Tumor necrosis factor alpha (TNF-α), a central mediator of the inflammatory response, is released from basophilic cells and other cells in response to a variety of proinflammatory stimuli. Vialinin A is a potent inhibitor of TNF-α production and is released from RBL-2H3 cells. Ubiquitin-specific peptidase 5 (USP5), a deubiquitinating enzyme, was identified as a target molecule of vialinin A and its enzymatic activity was inhibited by vialinin A. Here we report production of TNF-α is decreased in USP5 siRNA-knockdown RBL-2H3 cells, compared with control cells. The finding of the present study strongly suggests that USP5 is one of the essential molecules for the production of TNF-α in RBL-2H3.
Hydrazones are natural and synthetic compounds containing a C=N-N moiety. Here we found that the opportunistic pathogen Pseudomonas aeruginosa PAO1 produced NAD+- or NADP+-dependent hydrazone dehydrogenase (HDH), which converts hydrazones to the corresponding hydrazides and acids rather than to the simple hydrolytic product aldehydes. Gene cloning indicated that the HDH is part of the group X aldehyde dehydrogenase (ALDH) family, which is distributed among bacteria, although the physiological roles of the ALDH family remain unknown. The PAO1 strain upregulated HDH in the presence of the hydrazone adipic acid bis(ethylidene hydrazide) (AEH). Gene disruption of the HDH-encoding hdhA (PA4022) decreased growth rates in culture medium containing AEH as the sole carbon source, and this effect was more obvious in the double gene disruption of hdhA and its orthologous exaC (PA1984), indicating that these genes are responsible for hydrazone utilization. Recombinant proteins of group X ALDHs from Escherichia coli, Paracoccus denitrificans, and Ochrobactrum anthropi also acted as HDHs in that they produced HDH activity in the cells and degraded hydrazones. These findings indicated the physiological roles of group X ALDHs in bacteria and showed that they comprise a distinct ALDH subfamily.
Fosmidomycin, a potent inhibitor of 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), has antibacterial and antimalaria activity. Due to its poor pharmacokinetics, more lipophilic DXR inhibitors are needed. However, the hydrophobic binding site(s) in DXR remains elusive. Here, pyridine/quinoline containing phosphonates are identified to be DXR inhibitors with IC50 values as low as 840 nM. We also report three DXR:inhibitor structures, revealing a novel binding mode. The indole group of Trp211 is found to move ~4.6 Å to open up a mainly hydrophobic pocket, where the pyridine/quinoline rings of the inhibitors are located and have strong π-π stacking/charge-transfer interactions with the indole. Docking studies demonstrate our structures could be used to predict the binding modes of other lipophilic DXR inhibitors. Overall, this work shows an important role of Trp211 in inhibitor recognition and provides a structural basis for future drug design and development.
1-Deoxy-D-xylulose-5-phosphate reductoisomerase; protein crystallography; anti-infective; drug design; inhibitor recognition
Fosmidomycin, a potent inhibitor of 1-deoxy-d-xylulose-5-phosphate reductoisomerase (DXR), has antibacterial and antimalaria activity. Due to its poor pharmacokinetics, more lipophilic DXR inhibitors are needed. However, the hydrophobic binding site(s) in DXR remains elusive. Here, pyridine/quinoline containing phosphonates are identified to be DXR inhibitors with IC50 values as low as 840 nM. We also report three DXR/inhibitor structures, revealing a novel binding mode. The indole group of Trp211 is found to move ∼4.6 Å to open up a mainly hydrophobic pocket, where the pyridine/quinoline rings of the inhibitors are located and have strong π−π stacking/charge-transfer interactions with the indole. Docking studies demonstrate our structures could be used to predict the binding modes of other lipophilic DXR inhibitors. Overall, this work shows an important role of Trp211 in inhibitor recognition and provides a structural basis for future drug design and development.
1-Deoxy-d-xylulose-5-phosphate reductoisomerase; protein crystallography; anti-infective; drug design; inhibitor recognition
Because the Japanese native cattle Kuchinoshima-Ushi have been isolated in a small island and their lineage has been intensely protected, it has been assumed to date that numerous and valuable genomic variations are conserved in this cattle breed.
In this study, we evaluated genetic features of this breed, including single nucleotide polymorphism (SNP) information, by whole-genome sequencing using a Genome Analyzer II. A total of 64.2 Gb of sequence was generated, of which 86% of the obtained reads were successfully mapped to the reference sequence (Btau 4.0) with BWA. On an average, 93% of the genome was covered by the reads and the number of mapped reads corresponded to 15.8-fold coverage across the covered region. From these data, we identified 6.3 million SNPs, of which more than 5.5 million (87%) were found to be new. Out of the SNPs annotated in the bovine sequence assembly, 20,432 were found in protein-coding regions containing 11,713 nonsynonymous SNPs in 4,643 genes. Furthermore, phylogenetic analysis using sequence data from 10 genes (more than 10 kbp) showed that Kuchinoshima-Ushi is clearly distinct from European domestic breeds of cattle.
These results provide a framework for further genetic studies in the Kuchinoshima-Ushi population and research on functions of SNP-containing genes, which would aid in understanding the molecular basis underlying phenotypic variation of economically important traits in cattle and in improving intrinsic defects in domestic cattle breeds.
Mhp1, a hydantoin transporter from M. liquefaciens, was purified and crystallized. Diffraction data were collected to 2.85 Å resolution; the crystal belonged to the orthorhombic space group P212121.
The integral membrane protein Mhp1 from Microbacterium liquefaciens transports hydantoins and belongs to the nucleobase:cation symporter 1 family. Mhp1 was successfully purified and crystallized. Initial crystals were obtained using the hanging-drop vapour-diffusion method but diffracted poorly. Optimization of the crystallization conditions resulted in the generation of orthorhombic crystals (space group P212121, unit-cell parameters a = 79.7, b = 101.1, c = 113.8 Å). A complete data set has been collected from a single crystal to a resolution of 2.85 Å with 64 741 independent observations (94% complete) and an R
merge of 0.12. Further experimental phasing methods are under way.
transporters; nucleobase:cation symporter 1 family; membrane proteins; hydantoins
The ‘Nucleobase-Cation-Symport-1’, NCS1, transporters are essential components of salvage pathways for nucleobases and related metabolites. Here, we report the 2.85 Å resolution structure of the NCS1 benzyl-hydantoin transporter, Mhp1, from Microbacterium liquefaciens. Mhp1 contains 12 transmembrane helices, ten of which are arranged in two inverted repeats of 5 helices. The structures of the outward-facing open and substrate-bound occluded conformations were solved showing how the outward-facing cavity closes upon binding of substrate. Comparisons with the leucine (LeuTAa) and the galactose (vSGLT) transporters reveal that the outward- and inward-facing cavities are symmetrically arranged on opposite sides of the membrane. The reciprocal opening and closing of these cavities is synchronised by the inverted repeat helices 3 and 8, providing the structural basis of the ‘alternating access’ model for membrane transport.
The crystallization and preliminary X-ray studies of the aminoglycoside antibiotic-modifying enzyme hygromycin B phosphotransferase from E. coli are reported.
Aminoglycoside antibiotics, such as hygromycin, kanamycin, neomycin, spectinomycin and streptomycin, inhibit protein synthesis by acting on bacterial and eukaryotic ribosomes. Hygromycin B phosphotransferase (Hph; EC 22.214.171.124) converts hygromycin B to 7′′-O-phosphohygromycin using a phosphate moiety from ATP, resulting in the loss of its cell-killing activity. The Hph protein has been crystallized for the first time using a thermostable mutant and the hanging-drop vapour-diffusion method. The crystal provided diffraction data to a resolution of 2.1 Å and belongs to space group P3221, with unit-cell parameters a = b = 71.0, c = 125.0 Å. Crystals of complexes of Hph with hygromycin B and AMP-PNP or ADP have also been obtained in the same crystal form as that of the apoprotein.
aminoglycoside antibiotics; hygromycin B phosphotransferase; MAD; selenomethionine
The crystal structure of 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) from Escherichia coli complexed with Mg2+, NADPH and fosmidomycin was determined at 2.2 Å resolution. The structure showed a well defined loop conformation at the active site of DXR.
The crystal structure of 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) from Escherichia coli complexed with Mg2+, NADPH and fosmidomycin was solved at 2.2 Å resolution. DXR is the key enzyme in the 2-C-methyl-d-erythritol 4-phosphate pathway and is an effective target of antimalarial drugs such as fosmidomycin. In the crystal structure, electron density for the flexible loop covering the active site was clearly observed, indicating the well ordered conformation of DXR upon substrate binding. On the other hand, no electron density was observed for the nicotinamide-ribose portion of NADPH and the position of Asp149 anchoring Mg2+ was shifted by NADPH in the active site.
antimalarial drugs; fosmidomycin; MEP pathway
The tRNase domain of colicin D, which is specific to tRNAArgs, has been crystallized. A diffraction data set has been collected to a resolution of 1.05 Å.
The tRNase domain of colicin D, which cleaves only tRNAArgs at the 3′ side of their anticodon loops, has been expressed in Escherichia coli with its inhibitor protein and purified to a form free from the inhibitor using a low-pH buffer. Crystals were obtained by the hanging-drop vapour-diffusion method at 278 K from a buffer containing 100 mM Tris–HCl pH 8.5, 22% PEG MME 2000 and 1 mM nickel(II) chloride. Diffraction data to 1.05 Å resolution were collected at BL41XU, SPring-8. The crystals belong to space group P212121, with unit-cell parameters a = 34.7, b = 65.5, c = 96.5 Å.
arginine tRNAs; atomic resolution; colicin D; tRNases
Recombinant β-glucosidase from wheat seedlings complexed with a substrate aglycone has been crystallized in a hexameric active form. A diffraction data set has been collected at 1.7 Å.
The wheat β-glucosidase TaGlu1b, which is only active in a hexameric form, was tagged with 6×His at the N-terminus, overexpressed in Escherichia coli and purified in two steps. The protein complexed with a substrate aglycone was crystallized at 293 K from a solution containing 10 mM HEPES pH 7.2, 1 M LiSO4 and 150 mM NaCl using the hanging-drop vapour-diffusion method. Diffraction data were collected to 1.7 Å at the Photon Factory. The crystal belongs to space group P4132, with unit-cell parameters a = b = c = 194.65 Å, α = β = γ = 90°. The asymmetric unit was confirmed by molecular-replacement solution to contain one monomer, giving a solvent content of 72.1%.
β-glucosidases; DIMBOA; hexamers; wheat
Colicin E5—a tRNase toxin—specifically cleaves QUN (Q: queuosine) anticodons of the Escherichia coli tRNAs for Tyr, His, Asn and Asp. Here, we report the crystal structure of the C-terminal ribonuclease domain (CRD) of E5 complexed with a substrate analog, namely, dGpdUp, at a resolution of 1.9 Å. Thisstructure is the first to reveal the substrate recognition mechanism of sequence-specific ribonucleases. E5-CRD realized the strict recognition for both the guanine and uracil bases of dGpdUp forming Watson–Crick-type hydrogen bonds and ring stacking interactions, thus mimicking the codons of mRNAs to bind to tRNA anticodons. The docking model of E5-CRD with tRNA also suggests its substrate preference for tRNA over ssRNA. In addition, the structure of E5-CRD/dGpdUp along with the mutational analysis suggests that Arg33 may play an important role in the catalytic activity, and Lys25/Lys60 may also be involved without His in E5-CRD. Finally, the comparison of the structures of E5-CRD/dGpdUp and E5-CRD/ImmE5 (an inhibitor protein) complexes suggests that the binding mode of E5-CRD and ImmE5 mimics that of mRNA and tRNA; this may represent the evolutionary pathway of these proteins from the RNA–RNA interaction through the RNA–protein interaction of tRNA/E5-CRD.
Colicin E5 is a novel Escherichia coli ribonuclease that specifically cleaves the anticodons of tRNATyr, tRNAHis, tRNAAsn and tRNAAsp. Since this activity is confined to its 115 amino acid long C-terminal domain (CRD), the recognition mechanism of E5-CRD is of great interest. The four tRNA substrates share the unique sequence UQU within their anticodon loops, and are cleaved between Q (modified base of G) and 3′ U. Synthetic minihelix RNAs corresponding to the substrate tRNAs were completely susceptible to E5-CRD and were cleaved in the same manner as the authentic tRNAs. The specificity determinant for E5-CRD was YGUN at −1 to +3 of the ‘anticodon’. The YGU is absolutely required and the extent of susceptibility of minihelices depends on N (third letter of the anticodon) in the order A > C > G > U accounting for the order of susceptibility tRNATyr > tRNAAsp > tRNAHis, tRNAAsn. Contrastingly, we showed that GpUp is the minimal substrate strictly retaining specificity to E5-CRD. The effect of contiguous nucleotides is inconsistent between the loop and linear RNAs, suggesting that nucleotide extension on each side of GpUp introduces a structural constraint, which is reduced by a specific loop structure formation that includes a 5′ pyrimidine and 3′ A.