Tris(Imidazolyl) Dicopper(I) Complex and its Reactivity to Exert Catalytic Oxidation of Sterically Hindered Phenol Substrates via a [Cu2O]2+ Core

The Cu ion ligated with histidine residues is a common active site motif of various Cu-containing metalloenzymes exerting versatile catalytic oxidation reactions. Due to the scarce of structurally characterized biomimetic...

Kinetic and Structural Characterization of the First B3 Metallo-β-Lactamase with an Active Site Glutamic Acid

The structural diversity in metallo-β-lactamases (MBLs), especially in the vicinity of the active site, has been a major hurdle in the development of clinically effective inhibitors. Representatives from three variants of the B3 MBL subclass, containing either the canonical HHH/DHH active site motif (present in the majority of MBLs in this subclass) or the QHH/DHH (B3-Q) or HRH/DQK (B3-RQK) variations were reported previously. Here, we describe the structure and kinetic properties of the first example (SIE-1) of a fourth variant containing the EHH/DHH active site motif (B3-E). SIE-1 was identified in the hexachlorocyclohexane-degrading bacterium Sphingobium indicum , and kinetic analyses demonstrate that although it is active against a wide range of antibiotics its efficiency is lower than that of other B3 MBLs, but with improved efficiency towards cephalosporins relative to other β-lactam substrates. The overall fold of SIE-1 is characteristic of the MBLs; the notable variation is observed in the Zn1 site due to the replacement of the canonical His116 by a glutamate. The unusual preference of SIE-1 for cephalosporins and its occurrence in a widespread environmental organism suggests scope for increased MBL-mediated β-lactam resistance. It is thus relevant to include SIE-1 into MBL inhibitor design studies to widen the therapeutic scope of much needed anti-resistance drugs.

Cloning, Expression, Characterization, and Antioxidant Protection of Glutaredoxin3 From Psychrophilic Bacterium Psychrobacter sp. ANT206

Glutaredoxins (Grxs) are proteins that catalyze the glutathione (GSH)-dependent reduction of protein disulfides. In this study, a Grx-related gene (264 bp), encoding a Ps-Grx3, was cloned from Psychrobacter sp. ANT206. Sequence analysis indicated the presence of the active site motif CPYC in this protein. Homology modeling showed that Ps-Grx3 had fewer hydrogen bonds and salt bridges, as well as a lower Arg/(Arg + Lys) ratio than its mesophilic homologs, indicative of an improved catalytic ability at low temperatures. Site-directed mutagenesis demonstrated that the Cys13, Pro14, and Cys16 sites were essential for the catalytic activity of Ps-Grx3, while circular dichroism (CD) spectroscopy confirmed that point mutations in these amino acid residues led to the loss or reduction of enzyme activity. Furthermore, analysis of the biochemical properties of Ps-Grx3 showed that the optimum temperature of this enzyme was 25 °C. Importantly, Ps-Grx3 was more sensitive to tBHP and CHP than to H2O2, and retained approximately 40% activity even when the H2O2 concentration was increased to 1 mm Regarding substrate specificity, Ps-Grx3 had a higher affinity for HED, L-cystine, and DHA than for S-sulfocysteine and BSA. We also investigated the DNA-protective ability of Ps-Grx3 using the pUC19 plasmid, and found that Ps-Grx3 could protect supercoiled DNA from oxidation-induced damage at 15°C for 1.5 h. This study provides new insights into the structure and catalytic activity of a cold-adapted Grx3.

Molecular characterization of a prolyl endopeptidase from a feather-degrading thermophile Meiothermus ruber H328

Prolyl endopeptidase from an aerobic and Gram-negative thermophile Meiothermus ruber H328 (MrPEP) was purified in native and recombinant forms, but both preparations had comparable characteristics. Production of the native MrPEP was increased 10-fold by adding intact chicken feathers. The gene for MrPEP (mrH_2860) was cloned from the genome of strain H328 and found to have no signal sequence at the N-terminus. MrPEP is composed of two major domains: the β-propeller domain and the peptidase domain with a typical active site motif and catalytic triad. Based on extensive investigations with different types of peptide substrates and FRETS-25Xaa libraries, MrPEP showed strict preferences for Pro residue at the P1 position but broader preferences at the P2 and P3 positions in substrate specificity with stronger affinity for residues at the P3 position of substrate peptides that are longer than four residues in length. In conclusion, the molecular characterization of MrPEP resembles its animal counterparts more closely than bacterial counterparts in function and structure.

Structure and function of virion RNA polymerase of crAss-like phage

CrAss-like phages are a recently described family-level group of viruses that includes the most abundant virus in the human gut1,2. Genomes of all crAss-like phages encode a large virion-packaged protein2,3 that contains a DFDxD sequence motif, which forms the catalytic site in cellular multisubunit RNA polymerases (RNAPs)4. Using Cellulophaga baltica crAss-like phage phi14:2 as a model system, we show that this protein is a novel DNA-dependent RNAP that is translocated into the host cell along with the phage DNA and transcribes early phage genes. We determined the crystal structure of this 2,180-residue enzyme in a self-inhibited, likely pre-virion-packaged state. This conformation is attained with the help of a Cleft-blocking domain that interacts with the active site motif and occupies the RNA-DNA hybrid binding grove. Structurally, phi14:2 RNAP is most similar to eukaryotic RNAPs involved in RNA interference5,6, although most of phi14:2 RNAP structure (nearly 1,600 residues) maps to a new region of protein folding space. Considering the structural similarity, we propose that eukaryal RNA interference polymerases take their origin in a phage, which parallels the emergence of the mitochondrial transcription apparatus7.

Crystal structure of human anterior gradient protein 3

Oxidative protein folding in the endoplasmic reticulum is catalyzed by the protein disulfide isomerase family of proteins. Of the 20 recognized human family members, the structures of eight have been deposited in the PDB along with domains from six more. Three members of this family, ERp18, anterior gradient protein 2 (AGR2) and anterior gradient protein 3 (AGR3), are single-domain proteins which share sequence similarity. While ERp18 has a canonical active-site motif and is involved in native disulfide-bond formation, AGR2 and AGR3 lack elements of the active-site motif found in other family members and may both interact with mucins. In order to better define its function, the structure of AGR3 is required. Here, the recombinant expression, purification, crystallization and crystal structure of human AGR3 are described.

Cation-exchanged zeolites for the selective oxidation of methane to methanol

Development of an ideal methane activation catalyst presents a trade-off between stability and reactivity of the active site that can be achieved by tuning the transition metal cation, active site motif and the zeolite topology.

Dengue virus hijacks a noncanonical oxidoreductase function of a cellular oligosaccharyltransferase complex

Dengue virus (DENV) is the most common arboviral infection globally, infecting an estimated 390 million people each year. We employed a genome-wide CRISPR screen to identify host dependency factors required for DENV propagation, and identified the oligosaccharyltransferase (OST) complex as an essential host factor for DENV infection. Mammalian cells express two OSTs containing either STT3A or STT3B. We found that the canonical catalytic function of the OSTs as oligosaccharyltransferases is not necessary for DENV infection, as cells expressing catalytically inactive STT3A or STT3B are able to support DENV propagation. However, the OST subunit MAGT1, which associates with STT3B, is also required for DENV propagation. MAGT1 expression requires STT3B, and a catalytically inactive STT3B also rescues MAGT1 expression, supporting the hypothesis that STT3B serves to stabilize MAGT1 in the context of DENV infection. We found that the oxidoreductase CxxC active site motif of MAGT1 was necessary for DENV propagation as cells expressing an AxxA MAGT1 mutant were unable to support DENV infection.Interestingly, cells expressing single-cysteine CxxA or AxxC mutants of MAGT1 were able to support DENV propagation. Utilizing the engineered peroxidase APEX2, we demonstrate the close proximity between MAGT1 and NS1 or NS4B during DENV infection. These results reveal that the oxidoreductase activity of the STT3B-containing OST is necessary for DENV infection, which may guide the development of antivirals targeting DENV.

Arabidopsis thaliana dehydroascorbate reductase 2: Conformational flexibility during catalysis

Dehydroascorbate reductase (DHAR) catalyzes the glutathione (GSH)-dependent reduction of dehydroascorbate and plays a direct role in regenerating ascorbic acid, an essential plant antioxidant vital for defense against oxidative stress. DHAR enzymes bear close structural homology to the glutathione transferase (GST) superfamily of enzymes and contain the same active site motif, but most GSTs do not exhibit DHAR activity. The presence of a cysteine at the active site is essential for the catalytic functioning of DHAR, as mutation of this cysteine abolishes the activity. Here we present the crystal structure of DHAR2 from Arabidopsis thaliana with GSH bound to the catalytic cysteine. This structure reveals localized conformational differences around the active site which distinguishes the GSH-bound DHAR2 structure from that of DHAR1. We also unraveled the enzymatic step in which DHAR releases oxidized glutathione (GSSG). To consolidate our structural and kinetic findings, we investigated potential conformational flexibility in DHAR2 by normal mode analysis and found that subdomain mobility could be linked to GSH binding or GSSG release.

Structure of the ectodomain of the electron transporter Rv2874 fromMycobacterium tuberculosisreveals a thioredoxin-like domain combined with a carbohydrate-binding module

The members of the CcdA family are integral membrane proteins that use a disulfide cascade to transport electrons from the thioredoxin–thioredoxin reductase system in the interior of the cell into the extracytoplasmic space. The core transmembrane portion of this family is often elaborated with additional hydrophilic domains that act as adapters to deliver reducing potential to targets outside the cellular membrane. To investigate the function of family members inMycobacterium tuberculosis, the structure of the C-terminal ectodomain from Rv2874, one of three CcdA-family members present in the genome, was determined. The crystal structure, which was refined at 1.9 Å resolution withR= 0.195 andRfree= 0.219, reveals the predicted thioredoxin-like domain with its conserved Cys-X-X-Cys active-site motif. Unexpectedly, this domain is combined with a second domain with a carbohydrate-binding module (CBM) fold, this being the first reported example of a CBM in association with a thioredoxin-like domain fold. A cavity in the CBM adjacent to the thioredoxin active site suggests a likely carbohydrate-binding site, representing a broadening of the substrate range for CcdA-family members and an expansion of the thioredoxin-domain functionality to carbohydrate modification.