The membrane-peripheral subunits of transhydrogenase from Entamoeba histolytica are functional only when dimerized.
Unlike their bacterial and mammalian counterparts, the NADP(H)- and NAD(H)-binding components of proton-translocating transhydrogenase from the protozoan parasite Entamoeba histolytica (denoted ehdIII and ehdI, respectively) are tethered by a polypeptide linker. The recombinant tethered fragment, ehdIII-ehdI, was prepared without its membrane-spanning dII component. Dimers of ehdIII-ehdI catalyzed transhydrogenation, but monomers were inactive. The addition of ehdIII to ehdIII-ehdI monomers did not lead to an increase in the rate of transhydrogenation, showing that this inactivity is not the result of an unfavorable topology introduced by the linker. The addition of a bacterial dI to ehdIII-ehdI led to an increase in the rate of transhydrogenation, showing that the linker is flexible. A hybrid protein in which ehdIII is tethered to the bacterial dI (denoted ehdIII-rrdI) more readily formed active dimers. Data from small angle x-ray scattering by the hybrid dimers were fitted to models derived from the high-resolution crystal structure of the bacterial dI(2)dIII(1) complex (Cotton, N. P. J., White, S. A., Peake, S. J., McSweeney, S., and Jackson, J. B. (2001) Structure 9, 165-T176). The results show that the ehdIII-rrdI dimer is asymmetric; one dIII associates with dI, as in the bacterial complex, but the other is displaced. The results provide evidence for the alternating site, binding change model for proton translocation by intact transhydrogenase.
Weston
Chris J
CJ
School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom.
Venning
Jamie D
JD
Jackson
J Baz
JB
D000818:Animals; D019281:Dimerization; D004748:Entamoeba histolytica / Q000201:enzymology*; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009250:NADP Transhydrogenases / Q000502:physiology*; D011487:Protein Conformation; D012542:Scattering, Radiation; D013329:Structure-Activity Relationship
D009243:NAD; D009249:NADP; D009250:NADP Transhydrogenases