In contrast to the F-type ATPases, which use a proton gradient to generate ATP, the V-type enzymes use ATP to actively transport protons into organelles and extracellular compartments. We describe here the structure of the H-subunit (also called Vma13p) of the yeast enzyme. This is the first structure of any component of a V-type ATPase. The H-subunit is not required for assembly but plays an essential regulatory role. Despite the lack of any apparent sequence homology the structure contains five motifs similar to the so-called HEAT or armadillo repeats seen in the importins. A groove, which is occupied in the importins by the peptide that targets proteins for import into the nucleus, is occupied here by the 10 amino-terminal residues of subunit H itself. The structural similarity suggests how subunit H may interact with the ATPase itself or with other proteins. A cleft between the amino-and carboxyl-terminal domains also suggests another possible site of interaction with other factors.T he vacuolar proton-translocating ATPases play an important role in the acidification of extracelluar compartments and organelles and are found in most eukaryotic cells. In contrast to the F-type ATPases, which generate ATP from a proton gradient across a membrane, the V-type enzymes use ATP to acidify compartments for receptor-mediated endocytosis, intracellular trafficking, and protein degradation (1, 2). The enzyme is composed of two functionally distinct complexes V 1 and V 0 (Fig. 1). V 0 is integral to the membrane and is thought to consist of at least five different subunits with a total molecular mass of about 260 kDa. This complex is involved in translocation of protons across the membrane. The V 1 complex is more hydrophilic and is composed of at least eight different subunits totaling a molecular mass of about 570 kDa. Because of the relatively high sequence identity between the catalytic subunits ␣ and ␤ of the F-type ATPases and subunits B and A of the V-type ATPases, the V 1 complex is thought to use ATP to drive the proton translocation across the membrane. The rotary catalytic mechanism of ATP hydrolysis and proton transportation (3, 4) is thought to be very similar among the two classes of enzymes, but the different subunit stoichiometry and architectural appearance in electron micrographs suggests that the V-type ATPases are more complex (5, 6).Because the V-type enzymes are involved in key biochemical processes, such as the regulation of pH, and also because they consume rather than generate energy, their function is likely to be under tight control. Previous studies have revealed that most components of the V-type ATPase are essential for assembly of the enzyme complex (1). Characterization of subunit H, also known as Vma13p, however, indicates that this polypeptide is essential for the activity of the enzyme but not for targeting or assembly (7). The regulatory function of subunit H recently was demonstrated by Parra et al. (8). Binding of the subunit to isolated, cytosolic V 1 particles inhibits CaATP hydro...

Bacterial microcompartments (BMCs) are specialized organelles that use proteinaceous membranes to confine chemical reaction spaces. The ethanolamine ammonialyase microcompartment of Escherichia coli represents such a class of cytosolic organelles that enables bacteria to survive on small organic molecules such as ethanolamine as the sole source for carbon and nitrogen. We present here the crystal structure of the shell protein EutL at 2.2-Å resolution. With 219 residues, it is the largest representative of this BMC's shell proteins. In the crystal, EutL forms a trimer that exhibits a hexagonally shaped tile structure. The tiles arrange into a tightly packed 2D array that is likely to resemble the proteinaceous membrane of the intact BMC. In contrast to other BMC shell proteins, which have only 1 pore per tile, EutL exhibits 3 pores per tile, thereby significantly increasing the overall porosity of this protein membrane. Each of the individual pores is lined with negatively charged residues and aromatic residues that are proposed to facilitate passive transport of specific solutes. The characteristic shape of the hexagonal tile, which is also found in the microcompartments of carbon-fixating bacteria, may present an inherent and fundamental building unit that may provide a general explanation for the formation of differently sized microcompartments.metabolosome ͉ carboxysome ͉ bacterial organelle

The ethanolamine-utilizing bacterial microcompartment (Eut-BMC) of Escherichia coli is a polyhedral organelle that harbors specific enzymes for the catabolic degradation of ethanolamine. The compartment is composed of a proteinaceous shell structure that maintains a highly specialized environment for the biochemical reactions inside. Recent structural investigations have revealed hexagonal assemblies of shell proteins that form a tightly packed two-dimensional lattice that is likely to function as a selectively permeable protein membrane, wherein small channels are thought to permit controlled exchange of specific solutes. Here, we show with two nonisomorphous crystal structures that EutM also forms a two-dimensional protein membrane. As its architecture is highly similar to the membrane structure of EutL, it is likely that the structure represents a physiologically relevant form. Thus far, of all Eut proteins, only EutM and EutL have been shown to form such proteinaceous membranes. Despite their similar architectures, however, both proteins exhibit dramatically different pore structures. In contrast to EutL, the pore of EutM appears to be positively charged, indicating specificity for different solutes. Furthermore, we also show that the central pore structure of the EutL shell protein can be triggered to open specifically upon exposure to zinc ions, suggesting a specific gating mechanism.

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A new screening method for 6beta-hydroperoxycholest-4-en-3-one (HCEO)-forming cholesterol oxidase was devised in this study. As the result of the screening, a novel cholesterol oxidase producer (strain DS-1) was isolated and identified as Chromobacterium sp. Extracellular cholesterol oxidase of strain DS-1 was purified from the culture supernatant. The molecular mass of the purified enzyme was 58 kDa. This enzyme showed a visible adsorption spectrum having peaks at 355 and 450 nm, like a typical flavoprotein. The enzyme oxidized cholesterol to HCEO, with the consumption of 2 mol of O2 and the formation of 1 mol of H2O2 for every 1 mol of cholesterol oxidized. The enzyme oxidized 3beta-hydroxysteroids such as cholesterol, beta-cholestanol, and pregnenolone at high rates. The Km value for cholesterol was 26 microM. The enzyme was stable at pH 3 to 11 and most active at pH 7.0-7.5, showing optimal activity at pH 7.0 and 65 degrees C. The enzyme retained about 80% of its activity after incubation for 30 min at 85 degrees C. The thermal stability of the enzyme was the highest among the cholesterol oxidases tested. Moreover, the enzyme was more stable in the presence of various organic solvents and detergents than commercially available cholesterol oxidases.