Oxalate oxidase (EC 1.2.3.4) catalyzes the conversion of oxalate and dioxygen to hydrogen peroxide and carbon dioxide. In this study, glycolate was used as a structural analogue of oxalate to investigate substrate binding in the crystalline enzyme. The observed monodentate binding of glycolate to the active site manganese ion of oxalate oxidase is consistent with a mechanism involving C-C bond cleavage driven by superoxide anion attack on a monodentate coordinated substrate. In this mechanism, the metal serves two functions: to organize the substrates (oxalate and dioxygen) and to transiently reduce dioxygen. The observed structure further implies important roles for specific active site residues (two asparagines and one glutamine) in correctly orientating the substrates and reaction intermediates for catalysis. Combined spectroscopic, biochemical, and structural analyses of mutants confirms the importance of the asparagine residues in organizing a functional active site complex.Oxalate oxidase (OXO; EC 1.2.3.4) 6 catalyzes the oxidation of oxalate, reducing dioxygen to hydrogen peroxide and forming 2 mol of carbonOxalate oxidase is widespread in nature and has been found in bacteria (4), fungi (1, 5), and various plant tissues (6). It has been detected in barley seedling roots during germination and in the leaves of mature barley plants in response to powdery mildew infection (6, 7), suggesting a role in plant signaling and defense. The enzyme has been purified to homogeneity from barley seedling roots and its N-terminal sequence determined, allowing the corresponding cDNA to be isolated and the complete primary sequence to be determined (3,8). These developments led to the recognition that the enzyme, OXO, is identical to an important marker of grain development during germination of wheat called germin (3,8). OXO is a member of a functionally diverse protein superfamily known as the cupins (9) or double stranded ␤-helix proteins (10). Barley OXO forms a hexamer that has extreme stability to heat and proteolysis (11).Spectroscopic studies demonstrated that OXO requires manganese for catalysis (12) and subsequent crystallographic studies on the barley enzyme revealed the structure of the hexamer (Fig. 1a) and confirmed the presence of a mononuclear manganese center buried deep within its jellyroll ␤-barrel domain (13). The manganese is bound by the side chains of three histidines and one glutamate residue, as well as two water molecules that occupy adjacent positions in the roughly octahedral metal complex (Fig. 1b). Based on the lack of obvious optical absorption and the presence of a characteristic EPR spectrum, the manganese ion has been assigned as the reduced Mn(II) oxidation state in the resting enzyme (12). Spectroscopic studies using recombinant OXO expressed in Pichia pastoris confirmed the presence of Mn(II) in the resting recombinant enzyme and provided the first spectroscopic evidence for oxalate binding to the manganese (14). The EPR signal of the anaerobic substrate complex, like that of the nativ...

SummaryAs core components of the microRNA-induced silencing complex (miRISC), Argonaute (AGO) proteins interact with TNRC6 proteins, recruiting other effectors of translational repression/mRNA destabilization. Here, we show that LIMD1 coordinates the assembly of an AGO-TNRC6 containing miRISC complex by binding both proteins simultaneously at distinct interfaces. Phosphorylation of AGO2 at Ser 387 by Akt3 induces LIMD1 binding, which in turn enables AGO2 to interact with TNRC6A and downstream effector DDX6. Conservation of this serine in AGO1 and 4 indicates this mechanism may be a fundamental requirement for AGO function and miRISC assembly. Upon CRISPR-Cas9-mediated knockout of LIMD1, AGO2 miRNA-silencing function is lost and miRNA silencing becomes dependent on a complex formed by AGO3 and the LIMD1 family member WTIP. The switch to AGO3 utilization occurs due to the presence of a glutamic acid residue (E390) on the interaction interface, which allows AGO3 to bind to LIMD1, AJUBA, and WTIP irrespective of Akt signaling.

X-ray crystal structures of the metcyano form of dehaloperoxidase-hemoglobin (DHP A) from Amphitrite ornata (DHPCN) and the C73S mutant of DHP A (C73SCN) were determined using synchrotron radiation in order to further investigate the geometry of diatomic ligands coordinated to the heme iron. The DHPCN structure was also determined using a rotating-anode source. The structures show evidence of photoreduction of the iron accompanied by dissociation of bound cyanide ion (CN(-)) that depend on the intensity of the X-ray radiation and the exposure time. The electron density is consistent with diatomic molecules located in two sites in the distal pocket of DHPCN. However, the identities of the diatomic ligands at these two sites are not uniquely determined by the electron-density map. Consequently, density functional theory calculations were conducted in order to determine whether the bond lengths, angles and dissociation energies are consistent with bound CN(-) or O(2) in the iron-bound site. In addition, molecular-dynamics simulations were carried out in order to determine whether the dynamics are consistent with trapped CN(-) or O(2) in the second site of the distal pocket. Based on these calculations and comparison with a previously determined X-ray crystal structure of the C73S-O(2) form of DHP [de Serrano et al. (2007), Acta Cryst. D63, 1094-1101], it is concluded that CN(-) is gradually replaced by O(2) as crystalline DHP is photoreduced at 100 K. The ease of photoreduction of DHP A is consistent with the reduction potential, but suggests an alternative activation mechanism for DHP A compared with other peroxidases, which typically have reduction potentials that are 0.5 V more negative. The lability of CN(-) at 100 K suggests that the distal pocket of DHP A has greater flexibility than most other hemoglobins.

Like other Nedd4 ligases, Saccharomyces cerevisiae E3 Rsp5p utilizes adaptor proteins to interact with some substrates. Previous studies have indentified Bul1p and Bul2p as adaptor proteins that facilitate the ligase-substrate interaction. Here, we show the identification of a third member of the Bul family, Bul3p, the product of two adjacent open reading frames separated by a stop codon that undergoes readthrough translation. Combinatorial analysis of BUL gene deletions reveals that they regulate some, but not all, of the cellular pathways known to involve Rsp5p. Surprisingly, we find that Bul proteins can act antagonistically to regulate the same ubiquitin-dependent process, and the nature of this antagonistic activity varies between different substrates. We further show, using in vitro ubiquitination assays, that the Bul proteins have different specificities for WW domains and that the two forms of Bul3p interact differently with Rsp5p, potentially leading to alternate functional outcomes. These data introduce a new level of complexity into the regulatory interactions that take place between Rsp5p and its adaptors and substrates and suggest a more critical role for the Bul family of proteins in controlling adaptor-mediated ubiquitination.

The design and assembly of peptide‐based materials has advanced considerably, leading to a variety of fibrous, sheet, and nanoparticle structures. A remaining challenge is to account for and control different possible supramolecular outcomes accessible to the same or similar peptide building blocks. Here a de novo peptide system is presented that forms nanoparticles or sheets depending on the strategic placement of a “disulfide pin” between two elements of secondary structure that drive self‐assembly. Specifically, homodimerizing and homotrimerizing de novo coiled‐coil α‐helices are joined with a flexible linker to generate a series of linear peptides. The helices are pinned back‐to‐back, constraining them as hairpins by a disulfide bond placed either proximal or distal to the linker. Computational modeling indicates, and advanced microscopy shows, that the proximally pinned hairpins self‐assemble into nanoparticles, whereas the distally pinned constructs form sheets. These peptides can be made synthetically or recombinantly to allow both chemical modifications and the introduction of whole protein cargoes as required.