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161.
Colloc'h N Gabison L Monard G Altarsha M Chiadmi M Marassio G Sopkova-de Oliveira Santos J El Hajji M Castro B Abraini JH Prangé T 《Biophysical journal》2008,95(5):2415-2422
The localization of dioxygen sites in oxygen-binding proteins is a nontrivial experimental task and is often suggested through indirect methods such as using xenon or halide anions as oxygen probes. In this study, a straightforward method based on x-ray crystallography under high pressure of pure oxygen has been developed. An application is given on urate oxidase (UOX), a cofactorless enzyme that catalyzes the oxidation of uric acid to 5-hydroxyisourate in the presence of dioxygen. UOX crystals in complex with a competitive inhibitor of its natural substrate are submitted to an increasing pressure of 1.0, 2.5, or 4.0 MPa of gaseous oxygen. The results clearly show that dioxygen binds within the active site at a location where a water molecule is usually observed but does not bind in the already characterized specific hydrophobic pocket of xenon. Moreover, crystallizing UOX in the presence of a large excess of chloride (NaCl) shows that one chloride ion goes at the same location as the oxygen. The dioxygen hydrophilic environment (an asparagine, a histidine, and a threonine residues), its absence within the xenon binding site, and its location identical to a water molecule or a chloride ion suggest that the dioxygen site is mainly polar. The implication of the dioxygen location on the mechanism is discussed with respect to the experimentally suggested transient intermediates during the reaction cascade. 相似文献
162.
Arene cis-diols are interesting chemicals because of their chiral structures and great potentials in industrial synthesis of useful
chiral chemical products. Pseudomonas putida KT2442 was genetically modified to transform benzoic acid (benzoate) to 1,2-dihydroxy-cyclohexa-3,5-diene-1-carboxylic acid
(DHCDC) or named benzoate cis-diol. BenD gene encoding cis-diol dehydrogenase was deleted to generate a mutant named P. putida KTSY01. Genes benABC encoding benzoate dioxygenase were cloned into plasmid pSYM01 and overexpressed in P. putida KTSY01. The recombinant bacteria P. putida KTSY01 (pSYM01) showed strong ability to transform benzoate to DHCDC. DHCDC of 2.3 g/L was obtained with a yield of 73% after
24 h of cultivation in shake flasks incubated under optimized growth conditions. Transformation of benzoate carried out in
a 6-L fermentor using a benzoate fed-batch process produced over 17 g/L DHCDC after 48 h of fermentation. The average DHCDC
production rate was 0.356 g L−1 h−1. DHCDC purified from the fermentation broth showed a purity of more than 95%, and its chemical structure was confirmed by
nuclear magnetic resonance. 相似文献
163.
Ju T Goldsmith RB Chai SC Maroney MJ Pochapsky SS Pochapsky TC 《Journal of molecular biology》2006,363(4):823-834
Acireductone dioxygenase (ARD) catalyzes different reactions between O2 and 1,2-dihydroxy-3-oxo-5-(methylthio)pent-1-ene (acireductone) depending upon the metal bound in the active site. Ni2+ -ARD cleaves acireductone to formate, CO and methylthiopropionate. If Fe2+ is bound (ARD'), the same substrates yield methylthioketobutyrate and formate. The two forms differ in structure, and are chromatographically separable. Paramagnetism of Fe2+ renders the active site of ARD' inaccessible to standard NMR methods. The structure of ARD' has been determined using Fe2+ binding parameters determined by X-ray absorption spectroscopy and NMR restraints from H98S ARD, a metal-free diamagnetic protein that is isostructural with ARD'. ARD' retains the beta-sandwich fold of ARD, but a structural entropy switch increases order at one end of a two-helix system that bisects the beta-sandwich and decreases order at the other upon interconversion of ARD and ARD', causing loss of the C-terminal helix in ARD' and rearrangements of residues involved in substrate orientation in the active site. 相似文献
164.
A quantitative real-time polymerase chain reaction (PCR) assay was developed for monitoring naphthalene degradation during bioremediation processes. The phylogenetic affiliations of known naphthalene-hydroxylating dioxygenase genes were determined to target functionally related bacteria, and degenerate primers were designed on the basis of the close relationships among dioxygenase genes identified from naphthalene-degrading Proteobacteria. Evaluation of the amplification specificity demonstrated that the developed real-time PCR assay represents a rapid, precise means for the group-specific enumeration of naphthalene-degrading bacteria. According to validation with bacterial pure cultures, the assay discriminated between the targeted group of naphthalene dioxygenase sequences and genes in other naphthalene or aromatic hydrocarbon-degrading bacterial strains. Specific amplification of gene fragments sharing a high sequence similarity with the genes included in the assay design was also observed in soil samples recovered from large-scale remediation processes. The target genes could be quantified reproducibly at over five orders of magnitude down to 3 × 102 gene copies. To investigate the suitability of the assay in monitoring naphthalene biodegradation, the assay was applied in enumerating the naphthalene dioxygenase genes in a soil slurry microcosm. The results were in good agreement with contaminant mineralization and dot blot quantification of nahAc gene copies. Furthermore, the real-time PCR assay was found to be more sensitive than hybridization-based analysis. 相似文献
165.
Fullerene, C60 was functionalized to possess one or two fluorophore entities. The fluorophore–fullerene dyads thus synthesized contain either
a naphthalene, pyrene, or fluorene entity while the triads contain either a pyrene or fluorene entity in addition to a naphthalene
entity. The redox behavior of these dyads and triads were probed by cyclic voltammetric technique, while the geometry and
electronic structures were deduced from ab initio B3LYP/3-21G(*) method. Steady-state emission studies revealed the occurrence of energy transfer from the singlet excited
fluorophore to the fullerene entity in the case of the dyads while the occurrence of step-by-step sequential energy transfer
is envisioned in the case of the triads. A better ‘antenna-effect’ owing to the extended range of excitation wavelength to
induce energy transfer to the appended fullerene has been achieved in the case of the triads. 相似文献
166.
Filonov A. E. Akhmetov L. I. Puntus I. F. Esikova T. Z. Gafarov A. B. Izmalkova T. Yu. Sokolov S. L. Kosheleva I. A. Boronin A. M. 《Microbiology》2005,74(4):453-458
A genetically tagged, plasmid-containing, naphthalene-degrading strain, Pseudomonas putida KT2442(pNF142:: TnMod-OTc), has been constructed. The presence of the gfp gene (which codes for green fluorescent protein) and the kanamycin and rifampicin resistance genes in the chromosome of this strain allows the strain’s fate in model soil systems to be monitored, whereas a minitransposon, inserted into naphthalene biodegradation plasmid pNF142 and containing the tetracycline resistance gene, makes it possible to follow the horizontal transfer of this plasmid between various bacteria. Plasmid pNF142::TnMod-OTc is stable in strain P. putida KT2442 under nonselective conditions. The maximal specific growth rate of this strain on naphthalene is found to be higher than that of the natural host of plasmid pNF142. When introduced into a model soil system, the genetically tagged strain is stable and competitive for 40 days. The transfer of labeled plasmid pNF142::TnMod-OTc to natural soil bacteria, predominantly fluorescent pseudomonads, has been detected.__________Translated from Mikrobiologiya, Vol. 74, No. 4, 2005, pp. 526–532.Original Russian Text Copyright © 2005 by Filonov, Akhmetov, Puntus, Esikova, Gafarov, Izmalkova, Sokolov, Kosheleva, Boronin. 相似文献
167.
Andreas Karlsson Juan V. Parales Rebecca E. Parales David T. Gibson Hans Eklund S. Ramaswamy 《Journal of biological inorganic chemistry》2005,10(5):483-489
Nitric oxide (NO) is commonly used as an analogue for dioxygen in structural and spectroscopic studies of oxygen binding and oxygen activation. In this study, crystallographic structures of naphthalene dioxygenase (NDO) in complex with nitric oxide are reported. In the presence of the aromatic substrate indole, NO is bound end-on to the active-site mononuclear iron of NDO. The structural observations correlate well with spectroscopic measurements of NO binding to NDO in solution. However, the end-on binding of NO is in contrast to the recently reported structure of oxygen to the active-site iron of NDO that binds side-on. While NO is a good oxygen analogue with many similarities to O2, the different binding mode of NO to the active-site iron atom leads to different mechanistic implications. Hence, caution needs to be used in extrapolating NO as an analogue to O2 binding. 相似文献
168.
Joseph P. Emerson Michelle L. Wagner Mark F. Reynolds Lawrence Que Jr Michael J. Sadowsky Lawrence P. Wackett 《Journal of biological inorganic chemistry》2005,10(7):751-760
The manganese-dependent 3,4-dihydroxyphenylacetate 2,3-dioxygenase (MndD) from Arthrobacter globiformis CM-2 is an extradiol-cleaving catechol dioxygenase that catalyzes aromatic ring cleavage of 3,4-dihydroxyphenylacetate (DHPA).
Based on the recent crystal structure of the MndD–DHPA complex, a series of site-directed mutations were made at a conserved
second-sphere residue, histidine 200, to gain insight into and clarify the role this residue plays in the Mn(II)-dependent
catalytic mechanism. In this study, we report the activities and spectroscopic data of these H200 variants and their DHPA
and 4-nitrocatechol (4-NC) complexes. The data collected from wild-type and mutant MndDs are consistent with a role for H200
interacting with a manganese-bound dioxygen moiety and are inconsistent with other previously proposed roles involving proton
transfer. Spectroscopic observations, including unique low-field EPR signals found when DHPA and 4-NC are bound to the Mn(II)
center of MndD, are discussed and their relationship to dioxygen activation catalyzed in MndD is explored.
Electronic Supplementary Material Supplementary material is available for this article at 相似文献
169.
Woo J Howell MH von Arnim AG 《Protein science : a publication of the Protein Society》2008,17(4):725-735
Renilla luciferase (RLUC) is a versatile tool for gene expression assays and in vivo biosensor applications, but its catalytic mechanism remains to be elucidated. RLUC is evolutionarily related to the alpha/beta hydrolase family. Its closest known homologs are bacterial dehalogenases, raising the question of how a protein with a hydrolase fold can function as a decarboxylating oxygenase. Molecular docking simulations with the coelenterazine substrate against an RLUC homology model as well as a recently determined RLUC crystal structure were used to build hypotheses to identify functionally important residues, which were subsequently tested by site-directed mutagenesis, heterologous expression, and bioluminescence emission spectroscopy. The data highlighted two triads of residues that are critical for catalysis. The putative catalytic triad residues D120, E144, and H285 bear only limited resemblance to those found in the active site of aequorin, a coelenterazine-utilizing photoprotein, suggesting that the reaction scheme employed by RLUC differs substantially from the one established for aequorin. The role of H285 in catalysis was further supported by inhibition using diethylpyrocarbonate. Multiple substitutions of N53, W121, and P220--three other residues implicated in product binding in the homologous dehalogenase Sphingomonas LinB--also supported their involvement in catalysis. Together with luminescence spectra, our data lead us to propose that the conserved catalytic triad of RLUC is directly involved in the decarboxylation reaction of coelenterazine to produce bioluminescence, while the other active-site residues are used for binding of the substrate. 相似文献
170.
Quercetinase (quercetin 2,3-dioxygenase, EC 1.13.11.24) is produced by various filamentous fungi when grown on rutin as the sole carbon and energy source. From a rutin based liquid culture of Penicillium olsonii, we purified a quercetinase with a specific activity of 175U mg(-1). The enzyme is a monomeric glycoprotein of approximately 55 kDa, containing 0.9+/-0.1 copper atoms per protein. Its substrate specificity is restricted to the flavonol family of flavonoids. It is completely inhibited by diethyldithiocarbamate at a concentration of 100 nM and 1H-2-benzyl-3-hydroxy-4-oxoquinolin is a competitive inhibitor with a K(I) of 4 microM. The cDNA poquer1 was cloned and sequenced. It encodes a 365 amino acids long enzyme with a strong sequence identity with the Aspergillus japonicus quercetinase (Q7SIC2). Like the enzyme from A. japonicus, only one of the two cupin domains of the Penicillium olsonii quercetinase is able to bind a metal atom. 相似文献