Mannan-hydrolysis by hemicellulases: enzyme-polysaccharide interaction of a modular beta-mannanase

The enzymatic degradation of plant polysaccharides is a process of fundamental importance in nature which involves a wide range of enzymes. In this work, the structure and function of hemicellulose-degrading enzymes was investigated. The focus was on a beta-mannanase (TrMan5A) produced by the filamentous fungus Trichoderma reesei. This enzyme is composed of a catalytic module and a carbohydrate-binding module (CBM). In this thesis, the enzyme-polysaccharide interaction in both these modules was investigated. The results demonstrate that the CBM of TrMan5A is important for hydrolysis of complex mannan substrates containing cellulose. Furthermore, the increase in activity could be linked to binding of the CBM to the complex substrate. Binding studies revealed that the CBM binds to cellulose, but not to mannan. Studies of the enzyme/polysaccharide interaction in the active site cleft of the catalytic module of TrMan5A showed that a mutant of Arg 171 displayed activity in the same range as the wild-type enzyme toward polymeric substrates. However, the Arg171 mutant was impaired in hydrolysis of small substrates. Interestingly, this mutant also appears to have a more alkaline activity pH-optimum than the wild-type. The low or abolished activity observed with mutants of the predicted catalytic glutamates (Glu169 and Glu276) support their importance in hydrolysis. In addition to TrMan5A, the properties of a beta-mannanase (MeMan5A) from blue mussel and a beta-mannosidase (AnMan2A) from Aspergillus niger, were studied in this work. Investigations on the catalytic properties of the enzymes showed that all three enzymes are capable of degrading polymeric mannan. Furthermore, analysis by transmission electron microscopy revealed that TrMan5A and AnMan2A degrades highly crystalline mannan. Degradation of glucomannan and galactoglucomannan by several polysaccharide-degrading enzymes shows that these substrates can be hydrolysed by both mannoside- and glucoside-hydrolases. Furthermore, the results presented show that cellulases potentially are able to hydrolyse other components in the plant cell wall. Altogether, the results presented demonstrates the need to use complex substrates in order to reveal the mechanisms of plant polysaccharide degradation. In conclusion, this work has shown that the enzyme/polysaccharide interaction in the two modules of TrMan5A is important in determining the overall enzymatic activity and specificity

Relations Between Control Signal Properties and Robustness Measures

In this paper we consider control signal properties, such as maximum magnitude and activity, as well as system robustness measures. We derive an ideal controller and control signal for exponential disturbance rejection for a first order process with time delay. For the resulting closed-loop system, it is shown analytically that there are strong interconnections between robustness measures and control signal properties regarding load disturbance attenuation. The results imply that popular controller design methods implicitly take control signal properties into consideration

Structure of Hordeum vulgare NADPH-dependent thioredoxin reductase 2. Unwinding the reaction mechanism

Thioredoxins (Trxs) are protein disulfide reductases that regulate the intracellular redox environment and are important for seed germination in plants. Trxs are in turn regulated by NADPH-dependent thioredoxin reductases (NTRs), which provide reducing equivalents to Trx using NADPH to recycle Trxs to the active form. Here, the first crystal structure of a cereal NTR, HvNTR2 from Hordeum vulgare (barley), is presented, which is also the first structure of a monocot plant NTR. The structure was determined at 2.6 Å resolution and refined to an R (cryst) of 19.0% and an R (free) of 23.8%. The dimeric protein is structurally similar to the structures of AtNTR-B from Arabidopsis thaliana and other known low-molecular-weight NTRs. However, the relative position of the two NTR cofactor-binding domains, the FAD and the NADPH domains, is not the same. The NADPH domain is rotated by 25° and bent by a 38% closure relative to the FAD domain in comparison with AtNTR-B. The structure may represent an intermediate between the two conformations described pre­viously: the flavin-oxidizing (FO) and the flavin-reducing (FR) conformations. Here, analysis of interdomain contacts as well as phylogenetic studies lead to the proposal of a new reaction scheme in which NTR–Trx interactions mediate the FO to FR transformation

In silico approach to predict pancreatic β-cells classically secreted proteins

Pancreatic β-cells, residents of the islets of Langerhans, are the unique insulin-producers in the body. Their physiology is a topic of intensive studies aiming to understand the biology of insulin production and its role in diabetes pathology. However, investigations about these cells' subset of secreted proteins, the secretome, are surprisingly scarce and a list describing islet/β-cell secretome upon glucose-stimulation is not yet available. In silico predictions of secretomes are an interesting approach that can be employed to forecast proteins likely to be secreted. In this context, using the rationale behind classical secretion of proteins through the secretory pathway, a Python tool capable of predicting classically secreted proteins was developed. This tool was applied to different available proteomic data (human and rodent islets, isolated β-cells, β-cell secretory granules, and β-cells supernatant), filtering them in order to selectively list only classically secreted proteins. The method presented here can retrieve, organize, search and filter proteomic lists using UniProtKB as a central database. It provides analysis by overlaying different sets of information, filtering out potential contaminants and clustering the identified proteins into functional groups. A range of 70-92% of the original proteomes analyzed was reduced generating predicted secretomes. Islet and β-cell signal peptide-containing proteins, and endoplasmic reticulum-resident proteins were identified and quantified. From the predicted secretomes, exemplary conservational patterns were inferred, as well as the signaling pathways enriched within them. Such a technique proves to be an effective approach to reduce the horizon of plausible targets for drug development or biomarkers identification