRAPHIC EVIDENCE

The reaction catalyzed by Escherichia coli dihydrofolate reductase (ec DHFR) cycles through five detectable kinetic intermediates: holoenzyme , Michaelis complex, ternary product complex, tetrahydrofolate (THF) b inary complex, and THF NADPH complex. Isomorphous crystal structures a nalogous to these five intermediates and to the transition state (as r epresented by the methotrexate NADPH complex) have been used to assemb le a 2.1 Angstrom resolution movie depicting loop and subdomain moveme nts during the catalytic cycle (see Supporting Information). The struc tures suggest that the M20 loop is predominantly closed over the react ants in the holoenzyme, Michaelis, and transition state complexes. But , during the remainder of the cycle, when nicotinamide is not bound, t he loop occludes (protrudes into) the nicotinamide-ribose binding pock et. Upon changing from the closed to the occluded conformation, the ce ntral portion of the loop rearranges from beta-sheet to 3(10) helix. T he change may occur by way of an irregularly structured open loop conf ormation, which could transiently admit a water molecule into position to protonate N5 of dihydrofolate. From the Michaelis to the transitio n state analogue complex, rotation between two halves of ecDHFR, the a denosine binding subdomain and loop subdomain, closes the (p-aminobenz oyl)glutamate (pABG) binding crevice by approximate to 0.5 Angstrom. R esulting enhancement of contacts with the pABG moiety may stabilize pu ckering at C6 of the pteridine ring in the transition state. The subdo main rotation is further adjusted by cofactor-induced movements (appro ximate to 0.5 Angstrom) of helices B and C, producing a larger pABG cl eft in the THF NADPH analogue complex than in the THF analogue complex . Such movements may explain how THF release is assisted by NADPH bind ing. Subdomain rotation is not observed in vertebrate DHFR structures, but an analogous loop movement (residues 59-70) appears to similarly adjust the pABG cleft width, suggesting that these movements are impor tant for catalysis. Loop movement, also unobserved in vertebrate DHFR structures, may preferentially weaken NADP(+) vs NADPH binding in ecDH FR, an evolutionary adaptation to reduce product inhibition in the NAD P(+) rich environment of prokaryotes.