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Modification of the polyketide anthraquinone AQ-256 in the entomopathogenic Photorhabdus luminescens involves several O -methylations, but the biosynthetic gene cluster antA-I lacks corresponding tailoring enzymes. We here describe the identification of five putative, highly homologous O -methyltransferases encoded in the genome of P. luminescens. Activity assays in vitro and deletion experiments in vivo revealed that three of them account for anthraquinone tailoring by producing three monomethylated and two dimethylated species of AQ-256. X-ray structures of all five enzymes indicate high structural and mechanistic similarity. As confirmed by structure-based mutagenesis, a conserved histidine at the active site likely functions as a general base for substrate deprotonation and subsequent methyl transfer in all enzymes. Eight complex structures with AQ-256 as well as mono- and dimethylated derivatives confirm the substrate specificity patterns found in vitro and visualize how single amino acid differences in the active-site pockets impact substrate orientation and govern site-specific methylation. [Display omitted] • O -methyltransferases encoded outside the ant gene cluster methylate anthraquinones • 3 methyltransferases produce 3 mono- and 2 dimethylated anthraquinone derivatives • Structures of 3 active and 2 inactive anthraquinone tailoring enzymes are reported • Ligand complex structures illustrate snapshots of the tailoring reactions Anthraquinones can carry methyl groups, but the corresponding tailoring enzymes have remained elusive. Huber et al. identified a tandem array of five methyltransferase genes, of which three are required for anthraquinone modification. Activity assays and X-ray structures of all five enzymes reveal substrate specificities and illustrate the methylation sequence. [ABSTRACT FROM AUTHOR]

Summary Inhibition of the 20S proteasome by bortezomib (Velcade) constitutes a successfully applied therapy for blood cancer. However, emerging resistance restricts its medicinal use. For example, mutations in the proteolytically active β5-subunit of the proteasome, the main target of inhibitors, were reported to impair drug binding and thus to reduce therapeutic efficacy. Using yeast as a model system, we describe here a systematic evaluation of these mutations by cell growth analysis, proteasome inhibition assays, and X-ray crystallography. The 11 mutants examined display decreased proliferation rates, impaired proteolytic activity, and marked resistance to bortezomib as well as the α′,β′-epoxyketone inhibitors carfilzomib (Kyprolis) and ONX 0914, while the second-generation compound carfilzomib was the least affected. In total, 49 proteasome X-ray structures, including structural data on proteasome-carfilzomib complexes, reveal three distinct molecular mechanisms that hamper both drug binding and natural substrate turnover to an extent that is still compatible with cell survival. [ABSTRACT FROM AUTHOR]

The heterotrimeric CCAAT-binding complex (CBC) is a fundamental eukaryotic transcription factor recognizing the CCAAT box. In certain fungi, like Aspergilli , the CBC cooperates with the basic leucine zipper HapX to control iron metabolism. HapX functionally depends on the CBC, and the stable interaction of both requires DNA. To study this cooperative effect, X-ray structures of the CBC-HapX-DNA complex were determined. Downstream of the CCAAT box, occupied by the CBC, a HapX dimer binds to the major groove. The leash-like N terminus of the distal HapX subunit contacts the CBC, and via a flexible polyproline type II helix mediates minor groove interactions that stimulate sequence promiscuity. In vitro and in vivo mutagenesis suggest that the structural and functional plasticity of HapX results from local asymmetry and its ability to target major and minor grooves simultaneously. The latter feature may also apply to related transcription factors such as yeast Hap4 and distinct Yap family members. [Display omitted] • X-ray structures of CBC and HapX bound to two distinct DNA strands are reported • CBC and HapX together recognize three sequence motifs in minor and major grooves • Local asymmetry of the HapX homodimer allows for a 2:1 interaction with the CBC • The N terminus of the distal HapX contacts the minor groove via a polyproline helix Huber et al. determined the structures of the CBC-HapX-DNA transcription factor complex relevant for iron homeostasis in certain fungi. The data explain the cooperative recognition of and the functional need for three DNA motifs and exemplify how nature uses asymmetry to mediate 2:1 interactions between a homodimer and an interaction partner. [ABSTRACT FROM AUTHOR]

Summary: The CCAAT box is a frequent element of eukaryotic promoters, and its specific recognition by the conserved heterotrimeric CCAAT-binding complex (CBC) constitutes a key step in promoter organization and regulation of transcription. Here, we report the crystal structures of the CBC from Aspergillus nidulans in the absence and in complex with double-stranded DNA at 1.8 Å resolution. The histone-like subunits HapC and HapE induce nucleosome-like DNA bending by interacting with the sugar-phosphate backbone. Minor groove sensing and widening by subunit HapB tightly anchor the CBC to the CCAAT box, as shown by structural and biochemical data. Furthermore, crucial interactions of the DNA duplex with subunit HapB provide an explanation for the sequence specificity of the CBC. The herein-described mode of transcription factor binding answers the question of how histone proteins gained sequence specificity for the CCAAT box. [ABSTRACT FROM AUTHOR]

Summary The heptameric proteasome activator (PA) 28αβ is known to modulate class I antigen processing by docking onto 20S proteasome core particles (CPs). The exact stoichiometry and arrangement of its α and β subunits, however, is still controversial. Here we analyzed murine PA28 complexes regarding structure and assembly. Strikingly, PA28α, PA28β, and PA28αβ preparations form heptamers, but solely PA28α and PA28αβ associate with CPs. Co-expression of α and β yields one unique PA28αβ species with an unchangeable subunit composition. Structural data on PA28α, PA28β, and PA28αβ up to 2.9 Å resolution reveal a PA28α 4 β 3 complex with an alternating subunit arrangement and a single α-α interface. Differential scanning fluorimetry experiments and activity assays classify PA28α 4 β 3 as most stable and most active, indicating that this assembly might represent the physiologically relevant species. Together, our data resolve subunit composition and arrangement of PA28αβ and clarify how an asymmetric heptamer can be assembled from two highly homologous subunits. [ABSTRACT FROM AUTHOR]

After elucidation of the atomic details of 20S proteasomes, current research focuses on the regulatory 19S particle. In this issue of Structure, He et al. present the crystal structure of Rpn2 and use electron microscopy to examine differences between Rpn2 and Rpn1. [ABSTRACT FROM AUTHOR]

• Affinity between 20S constitutive- or immunoproteasome and PA28αβ directly determined. • 20S constitutive- and immunoproteasome bind to PA28αβ with the same affinity. • PA28αβ activates 20S constitutive- and immunoproteasome to the same extent. • Equal FAT10 degradation rate in constitutive- and immunoproteasome containing cells. The 20S immunoproteasome (IP) is an interferon(IFN)-γ − and tumor necrosis factor (TNF) −inducible variant of the 20S constitutive proteasome (CP) in which all its peptidolytically active subunits β1, β2, and β5 are replaced by their cytokine inducible homologues β1i (LMP2), β2i (MECL-1), and β5i (LMP7). These subunit replacements alter the cleavage specificity of the proteasome and the spectrum of proteasome-generated peptide ligands of MHC class I molecules. In addition to antigen processing, the IP has recently been shown to serve unique functions in the generation of pro-inflammatory T helper cell subtypes and cytokines as well as in the pathogenesis of autoimmune diseases, but the mechanistic involvement of the IP in these processes has remained elusive. In this study we investigated whether the IP differs from the CP in the interaction with two IFN-γ/TNF inducible factors: the 11S proteasome regulator PA28αβ and the ubiquitin-like modifier FAT10 (ubiquitin D). Using thermophoresis, we determined the affinity of PA28αβ for the CP and IP to be 12.2 nM +/− 2.8 nM and 15.3 nM +/− 2.7 nM, respectively, which is virtually identical. Also the activation of the peptidolytic activities of the IP and CP by PA28αβ did not differ. For FAT10 we determined the degradation kinetics in cycloheximide chase experiments in cells expressing almost exclusively IP or CP as well as in IFN-γ stimulated and unstimulated cells and found no differences between the degradation rates. Taken together, we conclude that neither differences in the binding strength to, nor activation by PA28αβ, nor a difference in the rate of FAT10-mediated degradation can account for distinct functional capabilities of the IP as compared to the CP. [ABSTRACT FROM AUTHOR]

Abstract: Xenobiotics are widely used as pesticides. The detoxification of xenobiotics frequently involves conjugation to glutathione prior to compartmentalization and catabolism. In plants, degradation of glutathione-S-conjugates is initiated either by aminoterminal or carboxyterminal amino acid cleavage catalyzed by a γ-glutamyl transpeptidase and phytochelatin synthase, respectively. In order to establish yeast as a model system for the analysis of the plant pathway, we used monochlorobimane as a model xenobiotic in Saccharomyces cerevisiae and mutants thereof. The catabolism of monochlorobimane is initiated by conjugation to form glutathione-S-bimane, which is then turned over into a γ-GluCys-bimane conjugate by the vacuolar serine carboxypeptidases CPC and CPY. Alternatively, the glutathione-S-bimane conjugate is catabolized by the action of the γ-glutamyl transpeptidase Cis2p to a CysGly-conjugate. The turnover of glutathione-S-bimane was impaired in yeast cells deficient in Cis2p and completely abolished by the additional inactivation of CPC and CPY in the corresponding triple knockout. Inducible expression of the Arabidopsis phytochelatin synthase AtPCS1 in the triple knockout resulted in the turnover of glutathione-S-bimane to the γ-GluCys-bimane conjugate as observed in plants. Challenge of AtPCS1-expressing yeast cells with zinc, cadmium, and copper ions, which are known to activate AtPCS1, enhanced γ-GluCys-bimane accumulation. Thus, initial catabolism of glutathione-S-conjugates is similar in plants and yeast, and yeast is a suitable system for a study of enzymes of the plant pathway. [Copyright &y& Elsevier]