Johanne Mørch Jensen

Inactivation of barley limit dextrinase inhibitor by thioredoxin-catalysed disulfide reduction

Barley limit dextrinase (LD) that catalyses hydrolysis of α‐1,6 glucosidic linkages in starch‐derived dextrins is inhibited by limit dextrinase inhibitor (LDI) found in mature seeds. LDI belongs to the chloroform/methanol soluble protein family (CM‐protein family) and has four disulfide bridges and one glutathionylated cysteine. Here, thioredoxin is shown to progressively reduce disulfide bonds in LDI accompanied by loss of activity. A preferential reduction of the glutathionylated cysteine, as indicated by thiol quantification and molecular mass analysis using electrospray ionisation mass spectrometry, was not related to LDI inactivation. LDI reduction is proposed to cause conformational destabilisation leading to loss of function.

Efficient secretory expression of functional barley limit dextrinase inhibitor by high cell-density fermentation of Pichia pastoris

The limit dextrinase inhibitor (LDI) from barley seeds acts specifically on limit dextrinase (LD), an endogenous starch debranching enzyme. LDI is a 14 kDa hydrophobic protein containing four disulfide bonds and one unpaired thiol group previously found to be either glutathionylated or cysteinylated. It is a member of the so-called CM-protein family that includes α-amylase and serine protease inhibitors, which have been extremely challenging to produce recombinantly in functional form and in good yields. Here, LDI is produced in very high yields by secretory expression by Pichia pastoris applying high cell-density fermentation in a 5L fed-batch bioreactor. Thus about 200mg of LDI, which showed twofold higher inhibitory activity towards LD than LDI from barley seeds, was purified from 1L of culture supernatant by His-tag affinity chromatography and gel filtration. Electrospray ionization mass spectrometry verified the identity of the produced glutathionylated LDI-His(6). At a 1:1M ratio the recombinant LDI completely inhibited hydrolysis of pullulan catalyzed by 5-10 nM LD. LDI retained stability in the pH 2-12 range and at pH 6.5 displayed a half-life of 53 and 33 min at 90 and 93°C, respectively. The efficient heterologous production of LDI suggests secretory expression by P. pastoris to be a promising strategy to obtain other recombinant CM-proteins.

Crystal Structure of Barley Limit Dextrinase:Limit Dextrinase Inhibitor (Ld:Ldi) Complex Reveals Insights Into Mechanism and Diversity of Cereal-Type Inhibitors.

Background: Barley limit dextrinase (LD), the sole starch debranching enzyme active during seed germination, is regulated by an endogenous inhibitor (LDI). Results: The crystal structure of the LD-LDI complex reveals a new and unexpected binding mode among cereal type inhibitors. Conclusion: A hydrophobic cluster drives the picomolar affinity of LDI. Significance: The molecular understanding of regulation of starch mobilization during germination is augmented. Molecular details underlying regulation of starch mobilization in cereal seed endosperm remain unknown despite the paramount role of this process in plant growth. The structure of the complex between the starch debranching enzyme barley limit dextrinase (LD), hydrolyzing α-1,6-glucosidic linkages, and its endogenous inhibitor (LDI) was solved at 2.7 Å. The structure reveals an entirely new and unexpected binding mode of LDI as compared with previously solved complex structures of related cereal type family inhibitors (CTIs) bound to glycoside hydrolases but is structurally analogous to binding of dual specificity CTIs to proteases. Site-directed mutagenesis establishes that a hydrophobic cluster flanked by ionic interactions in the protein-protein interface is vital for the picomolar affinity of LDI to LD as assessed by analysis of binding by using surface plasmon resonance and also supported by LDI inhibition of the enzyme activity. A phylogenetic analysis identified four LDI-like proteins in cereals among the 45 sequences from monocot databases that could be classified as unique CTI sequences. The unprecedented binding mechanism shown here for LDI has likely evolved in cereals from a need for effective inhibition of debranching enzymes having characteristic open active site architecture. The findings give a mechanistic rationale for the potency of LD activity regulation and provide a molecular understanding of the debranching events associated with optimal starch mobilization and utilization during germination. This study unveils a hitherto not recognized structural basis for the features endowing diversity to CTIs.

The Barley Grain Thioredoxin System – an Update

Thioredoxin (Trx) reduces disulfide bonds and play numerous important functions in plants. In cereal seeds, cytosolic h-type Trx facilitates the release of energy reserves during the germination process and is recycled by NADPH-dependent Trx reductase. This review presents a summary of the research conducted during the last 10 years to elucidate the structure and function of the barley seed Trx system at the molecular level combined with proteomic approaches to identify target proteins.

Methodology for Developing a Diesel Exhaust After Treatment Simulation Tool

(11/12/2018) Methodology for Developing a Diesel Exhaust After Treatment Simulation Tool A methodology for the development of catalyst models is presented. Also, a methodology of the implementation of such models into a modular simulation tool, which simulates the units in succession, is presented. A case study is presented illustrating how suitable models can be found and used for simulations. Such simulations illustrate the behavior of the individual units and the overall system. It is shown how, by simulating the units in succession, the entire after treatment system can be tested and optimized, because the integration makes it possible to observe the effect of the modules on one another.

α-Amylases. Interaction with Polysaccharide Substrates, Proteinaceous Inhibitors and Regulatory Proteins

Abstract α-Amylases occur widely in plants, animals, and microorganisms. They often act in synergy with other related and degradative enzymes and may also be regulated by proteinaceous inhibitors. Open questions exist on how α-amylases interact with polysaccharides. Several enzymes possess secondary carbohydrate binding sites situated on the surface at a certain distance of the active site cleft. The functions of such sites were studied in barley α-amylase isozymes by structure-guided mutational analysis and measurement of activity and binding parameters. Two surface sites were assigned distinct roles. One of the sites seems to participate in hydrolysis of polysaccharides by a multiple attack mechanism. Polysaccharide processing enzymes can also contain carbohydrate binding modules, e.g. starch binding domains that assist in the attack on macromolecular substrates and are useful in engineering of enzyme efficiency. The multidomain nature of these enzymes raises questions on the dynamics and structural properties in solution and in substrate complexes.