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PcyA, a ferredoxin-dependent bilin pigment reductase, catalyzes the site-specific reduction of the two vinyl groups of biliverdin (BV), producing phycocyanobilin. Previous neutron crystallography detected both the neutral BV and its protonated form (BVH

In mammals, the green heme metabolite biliverdin is converted to a yellow anti-oxidant by NAD(P)H-dependent biliverdin reductase (BVR), whereas in O2-dependent photosynthetic organisms it is converted to photosynthetic or light-sensing pigments by ferredoxin-dependent bilin reductases (FDBRs). In NADP+-bound and biliverdin-bound BVR-A, two biliverdins are stacked at the binding cleft; one is positioned to accept hydride from NADPH, and the other appears to donate a proton to the first biliverdin through a neighboring arginine residue. During the FDBR-catalyzed reaction, electrons and protons are supplied to bilins from ferredoxin and from FDBRs and waters bound within FDBRs, respectively. Thus, the protonation sites of bilin and catalytic residues are important for the analysis of site-specific reduction. The neutron structure of FDBR sheds light on this issue.

Peptidylarginine deiminase type III (PAD3) is an isozyme belonging to the PAD enzyme family that converts arginine to citrulline residue(s) within proteins. PAD3 is expressed in most differentiated keratinocytes of the epidermis and hair follicles, while S100A3, trichohyalin, and filaggrin are its principal substrates. In this study, the X-ray crystal structures of PAD3 in six states, including its complex with the PAD inhibitor Cl-amidine, were determined. This structural analysis identified a large space around Gly374 in the PAD3-Ca2+-Cl-amidine complex, which may be used to develop novel PAD3-selective inhibitors. In addition, similarities between PAD3 and PAD4 were found based on the investigation of PAD4 reactivity with S100A3 in vitro. A comparison of the structures of PAD1, PAD2, PAD3, and PAD4 implied that the flexibility of the structures around the active site may lead to different substrate selectivity among these PAD isozymes.

Vapor-phase catalytic dehydration of 1,5-pentanediol (1,5-PDO) was investigated over monoclinic ZrO2 catalysts modified with basic oxides. An unsaturated alcohol, 4-penten-1-ol (4P1OL), was produced together with the formation of tetrahydropyran, δ-valerolactone, 1,4-pentadiene, pentanal, 1-pentanol, and 5-hydroxypentanal, etc. Among the modified ZrO2 catalysts, only ZrO2 modified with MgO enhanced the selectivity to 4P1OL efficiently. The most active modified catalyst was found to have 20 mol% MgO and a calcination at 800 °C (MgO/ZrO2), and the selectivity of 4P1OL exceeded 83% at 400 °C. A pulse adsorption measurement of several chemicals clarified adsorptive interaction between a reactant and a catalyst at 220 °C: the interaction between 1,5-PDO and MgO/ZrO2 was stronger than the other adsorbates and catalysts. Another strong adsorptive interaction between 1,4-butanediol and CaO/ZrO2, which was effective in the dehydration of 1,4-butanediol to produce 3-buten-1-ol, was also observed.

Heme oxygenase catalyzes the degradation of heme to biliverdin, iron, and carbon monoxide. Here, we present crystal structures of the substrate-free, Fe3+-biliverdin-bound, and biliverdin-bound forms of HmuO, a heme oxygenase from Corynebacterium diphtheriae, refined to 1.80, 1.90, and 1.85 Å resolution, respectively. In the substrate-free structure, the proximal and distal helices, which tightly bracket the substrate heme in the substrate-bound heme complex, move apart, and the proximal helix is partially unwound. These features are supported by the molecular dynamic simulations. The structure implies that the heme binding fixes the enzyme active site structure, including the water hydrogen bond network critical for heme degradation. The biliverdin groups assume the helical conformation and are located in the heme pocket in the crystal structures of the Fe3+-biliverdin-bound and the biliverdin-bound HmuO, prepared by in situ heme oxygenase reaction from the heme complex crystals. The proximal His serves as the Fe3+-biliverdin axial ligand in the former complex and forms a hydrogen bond through a bridging water molecule with the biliverdin pyrrole nitrogen atoms in the latter complex. In both structures, salt bridges between one of the biliverdin propionate groups and the Arg and Lys residues further stabilize biliverdin at the HmuO heme pocket. Additionally, the crystal structure of a mixture of two intermediates between the Fe3+-biliverdin and biliverdin complexes has been determined at 1.70 Å resolution, implying a possible route for iron exit.

Dysiherbaine (DH) and neodysiherbaine A (NDH) selectively bind and activate two kainate-type ionotropic glutamate receptors, GluK1 and GluK2. The ligand-binding domains of human GluK1 and GluK2 were crystallized as bound forms with a series of DH analogues including DH, NDH, 8-deoxy-NDH, 9-deoxy-NDH and 8,9-dideoxy-NDH (MSVIII-19), isolated from natural sources or prepared by total synthesis. Since the DH analogues exhibit a wide range of binding affinities and agonist efficacies, it follows that the detailed analysis of crystal structure would provide us with a significant opportunity to elucidate structural factors responsible for selective binding and some aspects of gating efficacy. We found that differences in three amino acids (Thr503, Ser706 and Ser726 in GluK1 and Ala487, Asn690 and Thr710 in GluK2) in the ligand-binding pocket generate differences in the binding modes of NDH to GluK1 and GluK2. Furthermore, deletion of the C9 hydroxy group in NDH alters the ligand conformation such that it is no longer suited for binding to the GluK1 ligand-binding pocket. In GluK2, NDH pushes and rotates the side chain of Asn690 (substituted for Ser706 in GluK1) and disrupts an interdomain hydrogen bond with Glu409. The present data support the idea that receptor selectivities of DH analogues resulted from the differences in the binding modes of the ligands in GluK1/GluK2 and the steric repulsion of Asn690 in GluK2. All ligands, regardless of agonist efficacy, induced full domain closure. Consequently, ligand efficacy and domain closure did not directly coincide with DH analogues and the kainate receptors.

S100A3, a member of the EF-hand-type Ca(2+)-binding S100 protein family, is unique in its exceptionally high cysteine content and Zn(2+) affinity. We produced human S100A3 protein and its mutants in insect cells using a baculovirus expression system. The purified wild-type S100A3 and the pseudo-citrullinated form (R51A) were crystallized with ammonium sulfate in N,N-bis(2-hydroxyethyl)glycine buffer and, specifically for postrefolding treatment, with Ca(2+)/Zn(2+) supplementation. We identified two previously undocumented disulfide bridges in the crystal structure of properly folded S100A3: one disulfide bridge is between Cys30 in the N-terminal pseudo-EF-hand and Cys68 in the C-terminal EF-hand (SS1), and another disulfide bridge attaches Cys99 in the C-terminal coil structure to Cys81 in helix IV (SS2). Mutational disruption of SS1 (C30A+C68A) abolished the Ca(2+) binding property of S100A3 and retarded the citrullination of Arg51 by peptidylarginine deiminase type III (PAD3), while SS2 disruption inversely increased both Ca(2+) affinity and PAD3 reactivity in vitro. Similar backbone structures of wild type, R51A, and C30A+C68A indicated that neither Arg51 conversion by PAD3 nor SS1 alters the overall dimer conformation. Comparative inspection of atomic coordinates refined to 2.15-1.40 Å resolution shows that SS1 renders the C-terminal classical Ca(2+)-binding loop flexible, which are essential for its Ca(2+) binding properties, whereas SS2 structurally shelters Arg51 in the metal-free form. We propose a model of the tetrahedral coordination of a Zn(2+) by (Cys)(3)His residues that is compatible with SS2 formation in S100A3.

Heme oxygenases found in mammals, plants, and bacteria catalyze degradation of heme using the same mechanism. Roles of distal Asp (Asp-136) residue in HmuO, a heme oxygenase of Corynebacterium diphtheriae, have been investigated by site-directed mutagenesis, enzyme kinetics, resonance Raman spectroscopy, and x-ray crystallography. Replacements of the Asp-136 by Ala and Phe resulted in reduced heme degradation activity due to the formation of ferryl heme, showing that the distal Asp is critical in HmuO heme oxygenase activity. D136N HmuO catalyzed heme degradation at a similar efficiency to wild type and D136E HmuO, implying that the carboxylate moiety is not required for the heme catabolism by HmuO. Resonance Raman results suggest that the inactive ferryl heme formation in the HmuO mutants is induced by disruption of the interaction between a reactive Fe-OOH species and an adjacent distal pocket water molecule. Crystal structural analysis of the HmuO mutants confirms partial disappearance of this nearby water in D136A HmuO. Our results provide the first experimental evidence for the catalytic importance of the nearby water molecule that can be universally critical in heme oxygenase catalysis and propose that the distal Asp helps in positioning the key water molecule at a position suitable for efficient activation of the Fe-OOH species.

HmuO, a heme oxygenase of Corynebacterium diphtheriae, catalyzes degradation of heme using the same mechanism as the mammalian enzyme. The oxy form of HmuO, the precursor of the catalytically active ferric hydroperoxo species, has been characterized by ligand binding kinetics, resonance Raman spectroscopy, and x-ray crystallography. The oxygen association and dissociation rate constants are 5 μm-1 s-1 and 0.22 s-1, respectively, yielding an O2 affinity of 21 μm-1, which is ∼20 times greater than that of mammalian myoglobins. However, the affinity of HmuO for CO is only 3–4-fold greater than that for mammalian myoglobins, implying the presence of strong hydrogen bonding interactions in the distal pocket of HmuO that preferentially favor O2 binding. Resonance Raman spectra show that the Fe–O2 vibrations are tightly coupled to porphyrin vibrations, indicating the highly bent Fe–O–O geometry that is characteristic of the oxy forms of heme oxygenases. In the crystal structure of the oxy form the Fe–O–O angle is 110°, the O–O bond is pointed toward the heme α-meso-carbon by direct steric interactions with Gly-135 and Gly-139, and hydrogen bonds occur between the bound O2 and the amide nitrogen of Gly-139 and a distal pocket water molecule, which is a part of an extended hydrogen bonding network that provides the solvent protons required for oxygen activation. In addition, the O–O bond is orthogonal to the plane of the proximal imidazole side chain, which facilitates hydroxylation of the porphyrin α-meso-carbon by preventing premature O–O bond cleavage.

The 20S proteasome is the catalytic portion of the 26S proteasome. Constitutively expressed mammalian 20S proteasomes have three active subunits, beta 1, beta 2, and beta 5, which are replaced in the immunoproteasome by interferon-gamma-inducible subunits beta 1i, beta 2i, and beta 5i, respectively. Here we determined the crystal structure of the bovine 20S proteasome at 2.75 A resolution. The structures of alpha 2, beta 1, beta 5, beta 6, and beta 7 subunits of the bovine enzyme were different from the yeast enzyme but enabled the bovine proteasome to accommodate either the constitutive or the inducible subunits. A novel N-terminal nucleophile hydrolase activity was proposed for the beta 7 subunit. We also determined the site of the nuclear localization signals in the molecule. A model of the immunoproteasome was predicted from this constitutive structure.

The crystal structure of the Pyrus pyrifolia pistil ribonuclease (S(3)-RNase) responsible for gametophytic self-incompatibility was determined at 1.5-A resolution. It consists of eight helices and seven beta-strands, and its folding topology is typical of RNase T(2) family enzymes. Based on a structural comparison of S(3)-RNase with RNase Rh, a fungal RNase T(2) family enzyme, the active site residues of S(3)-RNase assigned were His(33) and His(88) as catalysts and Glu(84) and Lys(87) as stabilizers of an intermediate in the transition state. Moreover, amino acid residues that constitute substrate binding sites of the two RNases could be superimposed geometrically. A hypervariable (HV) region that has an S-allele-specific sequence comprises a long loop and short alpha-helix. This region is far from the active site cleft, exposed on the molecule's surface, and positively charged. Four positively selected (PS) regions, in which the number of nonsynonymous substitutions exceeds that of synonymous ones, are located on either side of the active site cleft, and accessible to solvent. These structural features suggest that the HV or PS regions may interact with a pollen S-gene product(s) to recognize self and non-self pollen.