Biochemical Characterization and Low-Resolution SAXS Molecular Envelope of GH1 ß-Glycosidase from Saccharophagus degradans.

The marine bacteria Saccharophagus degradans (also known as Microbulbifer degradans), are rod-shaped and gram-negative motile γ-proteobacteria, capable of both degrading a variety of complex polysaccharides and fermenting monosaccharides into ethanol. In order to obtain insights into structure-function relationships of the enzymes, involved in these biochemical processes, we characterized a S. degradans ß-glycosidase from glycoside hydrolase family 1 (SdBgl1B). SdBgl1B has the optimum pH of 6.0 and a melting temperature T m of approximately 50 °C. The enzyme has high specificity toward short D-glucose saccharides with ß-linkages with the following preferences ß-1,3 > ß-1,4 â‰« ß-1,6. The enzyme kinetic parameters, obtained using artificial substrates p-ß-NPGlu and p-ß-NPFuc and also the disaccharides cellobiose, gentiobiose and laminaribiose, revealed SdBgl1B preference for p-ß-NPGlu and laminaribiose, which indicates its affinity for glucose and also preference for ß-1,3 linkages. To better understand structural basis of the enzyme activity its 3D model was built and analysed. The 3D model fits well into the experimentally retrieved low-resolution SAXS-based envelope of the enzyme, confirming monomeric state of SdBgl1B in solution.

Purification, and biochemical and biophysical characterization of cellobiohydrolase I from Trichoderma harzianum IOC 3844.

Because of its elevated cellulolytic activity, the filamentous fungus Trichoderma harzianum has a considerable potential in biomass hydrolysis applications. Trichoderma harzianum cellobiohydrolase I (ThCBHI), an exoglucanase, is an important enzyme in the process of cellulose degradation. Here, we report an easy single-step ion-exchange chromatographic method for purification of ThCBHI and its initial biophysical and biochemical characterization. The ThCBHI produced by induction with microcrystalline cellulose under submerged fermentation was purified on DEAE-Sephadex A-50 media and its identity was confirmed by mass spectrometry. The ThCBHI biochemical characterization showed that the protein has a molecular mass of 66 kDa and pI of 5.23. As confirmed by smallangle X-ray scattering (SAXS), both full-length ThCBHI and its catalytic core domain (CCD) obtained by digestion with papain are monomeric in solution. Secondary structure analysis of ThCBHI by circular dichroism revealed alpha- helices and beta-strands contents in the 28% and 38% range, respectively. The intrinsic fluorescence emission maximum of 337 nm was accounted for as different degrees of exposure of ThCBHI tryptophan residues to water. Moreover, ThCBHI displayed maximum activity at pH 5.0 and temperature of 50 degrees C with specific activities against Avicel and p-nitrophenyl-ß-D-cellobioside of 1.25 U/mg and 1.53 U/mg, respectively.

Structural basis of GC-1 selectivity for thyroid hormone receptor isoforms.

BACKGROUND: Thyroid receptors, TRalpha and TRbeta, are involved in important physiological functions such as metabolism, cholesterol level and heart activities. Whereas metabolism increase and cholesterol level lowering could be achieved by TRbeta isoform activation, TRalpha activation affects heart rates. Therefore, beta-selective thyromimetics have been developed as promising drug-candidates for treatment of obesity and elevated cholesterol level. GC-1 [3,5-dimethyl-4-(4'-hydroxy-3'-isopropylbenzyl)-phenoxy acetic acid] has ability to lower LDL cholesterol with 600- to 1400-fold more potency and approximately two- to threefold more efficacy than atorvastatin (Lipitor(c)) in studies in rats, mice and monkeys. RESULTS: To investigate GC-1 specificity, we solved crystal structures and performed molecular dynamics simulations of both isoforms complexed with GC-1. Crystal structures reveal that, in TRalpha Arg228 is observed in multiple conformations, an effect triggered by the differences in the interactions between GC-1 and Ser277 or the corresponding asparagine (Asn331) of TRbeta. The corresponding Arg282 of TRbeta is observed in only one single stable conformation, interacting effectively with the ligand. Molecular dynamics support this model: our simulations show that the multiple conformations can be observed for the Arg228 in TRalpha, in which the ligand interacts either strongly with the ligand or with the Ser277 residue. In contrast, a single stable Arg282 conformation is observed for TRbeta, in which it strongly interacts with both GC-1 and the Asn331. CONCLUSION: Our analysis suggests that the key factors for GC-1 selectivity are the presence of an oxyacetic acid ester oxygen and the absence of the amino group relative to T3. These results shed light into the beta-selectivity of GC-1 and may assist the development of new compounds with potential as drug candidates to the treatment of hypercholesterolemia and obesity.