Lipopolysaccharides are important components of the bacterial cell envelope that among other things act as a protective barrier against the environment and toxic molecules such as antibiotics. One of ...the most widely disseminated pathways of polysaccharide biosynthesis is the inner membrane bound Wzy-dependent pathway. Here we present the 3.0 Å structure of the co-polymerase component of this pathway, WzzB from E. coli solved by single-particle cryo-electron microscopy. The overall architecture is octameric and resembles a box jellyfish containing a large bell-shaped periplasmic domain with the 2-helix transmembrane domain from each protomer, positioned 32 Å apart, encircling a large empty transmembrane chamber. This structure also reveals the architecture of the transmembrane domain, including the location of key residues for the Wzz-family of proteins and the Wzy-dependent pathway present in many Gram-negative bacteria, explaining several of the previous biochemical and mutational studies and lays the foundation for future investigations.
Nitrogen fixation is the vital biochemical process in which atmospheric molecular nitrogen is made available to the biosphere. The process is highly energetically costly and thus tightly regulated. ...The activity of the key enzyme, nitrogenase, is controlled by reversible mono‐ADP‐ribosylation of one of its components, the Fe protein. This protein provides the other component, the MoFe protein, with the electrons required for the reduction of molecular nitrogen. The Fe‐protein is ADP‐ribosylated and de‐ADP‐ribosylated by dinitrogenase reductase ADP‐ribosyl transferase and dinitrogenase reductase activating glycohydrolase, respectively. Here we review the current biochemical and structural knowledge of this central regulatory reaction.
Nitrogen fixation is the vital biochemical process in which atmospheric molecular nitrogen is made available to the biosphere. The process is highly energetically costly and thus tightly regulated. The activity of the key enzyme, nitrogenase is controlled by reversible mono‐ADP‐ribosylation of one of its components. Here we review the current biochemical and structural knowledge of this central regulatory reaction.
Lipoproteins are important components of the cell envelope and are responsible for many essential cellular functions. They are produced by the post-translational covalent attachment of lipids that ...occurs via a sequential 3-step process controlled by three integral membrane enzymes. The last step of this process, unique to Gram-negative bacteria, is the N-acylation of the terminal cysteine by Apolipoprotein N-acyltransferase (Lnt) to form the final mature lipoprotein. Here we report 2 crystal forms of Lnt from Escherichia coli. In one form we observe a highly dynamic arm that is able to restrict access to the active site as well as a covalent modification to the active site cysteine consistent with the thioester acyl-intermediate. In the second form, the enzyme crystallized in an open conformation exposing the active site to the environment. In total we observe 3 unique Lnt molecules that when taken together suggest the movement of essential loops and residues are triggered by substrate binding that could control the interaction between Lnt and the incoming substrate apolipoprotein. The results provide a dynamic context for residues shown to be central for Lnt function and provide further insights into its mechanism.
Recent developments of novel electron diffraction techniques have shown to be powerful for determination of atomic resolution structures from micron- and nano-sized crystals, too small to be studied ...by single-crystal X-ray diffraction. In this work, the structure of a rare lysozyme polymorph is solved and refined using continuous rotation MicroED data and standard X-ray crystallographic software. Data collection was performed on a standard 200 kV transmission electron microscope (TEM) using a highly sensitive detector with a short readout time. The data collection is fast (∼3 min per crystal), allowing multiple datasets to be rapidly collected from a large number of crystals. We show that merging data from 33 crystals significantly improves not only the data completeness, overall I/σ and the data redundancy, but also the quality of the final atomic model. This is extremely useful for electron beam-sensitive crystals of low symmetry or with a preferred orientation on the TEM grid.
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•The structure of a rare lysozyme polymorph is determined using MicroED data•Fast data collection allows MicroED datasets being collected from multiple crystals•Merging multiple MicroED datasets improves the data quality•Highly redundant multiple datasets give more accurate structure models
Electron diffraction can be used for structure determination of macromolecular crystals too small to be studied by X-ray diffraction. Xu et al. determined the atomic structure of a rare lysozyme crystal form using electron diffraction data, and demonstrated that high data redundancy from multiple crystals improves the final structural model.
A dynamic tool for nitrogen reduction Hogbom, Martin
Science (American Association for the Advancement of Science),
09/2014, Letnik:
345, Številka:
6204
Journal Article
Recenzirano
Odprti dostop
Carbon monoxide reveals new possibilities for substrate binding in nitrogenase
Also see Report by
Spatzal
et al.
Even though nitrogen makes up almost 80% of the atmosphere, it is a limiting nutrient ...for biomass production. The low reactivity of nitrogen gas (N
2
) is a result of its very strong, unpolarized triple bond. Nitrogenase is the only enzyme known that can break this bond to produce compounds such as ammonia (NH
3
) for use in biosynthetic pathways. The atomic structure of this amazing system has been known for more than two decades (
1
,
2
), but the chemical mechanism of this central reaction remains unknown. In a biochemical and structural tour de force, on page 1620 of this issue, Spatzal
et al.
(
3
) report the crystal structure of carbon monoxide (CO) bound to the catalytic metal cluster of the enzyme. This work revealed an unexpected structural rearrangement of the cofactor.
Micro-crystal electron diffraction (MicroED) has shown great potential for structure determination of macromolecular crystals too small for X-ray diffraction. However, specimen preparation remains a ...major bottleneck. Here, we report a simple method for preparing MicroED specimens, named Preassis, in which excess liquid is removed through an EM grid with the assistance of pressure. We show the ice thicknesses can be controlled by tuning the pressure in combination with EM grids with appropriate carbon hole sizes. Importantly, Preassis can handle a wide range of protein crystals grown in various buffer conditions including those with high viscosity, as well as samples with low crystal concentrations. Preassis is a simple and universal method for MicroED specimen preparation, and will significantly broaden the applications of MicroED.
The very-long-chain fatty acyl-CoA synthetase FadD13 from Mycobacterium tuberculosis activates fatty acids for further use in mycobacterial lipid metabolism. FadD13 is a peripheral membrane protein, ...with both soluble and membrane-bound populations in vivo. The protein displays a distinct positively charged surface patch, suggested to be involved in membrane association. In this paper, we combine structural analysis with liposome co-flotation assays and membrane association modeling to gain a more comprehensive understanding of the mechanisms behind membrane association. We show that FadD13 has affinity for negatively charged lipids, such as cardiolipin. Addition of a fatty acid substrate to the liposomes increases the apparent affinity of FadD13, consistent with our previous hypothesis that FadD13 can utilize the membrane to harbor its very-long-chain fatty acyl substrates. In addition, we unambiguously show that FadD13 adopts a dimeric arrangement in solution. The dimer interface partly buries the positive surface patch, seemingly inconsistent with membrane binding. Notably, when cross-linking the dimer, it lost its ability to bind and co-migrate with liposomes. To better understand the dynamics of association, we utilized two mutant variants of FadD13, one in which the positively charged patch was altered to become more negative and one more hydrophobic. Both variants were predominantly monomeric in solution. The hydrophobic variant maintained the ability to bind to the membrane, whereas the negative variant did not. Taken together, our data indicate that FadD13 exists in a dynamic equilibrium between the dimer and monomer, where the monomeric state can adhere to the membrane via the positively charged surface patch.
The manganese/iron-carboxylate proteins make up a recently discovered group of proteins that contain a heterodinuclear Mn/Fe redox cofactor. The chemical potential of the heterodinuclear metal site ...is just starting to be characterized, but available data suggest that it may have capabilities for similarly versatile chemistry as the extensively studied diiron-carboxylate cofactor. The presently identified members of the manganese/iron-carboxylate proteins are all sequence homologues of the radical-generating R2 subunit of class I ribonucleotide reductase, canonically a diiron protein. They are also commonly misannotated as such in databases. In spite of the sequence similarity, the manganese/iron-carboxylate proteins form at least two functionally distinct groups, radical-generating ribonucleotide reductase subunits and ligand-binding Mn/Fe proteins. Here, the presently available sequences for the manganese/iron-carboxylate proteins are gathered, grouped, and analyzed. The analysis provides sequence determinants that allow group identification of new sequences on the single-protein level. Key differences between the groups are mapped on the known representative structures, providing clues to the structural prerequisites for metal specificity, cofactor formation, and difference in function. The organisms that encode manganese/iron-carboxylate proteins are briefly discussed; their environmental preference suggests that the Mn/Fe heterodinuclear cofactor is preferred by extremophiles and pathogens with a particularly high relative presence in Archaea.
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