Crystallization and preliminary X-ray study of β-mannosidase from<i>Trichoderma reesei</i>

Aparício, Ricardo; Eneiskaya, E. V.; Kulminskaya, Anna A.; Savel'ev, Andrew N.; Golubev, A. M.; Neustroev, Kirill N.; Kobarg, Jörg; Polikarpov, Igor

doi:10.1107/s0907444999016625

Cited by 3 publications

(4 citation statements)

References 26 publications

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“…The purified protein was dialyzed and concentrated to 10 mgÁmL À1 prior to crystallization. Initial crystallization conditions were as in the macromolecular crystallization reagent kits I and II (Hampton Research) and further optimized [30]. Diffraction-quality crystals were grown from mother solution containing 26% poly(ethylene glycol) 400, 0.13 M CdCl 2 , and 0.1 M sodium acetate (pH 4.7).…”

Section: Methodsmentioning

confidence: 99%

Insights into the structure and function of fungal β‐mannosidases from glycoside hydrolase family 2 based on multiple crystal structures of the Trichoderma harzianum enzyme

et al. 2014

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Hemicellulose is an important part of the plant cell wall biomass, and is relevant to cellulosic ethanol technologies. b-Mannosidases are enzymes capable of cleaving nonreducing residues of b-D-mannose from b-D-mannosides and hemicellulose mannose-containing polysaccharides, such as mannans and galactomannans. b-Mannosidases are distributed between glycoside hydrolase (GH) families 1, 2, and 5, and only a handful of the enzymes have been structurally characterized to date. The only published X-ray structure of a GH family 2 mannosidase is that of the bacterial Bacteroides thetaiotaomicron enzyme. No structures of eukaryotic mannosidases of this family are currently available. To fill this gap, we set out to solve the structure of Trichoderma harzianum GH family 2 b-mannosidase and to refine it to 1.9-A resolution. Structural comparisons of the T. harzianum GH2 b-mannosidase highlight similarities in its structural architecture with other members of GH family 2, reveal the molecular mechanism of b-mannoside binding and recognition, and shed light on its putative galactomannan-binding site. DatabaseCoordinates and observed structure factor amplitudes have been deposited with the Protein Data Bank (4CVU and 4UOJ). The T. harzianum b-mannosidase 2A nucleotide sequence has GenBank accession number BankIt1712036 GeneMark.hmm KJ624918. AbbreviationsBtMan2A, Bacteroides thetaiotaomicron glycosyl hydrolase family 2 b-mannosidase; CBM, carbohydrate-binding module; CmMan5A, Cellvibrio mixtus glycosyl hydrolase family 5 b-mannosidase; GH, glycosyl hydrolase; GM, b-galactomannan; Man 2 , mannobiose; Man 3 , mannotriose; Man 4 , mannotetraose; PDB, Protein Data Bank; ThMan2A, Trichoderma harzianum glycosyl hydrolase family 2 b-mannosidase.

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Section: Methodsmentioning

confidence: 99%

Insights into the structure and function of fungal β‐mannosidases from glycoside hydrolase family 2 based on multiple crystal structures of the Trichoderma harzianum enzyme

et al. 2014

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“…Enhancement of SAXS EnVelope by using Crystallographic Data. T. reesei β-mannosidase was crystallized in the presence of CdCl 2 as described (7). Crystals diffracting to medium resolution (∼2.5 Å) grow in space groups P4 1 2 1 2 and P2 1 2 1 2 1 under very similar crystallization conditions.…”

Section: Methodsmentioning

confidence: 99%

“…Although the precise role of this site is not clear, this result might suggest that the β-mannosidase fold is comprised of more than one domain. To provide experimental structural information about T. reesei β-mannosidase, X-ray crystallographic (7), circular dichroism (CD), and small-angle X-ray scattering (SAXS) studies were initiated. In work presented here, a lowresolution envelope of the protein was retrieved by an ab initio procedure from the synchrotron SAXS 1 data.…”

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Structural Insights into the β-Mannosidase from T. reesei Obtained by Synchrotron Small-Angle X-ray Solution Scattering Enhanced by X-ray Crystallography

et al. 2002

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A molecular envelope of the beta-mannosidase from Trichoderma reesei has been obtained by combined use of solution small-angle X-ray scattering (SAXS) and protein crystallography. Crystallographic data at 4 A resolution have been used to enhance informational content of the SAXS data and to obtain an independent, more detailed protein shape. The phased molecular replacement technique using a low resolution SAXS model, building, and refinement of a free atom model has been employed successfully. The SAXS and crystallographic free atom models exhibit a similar globular form and were used to assess available crystallographic models of glycosyl hydrolases. The structure of the beta-galactosidase, a member of a family 2, clan GHA glycosyl hydrolases, shows an excellent fit to the experimental molecular envelope and distance distribution function of the beta-mannosidase, indicating gross similarities in their three-dimensional structures. The secondary structure of beta-mannosidase quantified by circular dichroism measurements is in a good agreement with that of beta-galactosidase. We show that a comparison of distance distribution functions in combination with 1D and 2D sequence alignment techniques was able to restrict the number of possible structurally homologous proteins. The method could be applied as a general method in structural genomics and related fields once protein solution scattering data are available.

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“…Cristalização da β-manosidaseAs condições de cristalização e análise preliminar de um cristal nativo de β-manosidase (Figura 3.1) foram publicadas sob a referência Aparicio, R., Eneiskaya, E. V.,Kulminskaya, A. A., Savel'ev, A. N., Golubev, A. M., Neustroev, K. N., Kobarg, J. and Polikarpov, I.…”

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Estudos estruturais sobre a [Beta]-manosidase de Trichoderma reesei e a Triose fosfato isomerase de músculo de coelho

Aparício¹,

Polikarpov²

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This PhD thesis summarizes the results obtained during the PhD program of the author. The main project consisted in structural studies of β-mannosidase from Trichoderma reesei and rabbit muscle Triose phosphate isomerase (TIM). Protein Crystallography and Small-Angle X-ray Scattering have been the mostly used techniques. The main results are the low resolution structure and a putative fold of β-mannosidase and three cristallographic TIM structures, determined in two crystal forms and different temperatures. One of the subunits in the highest resolution TIM structure (1.5Å) has the active site loop in the closed conformation despite the absence of a ligand in the active site, what had not been observed before. Both projects have led to new and important biological information. As a result of the doctorate program, five articles have been published and two further are in preparation.

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Crystallization and preliminary X-ray study of β-mannosidase fromTrichoderma reesei

Cited by 3 publications

References 26 publications

Insights into the structure and function of fungal β‐mannosidases from glycoside hydrolase family 2 based on multiple crystal structures of the Trichoderma harzianum enzyme

Insights into the structure and function of fungal β‐mannosidases from glycoside hydrolase family 2 based on multiple crystal structures of the Trichoderma harzianum enzyme

Structural Insights into the β-Mannosidase from T. reesei Obtained by Synchrotron Small-Angle X-ray Solution Scattering Enhanced by X-ray Crystallography

Estudos estruturais sobre a [Beta]-manosidase de Trichoderma reesei e a Triose fosfato isomerase de músculo de coelho

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