Virgínia Maria Rico Pires

Understanding the specific molecular interactions between proteins and β1,3‐1,4‐mixed‐linked d‐glucans is fundamental to harvest the full biological and biotechnological potential of these carbohydrates and of proteins that specifically recognize them. The family 11 carbohydrate‐binding module from Clostridium thermocellum (CtCBM11) is known for its binding preference for β1,3‐1,4‐mixed‐linked over β1,4‐linked glucans. Despite the growing industrial interest of this protein for the biotransformation of lignocellulosic biomass, the molecular determinants of its ligand specificity are not well defined. In this report, a combined approach of methodologies was used to unravel, at a molecular level, the ligand recognition of CtCBM11. The analysis of the interaction by carbohydrate microarrays and NMR and the crystal structures of CtCBM11 bound to β1,3‐1,4‐linked glucose oligosaccharides showed that both the chain length and the position of the β1,3‐linkage are important for recognition, and identified the tetrasaccharide Glcβ1,4Glcβ1,4Glcβ1,3Glc sequence as a minimum epitope required for binding. The structural data, along with site‐directed mutagenesis and ITC studies, demonstrated the specificity of CtCBM11 for the twisted conformation of β1,3‐1,4‐mixed‐linked glucans. This is mediated by a conformation–selection mechanism of the ligand in the binding cleft through CH‐π stacking and a hydrogen bonding network, which is dependent not only on ligand chain length, but also on the presence of a β1,3‐linkage at the reducing end and at specific positions along the β1,4‐linked glucan chain. The understanding of the detailed mechanism by which CtCBM11 can distinguish between linear and mixed‐linked β‐glucans strengthens its exploitation for the design of new biomolecules with improved capabilities and applications in health and agriculture.Database: Structural data are available in the Protein Data Bank under the accession codes 6R3M and 6R31.

The cellulosomal enzyme, RfGH5_1/2, of Ruminococcus flavefaciens contains an N-terminal module, a family 5 glycoside hydrolase GH5_4 with a putative endoglucanase activity, while C-terminal domain is a putative endo-mannanase (GH5_7). The two putative catalytic modules are separated by family 80 carbohydrate binding module (CBM80) having wide ligand specificity. The putative endo-mannanase module, GH5_7 (RfGH5_7), was cloned, expressed in Escherichia coli BL-21(DE3) cells and purified. SDS-PAGE analysis of purified RfGH5_7 showed molecular size ~ 35 kDa. Substrate specificity analysis of RfGH5_7 showed maximum activity against locust bean galactomannan (298.5 U/mg) followed by konjac glucomannan (256.2 U/mg) and carob galactomannan (177.2 U/mg). RfGH5_7 showed maximum activity at optimum pH 6.0 and temperature 60 °C. RfGH5_7 displayed stability in between pH 6.0 and 9.0 and thermostability till 50 °C. 10 mM Ca²⁺ ions increased the enzyme activity by 33%. The melting temperature of RfGH5_7 was 84 °C that was not affected by Ca²⁺ ions or chelating agents. RfGH5_7 showed, V_max, 389 U/mg and K_m, 0.92 mg/mL for locust bean galactomannan. TLC analysis revealed that RfGH5_7 hydrolysed locust bean galactomannan predominantly to mannose, mannobiose, mannotriose and higher degree of polymerization of manno-oligosaccharides indicating an endo-acting catalytic mechanism. This study revealed a highly active and thermostable endo-mannanase with considerable biotechnological potential.

Cellulosomes are highly sophisticated molecular nanomachines that participate in the deconstruction of complex polysaccharides, notably cellulose and hemicellulose. Cellulosomal assembly is orchestrated by the interaction of enzyme-borne dockerin (Doc) modules to tandem cohesin (Coh) modules of a non-catalytic primary scaffoldin. In some cases, as exemplified by the cellulosome of the major cellulolytic ruminal bacterium Ruminococcus flavefaciens, primary scaffoldins bind to adaptor scaffoldins that further interact with the cell surface via anchoring scaffoldins, thereby increasing cellulosome complexity. Here we elucidate the structure of the unique Doc of R. flavefaciens FD-1 primary scaffoldin ScaA, bound to Coh 5 of the adaptor scaffoldin ScaB. The RfCohScaB5-DocScaA complex has an elliptical architecture similar to previously described complexes from a variety of ecological niches. ScaA Doc presents a single-binding mode, analogous to that described for the other two Coh-Doc specificities required for cellulosome assembly in R. flavefaciens. The exclusive reliance on a single-mode of Coh recognition contrasts with the majority of cellulosomes from other bacterial species described to date, where Docs contain two similar Coh-binding interfaces promoting a dual-binding mode. The discrete Coh-Doc interactions observed in ruminal cellulosomes suggest an adaptation to the exquisite properties of the rumen environment.

BACKGROUND: The individual and combined effects of betaine and arginine supplemented to reduced protein diets were investigated on plasma metabolites, hepatic fatty acid composition and mRNA levels of lipid‐sensitive factors in commercial pigs. Betaine has previously been shown to reduce carcass fat deposition and arginine improves meat quality of finishing pigs. Forty male crossbred pigs were randomly assigned to one of five diets (n = 8): 160 g kg⁻¹ of crude protein (NPD), 130 g kg⁻¹ of crude protein (RPD), RPD with 3.3 g kg⁻¹ of betaine, RPD with 15 g kg⁻¹ of arginine, and RPD with 3.3 g kg⁻¹ of betaine and 15 g kg⁻¹ of arginine.RESULTS: The restriction of dietary protein increased total lipids (P < 0.001), total cholesterol (P < 0.001), high‐density lipoprotein‐cholesterol (P < 0.001) and low‐density lipoprotein cholesterol (P < 0.001). Betaine and arginine, individually or combined, reduced the majority of plasma lipids (P < 0.05) without affecting total fatty acids in the liver and the overall gene expression pattern.CONCLUSION: These findings suggest a positive effect of betaine and arginine, singly or combined, by reversing plasma lipids increase promoted by dietary protein restriction. © 2017 Society of Chemical Industry

[This corrects the article DOI: 10.1371/journal.pone.0106974.].