Crystal structure of a blue laccase from Lentinus tigrinus: evidences for intermediates in the molecular oxygen reductive splitting by multicopper oxidases

<p>Abstract</p> <p>Background</p> <p>Laccases belong to multicopper oxidases, a widespread class of enzymes implicated in many oxidative functions in pathogenesis, immunogenesis and morphogenesis of organisms and in the metabolic turnover of complex organic substances. They catalyze the coupling between the four one-electron oxidations of a broad range of substrates with the four-electron reduction of dioxygen to water. These catalytic processes are made possible by the contemporaneous presence of at least four copper ion sites, classified according to their spectroscopic properties: one type 1 (T1) site where the electrons from the reducing substrates are accepted, one type 2 (T2), and a coupled binuclear type 3 pair (T3) which are assembled in a T2/T3 trinuclear cluster where the electrons are transferred to perform the O₂reduction to H₂O.</p> <p>Results</p> <p>The structure of a laccase from the white-rot fungus <it>Lentinus (Panus) tigrinus</it>, a glycoenzyme involved in lignin biodegradation, was solved at 1.5 Å. It reveals a asymmetric unit containing two laccase molecules (A and B). The progressive reduction of the copper ions centers obtained by the long-term exposure of the crystals to the high-intensity X-ray synchrotron beam radiation under aerobic conditions and high pH allowed us to detect two sequential intermediates in the molecular oxygen reduction pathway: the "peroxide" and the "native" intermediates, previously hypothesized through spectroscopic, kinetic and molecular mechanics studies. Specifically the electron-density maps revealed the presence of an end-on bridging, μ-η₁:η₁peroxide ion between the two T3 coppers in molecule B, result of a two-electrons reduction, whereas in molecule A an oxo ion bridging the three coppers of the T2/T3 cluster (μ3-oxo bridge) together with an hydroxide ion externally bridging the two T3 copper ions, products of the four-electrons reduction of molecular oxygen, were best modelled.</p> <p>Conclusion</p> <p>This is the first structure of a multicopper oxidase which allowed the detection of two intermediates in the molecular oxygen reduction and splitting. The observed features allow to positively substantiate an accurate mechanism of dioxygen reduction catalyzed by multicopper oxidases providing general insights into the reductive cleavage of the O-O bonds, a leading problem in many areas of biology.</p

Transformation of high concentrations of chlorophenols by the white-rot basidiomycete Trametes versicolor immobilized on nylon mesh

Free-cell cultures of Trametes versicolor were compared with cultures immobilized on nylon mesh in a 2-litre bioreactor for transformation of pentachlorophenol (PCP) and 2,4-dichlorophenol (2,4-DCP), added at intervals to the liquid culture medium over a period of 816 hrs. Increasing amounts of PCP from 200 ppm to 2000 ppm added batchwise to cultures permitted acclimatization of the fungus to these toxic pollutants. A total addition of 2000 ppm of 2,4-DCP and 3400 ppm PCP were removed from the immobilized cultures with 85% of 2,4-DCP and 70% of PCP transformed by enzymes (laccase and Mn-peroxidase), 5% 2,4-DCP and 28% PCP adsorbed by the biomass and 10% 2,4-DCP and 2% PCP retained in the medium at the termination of the fermentation after 1020 hrs. In contrast free-cell cultures in the same medium with the same addition regime of PCP and 2,4-DCP, transformed 20% 2,4-DCP and 12% PCP by enzyme action, adsorbed 58% 2,4-DCP and 80% PCP by the biomass, and retained 22% 2,4-DCP and 8% PCP in the medium. The use of nylon mesh as an immobilization matrix for removal of PCP and 2,4-DCP facilitates more efficient removal of chlorophenols and can be adapted to scale-up for application of large volumes of chlorophenol-containing aqueous effluents

Скрининг, идентификация и характеристика бактерий Arthrobacter species, продуцирующих внеклеточную β-галактозидазу

β-Галактозидаза прокариот и, в частности, бактерий рода Arthrobacter – внутриклеточный фермент. Штамм Arthrobacter sp. БИМ B-2242 идентифицирован как A. sulfonivorans на основании анализа нуклеотидных последовательностей гена 16S рРНК. Штамм относится к грамположительным неподвижным бактериям с циклом морфогенеза клеток кокк – палочка – кокк, облигатным аэробам, мезофилам, положительным по признаку синтеза каталазы, протеазы, амилазы, уреазы и липазы. A. sulfonivorans ассимилирует лактозу, глюкозу, галактозу, рамнозу, сахарозу, арабинозу, сорбитол, пектин, крахмал в качестве источников углерода и энергии. Штамм утилизирует следуюшие источники азота: дрожжевой экстракт, пептон, бактопептон, триптон, желатин, казеин, казеинат натрия, мочевину, аминонокислоты, аммонийные соли неорганических кислот и нитраты. A. sulfonivorans продуцирует 2 изоформы истинно внеклеточных β-галактозидаз. Неочищенная β-галактозидаза проявляет максимум активности при температуре 42,5°C и pH 7,0, гидролизуя o-нитрофенил-β-D-галактопиранозид (oNPG). Удельная активность внеклеточной β-галактозидазы составила 101,2 ед/мг белка, что в 675,3 раза больше, чем внутриклеточной (0,15 ед/мг белка)

Reactions of "hybrid" Mn-peroxidase of the white rot fungus Panus tigrinus with benzylic alcohols in the presence of mediators

"Hybrid" Mn-peroxidase (hMnP) isolated from the white rot fungus Panus tigrinus 8/18 was studied with respect to its reactions with veratryl alcohol in the presence of typical laccase mediators in the reaction mixture. Eight compounds were tested as potential mediators in this reaction and only 1-hydroxybenzotriazole (HBT) and 3-hydroxy-1,2,3-benzotriazin-4(3H)-one (HBTO) were found to be effective. Up to 99% of 1 M veratryl alcohol was oxidized with formation of veratraldehyde as a reaction product over 24 h depending on the buffer system used. Except for veratryl alcohol, anisyl alcohol but not benzyl alcohol was oxidized in this reaction. Reactions with the participance of mediators were not catalytic, and the mediators were consumed during reaction with formation of dehydroxylated derivatives. Reactions with both HBT and HBTO resulted in temporal inactivation of hMnP. Kinetics of hMnP inactivation revealed it to be a pseudo-third order reaction. Investigation of the transformation of the absorption spectra of hMnP redox cycle intermediates in the presence of HBT or HBTO showed that the most likely reason of hMnP inactivation was its interaction with non-oxidized mediators

Crystallization and X ray diffraction studies of a two domain laccase from Streptomyces griseoflavus

Laccase (EC 1.10.3.2) is one of the most common copper-containing oxidases; it is found in many organisms and catalyzes the oxidation of primarily phenolic compounds by oxygen. Two-domain laccases have unusual thermostability, resistance to inhibitors and an alkaline optimum of activity. The causes of these properties in two-domain laccases are poorly understood. A recombinant two-domain laccase (SgfSL) was cloned from the genome of Streptomyces griseoflavus Ac-993, expressed in Escherichia coli and purified to homogeneity. The crystals of SgfSL belonged to the monoclinic space group P2(1), with unit-cell parameters a = 74.64, b = 94.72, c = 117.40 Å, β = 90.672°, and diffraction data were collected to 2.0 Å resolution using a synchrotron-radiation source. Two functional trimers per asymmetric unit correspond to a Matthews coefficient of 1.99 Å(3) Da(−1) according to the monomer molecular weight of 35.6 kDa