Crystallization and Preliminary Crystallographic Analysis of Laminarinase from Rhodothermus marinus: A Case of Pseudomerohedral Twinning

Golubev, A. M.; Rojas, Adriana L.; Nascimento, Alessandro S.; Bleicher, Lucas; Kulminskaya, Anna A.; Eneyskaya, Elena V.; Polikarpov, Igor

doi:10.2174/092986608786071139

Cited by 1 publication

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“…The laminarinase from R. marinus (RmLamR) was crystallized in space group P2 1 , with a dimer in the asymmetric unit . This dimer and all other possible quaternary assemblies formed by contacts in the crystal are unlikely to be found in solution, as indicated by theoretical estimates of the solvation free energy gain upon interface formation for each assembly obtained using the PISA server .…”

Section: Resultsmentioning

confidence: 99%

“…The laminarinase from R. marinus was cloned, expressed, purified, and crystallized as described . The X-ray diffraction images from the crystal were collected at the MX-1 beamline at the Brazilian National Synchrotron Light Laboratory (LNLS), in Campinas, Brazil .…”

Section: Methodsmentioning

confidence: 99%

“…The X-ray diffraction images from the crystal were collected at the MX-1 beamline at the Brazilian National Synchrotron Light Laboratory (LNLS), in Campinas, Brazil . The data set was indexed and integrated using MOSFLM, scaled in SCALA, and analyzed using phenix.xtriage. , During this process it was detected that the crystal presented a rare case of pseudomerohedral twinning . The structure of endo-β-1,3-glucanase from Nocardiopsis sp.…”

Section: Methodsmentioning

confidence: 99%

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Molecular Basis of the Thermostability and Thermophilicity of Laminarinases: X-ray Structure of the Hyperthermostable Laminarinase from Rhodothermus marinus and Molecular Dynamics Simulations

et al. 2011

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Glycosyl hydrolases are enzymes capable of breaking the glycosidic linkage of polysaccharides and have considerable industrial and biotechnological applications. Driven by the later applications, it is frequently desirable that glycosyl hydrolases display stability and activity under extreme environment conditions, such as high temperatures and extreme pHs. Here, we present X-ray structure of the hyperthermophilic laminarinase from Rhodothermus marinus (RmLamR) determined at 1.95 Å resolution and molecular dynamics simulation studies aimed to comprehend the molecular basis for the thermal stability of this class of enzymes. As most thermostable proteins, RmLamR contains a relatively large number of salt bridges, which are not randomly distributed on the structure. On the contrary, they form clusters interconnecting β-sheets of the catalytic domain. Not all salt bridges, however, are beneficial for the protein thermostability: the existence of charge-charge interactions permeating the hydrophobic core of the enzymes actually contributes to destabilize the structure by facilitating water penetration into hydrophobic cavities, as can be seen in the case of mesophilic enzymes. Furthermore, we demonstrate that the mobility of the side-chains is perturbed differently in each class of enzymes. The side-chains of loop residues surrounding the catalytic cleft in the mesophilic laminarinase gain mobility and obstruct the active site at high temperature. By contrast, thermophilic laminarinases preserve their active site flexibility, and the active-site cleft remains accessible for recognition of polysaccharide substrates even at high temperatures. The present results provide structural insights into the role played by salt-bridges and active site flexibility on protein thermal stability and may be relevant for other classes of proteins, particularly glycosyl hydrolases.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%