No abstract

A conserved putative dimerization GxxxG motif located in the unique membrane-spanning segment of the ATP synthase subunit e was altered in yeast both by insertion of an alanine residue and by replacement of glycine by leucine residues. These alterations led to the loss of subunit g and the loss of dimeric and oligomeric forms of the yeast ATP synthase. Furthermore, as in null mutants devoid of either subunit e or subunit g, mitochondria displayed anomalous morphologies with onion-like structures. By taking advantage of the presence of the endogenous cysteine 28 residue in the wild-type subunit e, disulfide bond formation between subunits e in intact mitochondria was found to increase the stability of an oligomeric structure of the ATP synthase in digitonin extracts. The data show the involvement of the dimerization motif of subunit e in the formation of supramolecular structures of mitochondrial ATP synthases and are in favour of the existence in the inner mitochondrial membrane of associations of ATP synthases whose masses are higher than those of ATP synthase dimers.

dTDP-D-glucose 4,6-dehydratase (RmlB) was first identified in the L-rhamnose biosynthetic pathway, where it catalyzes the conversion of dTDP-D-glucose into dTDP-4-keto-6-deoxy-D-glucose. The structures of RmlB from Salmonella enterica serovar Typhimurium in complex with substrate deoxythymidine 5'-diphospho-D-glucose (dTDP-D-glucose) and deoxythymidine 5'-diphosphate (dTDP), and RmlB from Streptococcus suis serotype 2 in complex with dTDP-D-glucose, dTDP, and deoxythymidine 5'-diphospho-D-pyrano-xylose (dTDP-xylose) have all been solved at resolutions between 1.8 A and 2.4 A. The structures show that the active sites are highly conserved. Importantly, the structures show that the active site tyrosine functions directly as the active site base, and an aspartic and glutamic acid pairing accomplishes the dehydration step of the enzyme mechanism. We conclude that the substrate is required to move within the active site to complete the catalytic cycle and that this movement is driven by the elimination of water. The results provide insight into members of the SDR superfamily.

Subunits e and g of Saccharomyces cerevisiae ATP synthase are required to maintain ATP synthase dimeric forms. Mutants devoid of these subunits display anomalous mitochondrial morphologies. An expression system regulated by doxycycline was used to modulate the expression of the genes encoding the subunits e and g. A decrease in the amount of subunit e induces a decrease in the amount of subunit g, but a decrease in the amount of subunit g does not affect subunit e. The loss of subunit e or g leads to the loss of supramolecular structures of ATP synthase, which is fully reversible upon removal of doxycycline. In the absence of doxycycline, mitochondria present poorly defined cristae. In the presence of doxycycline, onion-like structures are formed after five generations. When doxycycline is removed after five generations, cristae are mainly observed. The data demonstrate that the inner structure of mitochondria depends upon the ability of ATP synthase to make supramolecular structures.F 0 F 1 -ATP synthase is a molecular rotary motor that is responsible for aerobic synthesis of ATP. It exhibits a head piece (catalytic sector), a base piece (membrane sector), and two connecting stalks. The sector F 1 containing the head piece is a water-soluble unit that retains the ability to hydrolyze ATP when in soluble form. F 0 is embedded in the membrane and is mainly composed of hydrophobic subunits forming a specific proton-conducting pathway. When the F 1 and F 0 sectors are coupled, the enzyme functions as a reversible H ϩ -transporting ATPase or ATP synthase (1-4). The two connecting stalks are made of components from F 1 and F 0 . The central stalk is a part of the rotor of the enzyme. The second stalk, which is part of the stator, connects F 1 and hydrophobic membranous components of the enzyme probably via a flexible region (5). High resolution x-ray crystallographic data have led to solving the structure of F 1 (6 -9) from different sources. Stock et al. (10) reported the 3.9-Å resolution x-ray diffraction structure of the Saccharomyces cerevisiae F 1 associated with a c 10 -ring oligomer.In Escherichia coli, F 0 is composed of only 3 subunits, whereas the mitochondrial F 0 of mammals is composed of 10 different subunits (11). The same 10 components have been identified in the S. cerevisiae enzyme (12-14). Among these additional subunits not present in bacterial and chloroplast ATP synthases, subunits e and g are not involved in ATP synthesis function but are involved in the dimerization/oligomerization of the mitochondrial ATP synthase (13, 15) because the absence of subunits e and g in the respective null mutants abolishes the ability of ATP synthase to make supramolecular structures. Subunits e and g are small hydrophobic proteins with an N in -C out orientation in the inner mitochondrial membrane (12, 16) with a unique transmembrane span probably located at the interface between two ATP synthase monomers. Subunit e can form homodimers upon oxidation via its unique cysteine residue (17), and it has been reported...