The cytochrome bd oxidases are terminal oxidases that are present in bacteria and archaea. They reduce molecular oxygen (dioxygen) to water, avoiding the production of reactive oxygen species. In addition to their contribution to the proton motive force, they mediate viability under oxygen-related stress conditions and confer tolerance to nitric oxide, thus contributing to the virulence of pathogenic bacteria. Here we present the atomic structure of the bd oxidase from Geobacillus thermodenitrificans, revealing a pseudosymmetrical subunit fold. The arrangement and order of the heme cofactors support the conclusions from spectroscopic measurements that the cleavage of the dioxygen bond may be mechanistically similar to that in the heme-copper–containing oxidases, even though the structures are completely different.

SummaryGeranylgeranylglyceryl phosphate synthase (GGGPS) family enzymes catalyse the formation of an ether bond between glycerol-1-phosphate and polyprenyl diphosphates. They are essential for the biosynthesis of archaeal membrane lipids, but also occur in bacterial species, albeit with unknown physiological function. It has been known that there exist two phylogenetic groups (I and II) of GGGPS family enzymes, but a comprehensive study has been missing. We therefore visualized the variability within the family by applying a sequence similarity network, and biochemically characterized 17 representative GGGPS family enzymes regarding their catalytic activities and substrate specificities. Moreover, we present the first crystal structures of group II archaeal and bacterial enzymes. Our analysis revealed that the previously uncharacterized bacterial enzymes from group II have GGGPS activity like the archaeal enzymes and differ from the bacterial group I enzymes that are heptaprenylglyceryl phosphate synthases. The length of the isoprenoid substrate is determined in group II GGGPS enzymes by 'limiter residues' that are different from those in group I enzymes, as shown by site-directed mutagenesis. Most of the group II enzymes form hexamers. We could disrupt these hexamers to stable and catalytically active dimers by mutating a single amino acid that acts as an 'aromatic anchor'.

The great benefits that chemical pesticides have brought to agriculture are partly offset by widespread environmental damage to nontarget species and threats to human health. Microbial bioinsecticides are considered safe and highly specific alternatives but generally lack potency. Spindles produced by insect poxviruses are crystals of the fusolin protein that considerably boost not only the virulence of these viruses but also, in cofeeding experiments, the insecticidal activity of unrelated pathogens. However, the mechanisms by which spindles assemble into ultra-stable crystals and enhance virulence are unknown. Here we describe the structure of viral spindles determined by X-ray microcrystallography from in vivo crystals purified from infected insects. We found that a C-terminal molecular arm of fusolin mediates the assembly of a globular domain, which has the hallmarks of lytic polysaccharide monooxygenases of chitinovorous bacteria. Explaining their unique stability, a 3D network of disulfide bonds between fusolin dimers covalently crosslinks the entire crystalline matrix of spindles. However, upon ingestion by a new host, removal of the molecular arm abolishes this stabilizing network leading to the dissolution of spindles. The released monooxygenase domain is then free to disrupt the chitinrich peritrophic matrix that protects insects against oral infections. The mode of action revealed here may guide the design of potent spindles as synergetic additives to bioinsecticides.ost entomopoxviruses (EV) produce two types of intracellular crystals. Virus-containing spheroids are the main infectious form of EV (1) and are functionally analogous to polyhedra of cypovirus (2) and baculovirus (3, 4). In contrast, the function of the second type of crystals is less clear. These crystals of the viral fusolin protein, called "spindles" because of their characteristic shape, assemble in the endoplasmic reticulum of infected cells and for some species also occur embedded within the crystalline lattice of spheroids (5). Purified spindles are not infectious but strongly enhance the infectivity of EV by a mechanism that involves disruption of the peritrophic matrix, a physical barrier that protects the midgut epithelium of insects against oral pathogens (6, 7). Remarkably, in larval cofeeding experiments, spindles also enhance the insecticidal activity of unrelated oral pathogens such as baculovirus (8) and the Bacillus thuringiensis (Bt) toxin (9) by up to three orders of magnitude. This effect on virulence prompted their use as synergistic additives to common bioinsecticides, for instance by transgenic expression of spindles in plants to improve the effectiveness of baculovirus insecticides (10).Fusolin proteins have a signal sequence that targets them to the endoplasmic reticulum, and the mature protein has a mass of 36-44 kDa. Some fusolins are glycosylated, and the glycosylation site of the fusolin produced by Anomala cuprea EV (ACEV) is required for full virulence (11). Sequence analysis shows that the N-terminal regio...

Highlights d Allostery in the HisH-HisF bienzyme complex was regulated by light d Photo-sensitive unnatural amino acids in HisF were used to control HisH activity d HisH activity depends on the conformational organization of the catalytic H178

Nitrogen metabolism in bacteria and archaea is regulated by a ubiquitous class of proteins belonging to the P II family. P II proteins act as sensors of cellular nitrogen, carbon, and energy levels, and they control the activities of a wide range of target proteins by protein-protein interaction. The sensing mechanism relies on conformational changes induced by the binding of small molecules to P II and also by P II posttranslational modifications. In the diazotrophic bacterium Azospirillum brasilense, high levels of extracellular ammonium inactivate the nitrogenase regulatory enzyme DraG by relocalizing it from the cytoplasm to the cell membrane. Membrane localization of DraG occurs through the formation of a ternary complex in which the P II protein GlnZ interacts simultaneously with DraG and the ammonia channel AmtB. Here we describe the crystal structure of the GlnZ-DraG complex at 2.1 Å resolution, and confirm the physiological relevance of the structural data by site-directed mutagenesis. In contrast to other known P II complexes, the majority of contacts with the target protein do not involve the T-loop region of P II . Hence this structure identifies a different mode of P II interaction with a target protein and demonstrates the potential for P II proteins to interact simultaneously with two different targets. A structural model of the AmtB-GlnZDraG ternary complex is presented. The results explain how the intracellular levels of ATP, ADP, and 2-oxoglutarate regulate the interaction between these three proteins and how DraG discriminates GlnZ from its close paralogue GlnB.N itrogen is an indispensable element for life. In bacteria and plants, many aspects of the regulation of nitrogen metabolism are controlled by a class of ubiquitous proteins called P II signal transduction proteins (1-4). P II proteins are present in all domains of life and are one of the most widely distributed signal transduction proteins in nature: some prokaryotes encode multiple P II homologues (2, 3). P II proteins act as a central processing unit receiving signals of carbon, energy, and nitrogen levels, integrating and transforming them into different P II conformational states. The multitude of P II conformations control their ability to interact with, and thus regulate the activity of, a variety of target proteins including transporters, enzymes, and transcriptional regulators (4). P II proteins form compact barrel-shaped homotrimeric structures, in which each monomer is 12-14 kDa containing three functionally important loops: the T-, B-, and C-loops (4). The 20-residue long T-loop is structurally very flexible and participates in protein interactions in all the structures of P II -target complexes solved to date. The T-loop also contains the site of posttranslational modification if it is needed. In Proteobacteria, Y51 in the T-loop is subjected to reversible uridylylation in response to the cellular glutamine levels (5). The effector molecules ATP, ADP, and 2-oxoglutarate (2-OG) influence the conformational states of P II pr...

Rapid evolution of enzymes provides unique molecular insights into the remarkable adaptability of proteins and helps to elucidate the relationship between amino acid sequence, structure and function. We interrogated the evolution of the phosphotriesterase from Pseudomonas diminuta (PdPTE), which hydrolyzes synthetic organophosphates with remarkable catalytic efficiency. PTE is thought to be an evolutionarily “young” enzyme and it has been postulated that it has evolved from members of the phosphotriesterase-like lactonase (PLL) family that show promiscuous organophosphate degrading activity. Starting from a weakly promiscuous PLL scaffold (Dr0930 from Deinococcus radiodurans), we designed an extremely efficient organophosphate hydrolase (OPH) with broad substrate specificity using rational and random mutagenesis in combination with in vitro activity screening. The OPH activity for seven organophosphate substrates was simultaneously enhanced by up to five orders of magnitude, achieving absolute values of catalytic efficiencies up to 106 M−1 s−1. Structural and computational analyses identified the molecular basis for the enhanced OPH activity of the engineered PLL variants and demonstrated that OPH catalysis in PdPTE and the engineered PLL differ significantly in the mode of substrate binding.

Observations of the dose-rate effect in continuous X-ray diffraction data acquisition at room temperature are presented.

Baculoviruses are ubiquitous insect viruses well known for their use as bioinsecticides, gene therapy vectors, and protein expression systems. Overexpression of recombinant proteins in insect cell culture utilizes the strong promoter of the polyhedrin gene. In infected larvae, the polyhedrin protein forms robust intracellular crystals called polyhedra, which protect encased virions for prolonged periods in the environment. Polyhedra are produced by two unrelated families of insect viruses, baculoviruses and cypoviruses. The atomic structure of cypovirus polyhedra revealed an intricate packing of trimers, which are interconnected by a projecting N-terminal helical arm of the polyhedrin molecule. Baculovirus and cypovirus polyhedra share nearly identical lattices, and the Nterminal region of the otherwise unrelated baculovirus polyhedrin protein sequence is also predicted to be ␣-helical. These results suggest homology between the proteins and a common structural basis for viral polyhedra. Here, we present the 2.2-Å structure of baculovirus polyhedra determined by x-ray crystallography from microcrystals produced in vivo. We show that the underlying molecular organization is, in fact, very different. Although both polyhedra have nearly identical unit cell dimensions and share I23 symmetry, the polyhedrin molecules are structurally unrelated and pack differently in the crystals. In particular, disulfide bonds and domain-swapped N-terminal domains stabilize the building blocks of baculovirus polyhedra and interlocking C-terminal arms join unit cells together. We show that the N-terminal projecting helical arms have different structural roles in baculovirus and cypovirus polyhedra and conclude that there is no structural evidence for a common evolutionary origin for both classes of polyhedra.in vivo crystallization ͉ molecular arms ͉ occlusion body ͉ self-assembly ͉ virus evolution