Spectroscopic studies of the copper sites in wild-type Pseudomonas stutzeri N2O reductase and in an inactive protein isolated from a mutant deficient in copper-site biosynthesis

The 173–195 segment corresponding to the helix 2 of the C-globular prion protein domain could be one of several “spots” of intrinsic conformational flexibility. In fact, it possesses chameleon conformational behaviour and gathers several disease-associated point mutations. We have performed spectroscopic studies on the wild-type fragment 173–195 and on its D178N mutant dissolved in trifluoroethanol to mimic the in vivo system, both in the presence and in the absence of metal cations. NMR data showed that the structure of the D178N mutant is characterized by two short helices separated by a kink, whereas the wild-type peptide is fully helical. Both peptides retained these structural organizations, as monitored by CD, in the presence of metal cations. NMR spectra were however not in favour of the formation of definite ion-peptide complexes. This agrees with previous evidence that other regions of the prion protein are likely the natural target of metal cation binding.

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Pyridine-2,6-Bis(Thiocarboxylic Acid) Produced by Pseudomonas stutzeri KC Reduces and Precipitates Selenium and Tellurium Oxyanions

Applied and Environmental Microbiology ◽

10.1128/aem.72.5.3119-3129.2006 ◽

2006 ◽

Vol 72 (5) ◽

pp. 3119-3129 ◽

Cited By ~ 33

Author(s):

Anna M. Zawadzka ◽

Ronald L. Crawford ◽

Andrzej J. Paszczynski

Keyword(s):

Wild Type Strain ◽

Hydrolysis Product ◽

Pseudomonas Stutzeri ◽

Elemental Selenium ◽

Bacterial Cells ◽

Wild Type ◽

Chemical Structures ◽

Thiocarboxylic Acid ◽

Insoluble Compounds ◽

Initial Line

ABSTRACT The siderophore of Pseudomonas stutzeri KC, pyridine-2,6-bis(thiocarboxylic acid) (pdtc), is shown to detoxify selenium and tellurium oxyanions in bacterial cultures. A mechanism for pdtc's detoxification of tellurite and selenite is proposed. The mechanism is based upon determination using mass spectrometry and energy-dispersive X-ray spectrometry of the chemical structures of compounds formed during initial reactions of tellurite and selenite with pdtc. Selenite and tellurite are reduced by pdtc or its hydrolysis product H2S, forming zero-valent pdtc selenides and pdtc tellurides that precipitate from solution. These insoluble compounds then hydrolyze, releasing nanometer-sized particles of elemental selenium or tellurium. Electron microscopy studies showed both extracellular precipitation and internal deposition of these metalloids by bacterial cells. The precipitates formed with synthetic pdtc were similar to those formed in pdtc-producing cultures of P. stutzeri KC. Culture filtrates of P. stutzeri KC containing pdtc were also active in removing selenite and precipitating elemental selenium and tellurium. The pdtc-producing wild-type strain KC conferred higher tolerance against selenite and tellurite toxicity than a pdtc-negative mutant strain, CTN1. These observations support the hypothesis that pdtc not only functions as a siderophore but also is involved in an initial line of defense against toxicity from various metals and metalloids.

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Protein Engineering of Toluene-o-Xylene Monooxygenase from Pseudomonas stutzeri OX1 for Synthesizing 4-Methylresorcinol, Methylhydroquinone, and Pyrogallol

Applied and Environmental Microbiology ◽

10.1128/aem.70.6.3253-3262.2004 ◽

2004 ◽

Vol 70 (6) ◽

pp. 3253-3262 ◽

Cited By ~ 54

Author(s):

GÃ¯Â¿Â½nÃ¯Â¿Â½l Vardar ◽

Thomas K. Wood

Keyword(s):

Active Site ◽

High Performance ◽

Agar Plate ◽

Pseudomonas Stutzeri ◽

Saturation Mutagenesis ◽

Dna Shuffling ◽

Natural Substrate ◽

Wild Type ◽

Active Site Residues ◽

Derivatives Of

ABSTRACT Toluene-o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1 oxidizes toluene to 3- and 4-methylcatechol and oxidizes benzene to form phenol; in this study ToMO was found to also form catechol and 1,2,3-trihydroxybenzene (1,2,3-THB) from phenol. To synthesize novel dihydroxy and trihydroxy derivatives of benzene and toluene, DNA shuffling of the alpha-hydroxylase fragment of ToMO (TouA) and saturation mutagenesis of the TouA active site residues I100, Q141, T201, and F205 were used to generate random mutants. The mutants were initially identified by screening with a rapid agar plate assay and then were examined further by high-performance liquid chromatography and gas chromatography. Several regiospecific mutants with high rates of activity were identified; for example, Escherichia coli TG1/pBS(Kan)ToMO expressing the F205G TouA saturation mutagenesis variant formed 4-methylresorcinol (0.78 nmol/min/mg of protein), 3-methylcatechol (0.25 nmol/min/mg of protein), and methylhydroquinone (0.088 nmol/min/mg of protein) from o-cresol, whereas wild-type ToMO formed only 3-methylcatechol (1.1 nmol/min/mg of protein). From o-cresol, the I100Q saturation mutagenesis mutant and the M180T/E284G DNA shuffling mutant formed methylhydroquinone (0.50 and 0.19 nmol/min/mg of protein, respectively) and 3-methylcatechol (0.49 and 1.5 nmol/min/mg of protein, respectively). The F205G mutant formed catechol (0.52 nmol/min/mg of protein), resorcinol (0.090 nmol/min/mg of protein), and hydroquinone (0.070 nmol/min/mg of protein) from phenol, whereas wild-type ToMO formed only catechol (1.5 nmol/min/mg of protein). Both the I100Q mutant and the M180T/E284G mutant formed hydroquinone (1.2 and 0.040 nmol/min/mg of protein, respectively) and catechol (0.28 and 2.0 nmol/min/mg of protein, respectively) from phenol. Dihydroxybenzenes were further oxidized to trihydroxybenzenes with different regiospecificities; for example, the I100Q mutant formed 1,2,4-THB from catechol, whereas wild-type ToMO formed 1,2,3-THB (pyrogallol). Regiospecific oxidation of the natural substrate toluene was also checked; for example, the I100Q mutant formed 22% o-cresol, 44% m-cresol, and 34% p-cresol, whereas wild-type ToMO formed 32% o-cresol, 21% m-cresol, and 47% p-cresol.

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Spectroscopic Studies and Electronic Structure Description of the High Potential Type 1 Copper Site in Fungal Laccase: Insight into the Effect of the Axial Ligand

Journal of the American Chemical Society ◽

10.1021/ja991087v ◽

1999 ◽

Vol 121 (30) ◽

pp. 7138-7149 ◽

Cited By ~ 66

Author(s):

Amy E. Palmer ◽

David W. Randall ◽

Feng Xu ◽

Edward I. Solomon

Keyword(s):

Electronic Structure ◽

Spectroscopic Studies ◽

Axial Ligand ◽

High Potential ◽

Fungal Laccase ◽

Copper Site ◽

Structure Description ◽

Type 1 Copper ◽

Insight Into

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Activation and Inactivation of Pseudomonas stutzeriMethylbenzene Catabolism Pathways Mediated by a Transposable Element

Applied and Environmental Microbiology ◽

10.1128/aem.65.5.1876-1882.1999 ◽

1999 ◽

Vol 65 (5) ◽

pp. 1876-1882 ◽

Cited By ~ 24

Author(s):

Fabrizio Bolognese ◽

Cinzia di Lecce ◽

Enrica Galli ◽

Paola Barbieri

Keyword(s):

Transposable Element ◽

Type Strain ◽

Insertion Sequence ◽

Wild Type Strain ◽

Pseudomonas Stutzeri ◽

Inverted Repeats ◽

Wild Type ◽

Catabolic Pathway ◽

Catabolic Genes ◽

Target Dna

ABSTRACT The arrangement of the genes involved in o-xylene,m-xylene, and p-xylene catabolism was investigated in three Pseudomonas stutzeri strains: the wild-type strain OX1, which is able to grow on o-xylene but not on the meta and para isomers; the mutant M1, which grows on m-xylene and p-xylene but is unable to utilize the ortho isomer; and the revertant R1, which can utilize all the three isomers of xylene. A 3-kb insertion sequence (IS) termed ISPs1, which inactivates them-xylene and p-xylene catabolic pathway inP. stutzeri OX1 and the o-xylene catabolic genes in P. stutzeri M1, was detected. No IS was detected in the corresponding catabolic regions of the P. stutzeri R1 genome. ISPs1 is present in several copies in the genomes of the three strains. It is flanked by 24-bp imperfect inverted repeats, causes the direct duplication of 8 bp in the target DNA, and seems to be related to the ISL3 family.

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Electrochemical redox transformations of T1 and T2 copper sites in native Trametes hirsuta laccase at gold electrode

Biochemical Journal ◽

10.1042/bj20041015 ◽

2005 ◽

Vol 385 (3) ◽

pp. 745-754 ◽

Cited By ~ 121

Author(s):

Sergey SHLEEV ◽

Andreas CHRISTENSON ◽

Vladimir SEREZHENKOV ◽

Dosymzhan BURBAEV ◽

Alexander YAROPOLOV ◽

...

Keyword(s):

Electron Transfer ◽

Gold Electrode ◽

Direct Electron Transfer ◽

High Potential ◽

Redox Potentials ◽

Hydrogen Electrode ◽

Trametes Hirsuta ◽

Redox Transformations ◽

Copper Site ◽

Copper Sites

Mediatorless, electrochemically driven, redox transformations of T1 (type 1) and T2 copper sites in Trametes hirsuta laccase were studied by cyclic voltammetry and spectroelectrochemical redox titrations using bare gold electrode. DET (direct electron transfer) between the electrode and the enzyme was observed under anaerobic conditions. From analysis of experimental data it is concluded that the T2 copper site is in DET contact with gold. It was found that electron transfer between the gold surface and the T1 copper site progresses through the T2 copper site. From EPR measurements and electrochemical data it is proposed that the redox potential of the T2 site for high-potential ‘blue’ laccase is equal to about 400 mV versus NHE (normal hydrogen electrode) at pH 6.5. The hypothesis that the redox potentials of the T2 copper sites in low- and high-potential laccases/oxidases from totally different sources might be very similar, i.e. approx. 400 mV, is discussed.

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