Catalytic and structural effects of amino acid substitution at histidine 30 in human manganese superoxide dismutase: insertion of valine C gamma into the substrate access channel

Catalysis of the disproportionation of superoxide by human manganese superoxide dismutase (MnSOD) is characterized by an initial burst of catalysis followed by a much slower region that is zero order in superoxide and due to a product inhibition by peroxide anion. We have prepared site-specific mutants with replacements at His30, the side chain of which lies along the substrate access channel and is about 5.8 A from the metal. Using pulse radiolysis to generate superoxide, we have determined that kcat/K(m) was decreased and product inhibition increased for H30V MnSOD, both by 1-2 orders of magnitude, compared with wild type, H30N, and H30Q MnSOD. These effects are not attributed to the redox potentials, which are similar for all of these variants. An investigation of the crystal structure of H30V Mn(III)SOD compared with wild type, H30Q, and H30N Mn(III)SOD showed the positions of two gamma carbons of Val30 in the active site; Cgamma1 overlaps Cgamma of His30 in wild type, and Cgamma2 extends into the substrate access channel and occupies the approximate position of a water molecule in the wild type. The data suggest that Cgamma2 of the Val side chain has significantly interrupted catalysis by this overlap into the access channel with possible overlap with the substrate-product binding site. This is supported by comparison of the crystal structure of H30V MnSOD with that of azide bound to Mn(III)SOD from Thermus thermophilus and by visible absorption spectra showing that azide binding to the metal in H30V Mn(III)SOD is abolished. Moreover, the presence of Val30 caused a 100-fold decrease in the rate constant for dissociation of the product-inhibited complex compared with wild type.     

The Major Role of the Rab Ypt7p in Vacuole Fusion Is Supporting HOPS Membrane Association

Yeast vacuole fusion requires soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs), the Rab GTPase Ypt7p, vacuolar lipids, Sec17p and Sec18p, and the homotypic fusion and vacuole protein sorting complex (HOPS). HOPS is a multisubunit protein with direct affinities for SNAREs, vacuolar lipids, and the GTP-bound form of Ypt7p; each of these affinities contributes to HOPS association with the organelle. Using all-purified components, we have reconstituted fusion, but the Rab Ypt7p was not required. We now report that phosphorylation of HOPS by the vacuolar kinase Yck3p blocks HOPS binding to vacuolar lipids, making HOPS membrane association and the ensuing fusion depend on the presence of Ypt7p. In accord with this finding in the reconstituted fusion reaction, the inactivation of Ypt7p by the GTPase-activating protein Gyp1–46p only blocks the fusion of purified vacuoles when Yck3p is present and active. Thus, although Ypt7p may contribute to other fusion functions, its central role is to bind HOPS to the membrane.Rab proteins are small GTP-binding proteins involved in multiple steps of membrane traffic, including protein sorting, vesicle transport, and SNARE³-dependent membrane fusion (1). Rabs in their GTP-bound state bind proteins that are essential for mediating Rab function, which are therefore termed “effectors.” These effectors are diverse and perform various biochemical functions. For membrane fusion, Rabs and their effectors support tethering, the initial membrane contact that is needed for the subsequent assembly of trans-SNARE complexes between membranes (1, 2). A central question in organelle trafficking, which we now address, is whether Rabs are only required for binding their effectors to the membrane or whether they also activate the bound effector or provide some additional essential function for membrane fusion.We study membrane fusion using isolated yeast vacuoles (3). Yeast vacuole fusion requires the Rab GTPase Ypt7p, the heterohexameric HOPS complex, four vacuolar SNAREs, the SNARE disassembly chaperones Sec17p and Sec18p, and chemically minor yet functionally essential lipids, termed “regulatory” lipids. The HOPS complex is an effector of Ypt7p (4) and belongs to a group of functionally conserved large multisubunit tethering complexes, many of which are Rab effectors (5). The Vps39p subunit of HOPS is a nucleotide exchange factor for Ypt7p (6). HOPS is also a SNARE chaperone; its Vps33p subunit is a Sec1p/Munc18-1 family (SM) protein, HOPS binds multiple vacuolar SNAREs (7–9), and it proofreads SNARE complex structure (10). HOPS also binds to specific phosphoinositides (8), and these are among the regulatory lipids that are important for fusion (11–13).We have recently reconstituted membrane fusion using proteoliposomes of pure vacuolar proteins and lipids (13). HOPS and the regulatory lipids are crucial for rapid fusion of proteoliposome pairs bearing the three Q-SNAREs on one proteoliposome and the R-SNARE on the other and are absolutely required when all four SNAREs are present on each proteoliposome and Sec17p and Sec18p are present. Ypt7p is not required, showing that HOPS can stimulate SNARE-dependent fusion in vitro even in the absence of its Rab, although Ypt7p stimulates the fusion of these proteoliposomes.4Yeast vacuole fusion can be negatively regulated either by GTPase-activating proteins (GAPs) (14, 15) that promote GTP hydrolysis by Ypt7p or by the kinase Yck3p, which phosphorylates the Vps41p subunit of HOPS (16) and the vacuolar SNARE Vam3p (15). Yck3p is a palmitoylated (17), vacuole-localized kinase of the casein kinase I family (18). The complete fragmentation of vacuoles in vivo, indicating a block of fusion, requires both Ypt7p inactivation by a RabGAP and the presence of Yck3p (15). Yck3p is necessary for efficient vacuole inheritance (16) and normal vacuole morphology (19), suggesting that its function is part of the normal mechanism of vacuole segregation during the cell cycle. Although Yck3p clearly regulates vacuole fusion through phosphorylation of HOPS, it remains unclear which activities of HOPS are inhibited by Yck3p phosphorylation and whether Yck3p must also phosphorylate other vacuole fusion proteins such as Vam3p to block fusion.We now show that phosphorylation of the Vps41p subunit of HOPS by purified Yck3p reduces HOPS binding to membrane lipids, thereby making HOPS association with the membrane and the ensuing fusion of reconstituted proteoliposomes dependent on active Ypt7p. These data with proteoliposomes are supported by assays with purified vacuoles; the RabGAP Gyp1–46p only inhibits the in vitro fusion of yck3Δ vacuoles when purified Yck3p is added. As for Ypt7p and HOPS, the major function of other Rabs may also be to act as membrane receptors for their effectors.     

Capitalizing on multi-element interactions through bal-anced nutrition——A pathway to improve nitrogen use efficiency in China,India and North America

A viable option for increasing nitrogen (N) use efficiency and mitigation of negative impacts of N on the environment is to capitalize on multi-element interactions through implementation of nutrient management programs that provide balanced nutrition. Numerous studies have demonstrated the immediate efficacy of this approach in the developing regions like China and India as well as developed countries in North America. Based on 241 site-years of experiments in these countries, the first-year N recovery efficiency (RE) for the conventional or check treatments averaged 21% while the balanced treatments averaged 54% RE, for an average increase of 33% in RE due to balanced nutrition. Effective policies to promote adoption are most likely those that enable site-specific approaches to nutrient management decisions rather than sweeping, nation-wide incentives supporting one nutrient over another. Local farmers, advisers and officials need to be empowered with tools and information to help them define necessary changes in practices to create more balanced nutrient management.     

Autophagy: cells SNARE selves

Autophagosomes sequester cytosolic constituents and deliver this cargo to?lysosomes for destruction. Two groups now report that autophagosome maturation requires homotypic membrane fusion catalyzed by SNARE proteins.     

NADPH-dependent sulfite reductase flavoprotein adopts an extended conformation unique to this diflavin reductase

This is the first X-ray crystal structure of the monomeric form of sulfite reductase (SiR) flavoprotein (SiRFP-60) that shows the relationship between its major domains in an extended position not seen before in any homologous diflavin reductases. Small angle neutron scattering confirms this novel domain orientation also occurs in solution. Activity measurements of SiR and SiRFP variants allow us to propose a novel mechanism for electron transfer from the SiRFP reductase subunit to its oxidase metalloenzyme partner that, together, make up the SiR holoenzyme. Specifically, we propose that SiR performs its 6-electron reduction via intramolecular or intermolecular electron transfer. Our model explains both the significance of the stoichiometric mismatch between reductase and oxidase subunits in the holoenzyme and how SiR can handle such a large volume electron reduction reaction that is at the heart of the sulfur bio-geo cycle.     

CysG structure reveals tetrapyrrole-binding features and novel regulation of siroheme biosynthesis

Sulfur metabolism depends on the iron-containing porphinoid siroheme. In Salmonella enterica, the S-adenosyl-L-methionine (SAM)-dependent bismethyltransferase, dehydrogenase and ferrochelatase, CysG, synthesizes siroheme from uroporphyrinogen III (uro'gen III). The reactions mediated by CysG encompass two branchpoint intermediates in tetrapyrrole biosynthesis, diverting flux first from protoporphyrin IX biosynthesis and then from cobalamin (vitamin B(12)) biosynthesis. We determined the first structure of this multifunctional siroheme synthase by X-ray crystallography. CysG is a homodimeric gene fusion product containing two structurally independent modules: a bismethyltransferase and a dual-function dehydrogenase-chelatase. The methyltransferase active site is a deep groove with a hydrophobic patch surrounded by hydrogen bond donors. This asymmetric arrangement of amino acids may be important in directing substrate binding. Notably, our structure shows that CysG is a phosphoprotein. From mutational analysis of the post-translationally modified serine, we suggest a conserved role for phosphorylation in inhibiting dehydrogenase activity and modulating metabolic flux between siroheme and cobalamin pathways.     

Ground wētā in vines of the Awatere Valley,Marlborough: biology,density and distribution

The endemic New Zealand ground wētā (Hemiandrus sp. ‘promontorius’) has a Naturally Uncommon conservation status. This is because of the paucity of information on its density and distribution. Here, the biology, density and distribution of a population of this wētā found in and around vineyards in the Awatere Valley, Marlborough was studied. Wētā density was assessed in vineyards, paddocks and shrublands in this valley. Soil moisture, penetration resistance, pH and organic matter were recorded at locations with and without wētā. Wētā density in vineyards was significantly higher than in either paddocks or shrub habitats. In vineyards, the density of this insect was significantly higher under-vines than in the inter-rows. Higher numbers of this wētā were found in moist soils that required lower force to burrow. Females laid an average of 55 eggs between March and April, which hatched in September. These findings highlight the intersection between agriculture and conservation.     

Non-Boltzmann thermodynamic integration (NBTI) for macromolecular systems: relative free energy of binding of trypsin to benzamidine and benzylamine

The relative free energies of binding of trypsin to two amine inhibitors, benzamidine (BZD) and benzylamine (BZA), were calculated using non-Boltzmann thermodynamic integration (NBTI). Comparison of the simulations with the crystal structures of both complexes, trypsin-BZD and trypsin-BZA, shows that NBTI simulations better sample conformational space relative to thermodynamic integration (TI) simulations. The relative binding free energy calculated using NBTI was much closer to the experimentally determined value than that obtained using TI. The error in the TI simulation was found to be primarily due to incorrect sampling of BZA's conformation in the binding pocket. In contrast, NBTI produces a smooth mutation from BZD to BZA using a surrogate potential, resulting in a much closer agreement between the inhibitors' conformations and the omit electron density maps. This superior agreement between experiment and simulation, of both relative binding free energy differences and conformational sampling, demonstrates NBTI's usefulness for free energy calculations in macromolecular simulations.