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

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 angstrom 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 beta-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.

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

Thermophilic endo-1,3(4)-beta-glucanase (laminarinase) from Rhodothermus marinus was crystallized by the hanging-drop vapor diffusion method. The needle-like crystals belong to space group P2(1) and contain two protein molecules in the asymmetric unit with a solvent content of 51.75%. Diffraction data were collected to a resolution of 1.95 angstrom and resulted in a dataset with an overall R-merge of 10.4% and a completeness of 97.8%. Analysis of the structure factors revealed pseudomerohedral twinning of the crystals with a twin fraction of approximately 42%.

Karyotypes of a Cryptic Diploid Form of the Unisexual Leposoma percarinatum (Squamata, Gymnophthalmidae) and the Bisexual Leposoma ferreirai from the Lower Rio Negro, Amazonian Brazil

Karyotypes of Leposoma show a clear differentiation between species of the scincoides group from Brazilian Atlantic Forest (2n = 52, without distinctive size groups of chromosomes) and those of the parietale group from the Amazon (2n = 44, with 20M + 24m). In a previous study, we found that in the parietale group the parthenoform Leposoma percarinatum from the state of Mato Grosso, Brazil, exhibited a triploid karyotype (3n = 66) with 30 macrochromosomes and 36 microchromosomes. It was suggested that this karyotype arose after hybridization between a bisexual species with N = 22 (10M + 12m) and a hypothetical unisexual cryptic diploid form of the L. percarinatum complex. Herein, we describe the karyotypes for two species of the parietale group occurring sympatrically in the Arquipelago das Anavilhanas, lower Rio Negro, in Amazonian Brazil. The first represents a distinctive diploid parthenogenetic clone of the L. percarinatum complex, and the other is the recently described Leposoma ferreirai. Both species have 44 biarmed chromosomes clearly represented by 20 macrochromosomes and 24 microchromosomes and present Ag-NORs in one pair of the smallest sized microchromosomes; heteromorphism of size for these regions was detected in L. percarinatum. C-banding revealed blocks of constitutive heterochromatin on the telomeric and pericentromeric regions of macrochromosomes and some microchromosomes. The description of a diploid karyotype (2n = 44, 20M + 24m) for the L. percarinatum complex and its sympatric congener L. ferreirai provides new insight for a better understanding of the origin of parthenogenesis in the L. percarinatum complex.