KEGG(Kyoto Encyclopedia of Genes and Genomes)〔和文〕 (特集 ゲノム医学の現在と未来--基礎と臨床) -- (データベース)

Laccases of fungi attract considerable attention due to their possible involvement in the transformation of a wide variety of phenolic compounds including the polymeric lignin and humic substances. So far, more than a 100 enzymes have been purified from fungal cultures and characterized in terms of their biochemical and catalytic properties. Most ligninolytic fungal species produce constitutively at least one laccase isoenzyme and laccases are also dominant among ligninolytic enzymes in the soil environment. The fact that they only require molecular oxygen for catalysis makes them suitable for biotechnological applications for the transformation or immobilization of xenobiotic compounds.

Fungal laccases - occurrence and properties.

Nitrogen is an essential component of all living organisms and the main nutrient limiting life on our planet By far, the largest inventory of freely accessible nitrogen is atmospheric dinitrogen, but most organisms rely on more bioavailable forms of nitrogen, such as ammonium and nitrate, for growth The availability of these substrates depends on diverse nitrogen-transforming reactions that are carried out by complex networks of metabolically versatile microorganisms In this Review, we summarize our current understanding of the microbial nitrogen-cycling network, including novel processes, their underlying biochemical pathways, the involved microorganisms, their environmental importance and industrial applications

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The microbial nitrogen-cycling network

Based on its generally accessible I/II redox couple and bioavailability, copper plays a wide variety of roles in nature that mostly involve electron transfer (ET), O2 binding, activation and reduction, NO2− and N2O reduction and substrate activation. Copper sites that perform ET are the mononuclear blue Cu site that has a highly covalent CuII-S(Cys) bond and the binuclear CuA site that has a Cu2S(Cys)2 core with a Cu-Cu bond that keeps the site delocalized (Cu(1.5)2) in its oxidized state. In contrast to inorganic Cu complexes, these metalloprotein sites transfer electrons rapidly often over long distances, as has been previously reviewed.1–4 Blue Cu and CuA sites will only be considered here in their relation to intramolecular ET in multi-center enzymes. The focus of this review is on the Cu enzymes (Figure 1). Many are involved in O2 activation and reduction, which has mostly been thought to involve at least two electrons to overcome spin forbiddenness and the low potential of the one electron reduction to superoxide (Figure 2).5,6 Since the Cu(III) redox state has not been observed in biology, this requires either more than one Cu center or one copper and an additional redox active organic cofactor. The latter is formed in a biogenesis reaction of a residue (Tyr) that is also Cu catalyzed in the first turnover of the protein. Recently, however, there have been a number of enzymes suggested to utilize one Cu to activate O2 by 1e− reduction to form a Cu(II)-O2•− intermediate (an innersphere redox process) and it is important to understand the active site requirements to drive this reaction. The oxidases that catalyze the 4e−reduction of O2 to H2O are unique in that they effectively perform this reaction in one step indicating that the free energy barrier for the second two-electron reduction of the peroxide product of the first two-electron step is very low. In nature this requires either a trinuclear Cu cluster (in the multicopper oxidases) or a Cu/Tyr/Heme Fe cluster (in the cytochrome oxidases). The former accomplishes this with almost no overpotential maximizing its ability to oxidize substrates and its utility in biofuel cells, while the latter class of enzymes uses the excess energy to pump protons for ATP synthesis. In bacterial denitrification, a mononuclear Cu center catalyzes the 1e- reduction of nitrite to NO while a unique µ4S2−Cu4 cluster catalyzes the reduction of N2O to N2 and H2O, a 2e− process yet requiring 4Cu’s. Finally there are now several classes of enzymes that utilize an oxidized Cu(II) center to activate a covalently bound substrate to react with O2.



Figure 1

Copper active sites in biology.





Figure 2

Latimer Diagram for Oxygen Reduction at pH = 7.0 Adapted from References 5 and 6.



This review presents in depth discussions of all these classes of Cu enzymes and the correlations within and among these classes. For each class we review our present understanding of the enzymology, kinetics, geometric structures, electronic structures and the reaction mechanisms these have elucidated. While the emphasis here is on the enzymology, model studies have significantly contributed to our understanding of O2 activation by a number of Cu enzymes and are included in appropriate subsections of this review. In general we will consider how the covalency of a Cu(II)–substrate bond can activate the substrate for its spin forbidden reaction with O2, how in binuclear Cu enzymes the exchange coupling between Cu’s overcomes the spin forbiddenness of O2 binding and controls electron transfer to O2 to direct catalysis either to perform two e− electrophilic aromatic substitution or 1e− H-atom abstraction, the type of oxygen intermediate that is required for H-atom abstraction from the strong C-H bond of methane (104 kcal/mol) and how the trinuclear Cu cluster and the Cu/Tyr/Heme Fe cluster achieve their very low barriers for the reductive cleavage of the O-O bond.

Much of the insight available into these mechanisms in Cu biochemistry has come from the application of a wide range of spectroscopies and the correlation of spectroscopic results to electronic structure calculations. Thus we start with a tutorial on the different spectroscopic methods utilized to study mononuclear and multinuclear Cu enzymes and their correlations to different levels of electronic structure calculations.

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Copper Active Sites in Biology

Iterative model-building, structure refinement, and density modification with the PHENIX AutoBuild Wizard Thomas C. Terwilliger a* , Ralf W. Grosse-Kunstleve b , Pavel V. Afonine b , Nigel W. Moriarty b , Peter Zwart b , Li-Wei Hung a , Randy J. Read c , Paul D. Adams b* a b Los Alamos National Laboratory, Mailstop M888, Los Alamos, NM 87545, USA Lawrence Berkeley National Laboratory, One Cyclotron Road, Bldg 64R0121, Berkeley, CA 94720, USA. c Department of Haematology, University of Cambridge, Cambridge CB2 0XY, UK. * Email: terwill@lanl.gov or PDAdams@lbl.gov Running title: The PHENIX AutoBuild Wizard Abstract The PHENIX AutoBuild Wizard is a highly automated tool for iterative model- building, structure refinement and density modification using RESOLVE or TEXTAL model- building, RESOLVE statistical density modification, and phenix.refine structure refinement. Recent advances in the AutoBuild Wizard and phenix.refine include automated detection and application of NCS from models as they are built, extensive model completion algorithms, and automated solvent molecule picking. Model completion algorithms in the AutoBuild Wizard include loop-building, crossovers between chains in different models of a structure, and side-chain optimization. The AutoBuild Wizard has been applied to a set of 48 structures at resolutions ranging from 1.1 A to 3.2 A, resulting in a mean R-factor of 0.24 and a mean free R factor of 0.29. The R-factor of the final model is dependent on the quality of the starting electron density, and relatively independent of resolution. Keywords: Model building; model completion; macromolecular models; Protein Data Bank; structure refinement; PHENIX Introduction Iterative model-building and refinement is a powerful approach to obtaining a complete and accurate macromolecular model. The approach consists of cycles of building an atomic model based on an electron density map for a macromolecular structure, refining the structure, using the refined structure as a basis for improving the map, and building a new model. This type of approach has been carried out in a semi-automated fashion for many years, with manual model-building iterating with automated refinement (Jensen, 1997). More recently, with the development first of ARP/wARP (Perrakis et al., 1999), and later other procedures including RESOLVE iterative model-building and refinement (Terwilliger,

Iterative model-building, structure refinement, and density modification with the PHENIX AutoBuild Wizard

Several energy-transducing microbial enzymes have their peripheral subunits connected to the membrane through an integral membrane protein, that interacts with quinones but does not have redox cofactors, the so-called NrfD-like subunit. The periplasmic nitrite reductase (NrfABCD) was the first complex recognized to have a membrane subunit with these characteristics and consequently provided the family's name: NrfD. Sequence analyses indicate that NrfD homologs are present in many diverse enzymes, such as polysulfide reductase (PsrABC), respiratory alternative complex III (ACIII), dimethyl sulfoxide (DMSO) reductase (DmsABC), tetrathionate reductase (TtrABC), sulfur reductase complex (SreABC), sulfite dehydrogenase (SoeABC), quinone reductase complex (QrcABCD), nine-heme cytochrome complex (NhcABCD), group-2 [NiFe] hydrogenase (Hyd-2), dissimilatory sulfite-reductase complex (DsrMKJOP), arsenate reductase (ArrC) and multiheme cytochrome c sulfite reductase (MccACD). The molecular structure of ACIII subunit C (ActC) and Psr subunit C (PsrC), NrfD-like subunits, revealed the existence of ion-conducting pathways. We performed thorough primary structural analyses and built structural models of the NrfD-like subunits. We observed that all these subunits are constituted by two structural repeats composed of four-helix bundles, possibly harboring ion-conducting pathways and containing a quinone/quinol binding site. NrfD-like subunits may be the ion-pumping module of several enzymes. Our data impact on the discussion of functional implications of the NrfD-like subunit-containing complexes, namely in their ability to transduce energy.

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The Ion-Translocating NrfD-Like Subunit of Energy-Transducing Membrane Complexes.

A missense mutant of a Dps protein (DNA-binding protein from starved cells) from Marinobacter hydrocarbonoclasticus was used as a building block to develop a new supramolecular assembly complex which enhances the iron uptake, a physiological function of this mini-ferritin. The missense mutation was conducted in an exposed and flexible region of the N-terminal, wherein a threonine residue in position 10 was replaced by a cysteine residue (DpsT10C). This step enabled a click chemistry approach to the variant DpsT10C, where a thiol-ene coupling occurs. Two methods and two types of linker were used resulting in two different mini-ferritin supramolecular polymers, which have maintained secondary structure and native iron uptake physiological function. Electrophoretic assays and mass spectrometry were utilized to confirm that both functionalization and coupling reactions occured as predicted. The secondary structure has been investigated by circular dichroism and synchrotron radiation circular dichroism. Size and morphology were obtained by dynamic light scattering, size exclusion chromatography and atomic force microscopy, respectively. The iron uptake of the synthesized protein polymers was confirmed by UV-Vis spectroscopy loading assays.

Supramolecular protein polymers using mini-ferritin Dps as the building block

In recent years, type II NADH dehydrogenases (NDH-IIs) have emerged as potential drug targets for a wide range of human disease causative agents In this work, the NDH-II enzyme from the Gram-positive human pathogen Staphylococcus aureus was recombinantly expressed in Escherichia coli, purified, crystallized and a crystallographic data set was collected at a wavelength of 0873 A The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 818, b = 860, c = 2699 A, contained four monomers per asymmetric unit and diffracted to a resolution of 332 A A molecular-replacement solution was obtained and model building and refinement are currently under way

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Expression, purification, crystallization and preliminary X‐ray diffraction analysis of a type II NADH:quinone oxidoreductase from the human pathogen Staphylococcus aureus

Nucleotides are structural units relevant not only in nucleic acids but also as substrates or cofactors in key biochemical reactions. The size- and timescales of such nucleotide-protein interactions fall well within the scope of coarse-grained molecular dynamics, which holds promise of important mechanistic insight. However, the lack of specific parameters has prevented accurate coarse-grained simulations of protein interactions with most nucleotide compounds. In this work, we comprehensively develop coarse-grained parameters for key metabolites/cofactors (FAD, FMN, riboflavin, NAD, NADP, ATP, ADP, AMP, and thiamine pyrophosphate) in different oxidation and protonation states as well as for smaller molecules derived from them (among others, nicotinamide, adenosine, adenine, ribose, thiamine, and lumiflavin), summing up a total of 79 different molecules. In line with the Martini parameterization methodology, parameters were tuned to reproduce octanol-water partition coefficients. Given the lack of existing data, we set out to experimentally determine these partition coefficients, developing two methodological approaches, based on 31P-NMR and fluorescence spectroscopy, specifically tailored to the strong hydrophilicity of most of the parameterized compounds. To distinguish the partition of each relevant protonation species, we further potentiometrically characterized the protonation constants of key molecules. This work successfully builds a comprehensive and relevant set of computational models that will boost the biochemical application of coarse-grained simulations. It does so based on the measurement of partition and acid-base physicochemical data that, in turn, covers important gaps in nucleotide characterization.

Manuela M. Pereira

Papers

The Ion-Translocating NrfD-Like Subunit of Energy-Transducing Membrane Complexes.

Supramolecular protein polymers using mini-ferritin Dps as the building block

Expression, purification, crystallization and preliminary X‐ray diffraction analysis of a type II NADH:quinone oxidoreductase from the human pathogen Staphylococcus aureus

Coarse-Grained Parameterization of Nucleotide Cofactors and Metabolites: Protonation Constants, Partition Coefficients, and Model Topologies.

Insights into the antiporter-like subunits of respiratory complex I