Incorporation of copper ions into crystals of T2 copper-depleted laccase from<i>Botrytis aclada</i>

Осипов, Е.М.; Polyakov, K. M.; Tikhonova, Т. V.; Kittl, Roman; Dorovatovskii, Pavel V.; Shleev, Sergey; Popov, Vladimir O.; Ludwig, Roland

doi:10.1107/s2053230x1502052x

Cited by 17 publications

(9 citation statements)

References 23 publications

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“…S1). Another role of the T2 channel might be a way of copper ion restoration in T2depleted enzyme, as has been shown in the structural studies of two laccases (Glazunova et al, 2015;Osipov et al, 2015).…”

Section: Discussionmentioning

confidence: 84%

Structural study of the X-ray-induced enzymatic reduction of molecular oxygen to water bySteccherinum murashkinskyilaccase: insights into the reaction mechanism

Polyakov

Gavryushov

Ivanova

et al. 2017

Acta Cryst Sect D Struct Biol

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The laccase from Steccherinum murashkinskyi is a member of the large family of multicopper oxidases that catalyze the oxidation of a wide range of organic and inorganic substrates, accompanied by the reduction of dioxygen to water. The reducing properties of X-ray radiation and the high quality of the laccase crystals allow the study of the catalytic reduction of dioxygen to water directly in a crystal. A series of diffraction data sets with increasing absorbed radiation dose were collected from a single crystal of Steccherinum murashkinskyi laccase at 1.35 Å resolution. Changes in the active-site structure associated with the reduction of molecular oxygen to water on increasing the absorbed dose of ionizing radiation were detected. The structures in the series are mixtures of different states of the enzyme-substrate complex. Nevertheless, it was possible to interpret these structures as complexes of various oxygen ligands with copper ions in different oxidation states. The results allowed the mechanism of oxygen reduction catalyzed by laccases to be refined.

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Section: Discussionmentioning

confidence: 84%

Structural study of the X-ray-induced enzymatic reduction of molecular oxygen to water bySteccherinum murashkinskyilaccase: insights into the reaction mechanism

Polyakov

Gavryushov

Ivanova

et al. 2017

Acta Cryst Sect D Struct Biol

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“…Based on these observations, we conclude that it is most likely that Mt L is active in its monomeric form. Neither did the analysis of the dimeric assemblies of other laccases, using the PISA server, support the existence of asco-laccase dimers in solution ( Table 2 ), in contrast to the results from a SEC analysis of Ba L, which indicated the existence of a dimer [ 27 ]. The SEC and CE analyses of Mt L showed highly homogeneous preparations consistent with a single species in solution, although the existence of short-lived dimers cannot be excluded.…”

Section: Resultsmentioning

confidence: 97%

“…With the aim of elucidating the structure and function of asco-laccases, we have undertaken the structure determination of the asco-laccase from the thermophilic fungus Myceliophthora thermophila ( Mt L) [ 22 ], The structure is known of four other asco-laccases (AA1_3): Ma L from Melanocarpus albomyces [ 23 , 24 ], Ta L from Thielavia arenaria [ 25 ], Ba L from Botrytis aclada [ 26 , 27 ] and An L from Aspergillus niger [ 28 ] ( S1 Table ). A significant evolutionary distance between the different asco-laccases is reflected in significant structural differences.…”

Section: Introductionmentioning

confidence: 99%

A comparative structural analysis of the surface properties of asco-laccases

et al. 2018

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Laccases of different biological origins have been widely investigated and these studies have elucidated fundamentals of the generic catalytic mechanism. However, other features such as surface properties and residues located away from the catalytic centres may also have impact on enzyme function. Here we present the crystal structure of laccase from Myceliophthora thermophila (MtL) to a resolution of 1.62 Å together with a thorough structural comparison with other members of the CAZy family AA1_3 that comprises fungal laccases from ascomycetes. The recombinant protein produced in A. oryzae has a molecular mass of 75 kDa, a pI of 4.2 and carries 13.5 kDa N-linked glycans. In the crystal, MtL forms a dimer with the phenolic substrate binding pocket blocked, suggesting that the active form of the enzyme is monomeric. Overall, the MtL structure conforms with the canonical fold of fungal laccases as well as the features specific for the asco-laccases. However, the structural comparisons also reveal significant variations within this taxonomic subgroup. Notable differences in the T1-Cu active site topology and polar motifs imply molecular evolution to serve different functional roles. Very few surface residues are conserved and it is noticeable that they encompass residues that interact with the N-glycans and/or are located at domain interfaces. The N-glycosylation sites are surprisingly conserved among asco-laccases and in most cases the glycan displays extensive interactions with the protein. In particular, the glycans at Asn88 and Asn210 appear to have evolved as an integral part of the asco-laccase structure. An uneven distribution of the carbohydrates around the enzyme give unique properties to a distinct part of the surface of the asco-laccases which may have implication for laccase function–in particular towards large substrates.

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“…An alignment of the protein sequences of the six laccases (five from termites and one from fungal) is shown in Figure . The five termite‐laccases apparently shared more similarities among themselves in protein sequences than those with the fungal laccase, BaLac from Botrytis aclada (Osipov et al ., ). In addition, based on the copper‐binding‐site information of BaLac, the eight copper‐binding sites seemed to be extremely conserved among all of the six laccases.…”

Section: Resultsmentioning

confidence: 97%

Characterization of a laccase from a wood‐feeding termite, Coptotermes formosanus

Geng

Xie

et al. 2016

Insect Science

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Coptotermes formosanus Shiraki is a wood-feeding termite which secretes a series of lignolytic and cellulolytic enzymes for woody biomass degradation. However, the lignin modification mechanism in the termite is largely elusive, and the characteristics of most lignolytic enzymes in termites remain unknown. In this study, a laccase gene lac1 from C. formosanus was heterogeneously expressed in insect Sf9 cells. The purified Lac1 showed strong activities toward hydroquinone (305 mU/mg) and 2,6-dimethoxyphenol (2.9 mU/mg) with low K values, but not veratryl alcohol or 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid). Lac1 could function well from pH 4.5 to 7.5, and its activity was significantly inhibited by H O at above 4.85 mmol/L (P < 0.01). In addition, the lac1 gene was found to be mainly expressed in the salivary glands and foregut of C. formosanus, and seldom in the midgut or hindgut. These findings suggested that Lac1 is a phenol-oxidizing laccase like RflacA and RflacB from termite Reticulitermes flavipes, except that Lac1 was found to be more efficient in phenol oxidation, and it did not require H O for its function. It is suspected that this kind of termite laccase might only be able to directly oxidize low redox-potential substrates, and the high redox-potential groups in lignin might be oxidized by other enzymes in the termite or by using the Fenton reaction.

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Incorporation of copper ions into crystals of T2 copper-depleted laccase fromBotrytis aclada

Cited by 17 publications

References 23 publications

Structural study of the X-ray-induced enzymatic reduction of molecular oxygen to water bySteccherinum murashkinskyilaccase: insights into the reaction mechanism

Structural study of the X-ray-induced enzymatic reduction of molecular oxygen to water bySteccherinum murashkinskyilaccase: insights into the reaction mechanism

A comparative structural analysis of the surface properties of asco-laccases

Characterization of a laccase from a wood‐feeding termite, Coptotermes formosanus

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