Endo-probability and processivities measured on α-chitin.

<p>Endo-probability and processivities measured on α-chitin.</p

Progress curves of AA-CNW hydrolysis.

<p>(A) AA-CNWs (1 mg/mL) were hydrolysed with HCHT50, HCHT39, <i>Sm</i>ChiA, <i>Sm</i>ChiB or <i>Sm</i>ChiC at 37°C. The release of AA-labelled sugars and total soluble reducing ends were measured at defined time points (5, 10, 20, 40 and 60 min). Error bars show standard deviations and are from three independent experiments. (B) Progress curves at different concentrations of HCHT39.</p

Michaelis-Menten kinetics of HCHTs.

<p>α-chitin, amorphous chitin or CNWs were hydrolyzed with HCHT50 or HCHT39 at 37°C for 1 min. The solid lines represent the best fit according to the Michaelis-Menten equation. Error bars show standard deviations and are from three independent experiments.</p

Processivity and probability of endo initiation of HCHTs.

<p>AA-α-chitin (1 mg/mL) or reduced α-chitin (1 mg/mL) were hydrolyzed with HCHT50, HCHT39 (10 nM) or <i>Sm</i>ChiC (1 nM) at 37°C. (A) The release of soluble reducing groups (SRGs). Shown are the combined results with AA-α-chitin and reduced α-chitin. Error bars are from six independent experiments, three made with AA-α-chitin and three with reduced α-chitin as substrate. (B) The release of AA-labelled sugars from AA-α-chitin and the formation of insoluble reducing groups (IRG) on reduced α-chitin under otherwise identical conditions. (C) Data of the hydrolysis of reduced α-chitin from panels (A) and (B) plotted in the coordinates of total reducing groups (RG_tot = IRG + SRG) <i>versus</i> IRG. The solid lines represent the best fit of linear regression (only the data points shown within the solid lines were included in linear regression analysis). The slope of the solid line from linear regression equals to apparent processivity, P^app. (D) Discrimination between different populations of HCHT bound to α-chitin. The total concentration of HCHT was 10 nM and that of α-chitin was 1 mg/mL. The concentration of total bound HCHT ([HCHT]_bound) was found as a difference between the total concentration of the enzyme and the concentration of the enzyme free in solution. The concentration of HCHT with free active site was measured by following the MU-NAG₂ hydrolyzing activity of HCHT in the presence of α-chitin. The concentration of bound HCHT with active site occupied by chitin ([HCHT] _{bound OA}) was found as a difference between the total concentration of the enzyme and that with free active site. The concentration of bound HCHT with free active site ([HCHT] _{bound FA}) was found as a difference between the [HCHT]_bound and [HCHT] _{bound OA}. Error bars show standard deviations and are from three independent experiments.</p

Michaelis-Menten kinetic parameter values on different chitin substrates.

<p>Michaelis-Menten kinetic parameter values on different chitin substrates.</p

NAG₂ inhibition of HCHT.

<p>(A) Activity of HCHT on MU-NAG₂ substrate as a function of substrate concentration. (B) NAG₂ inhibition of HCHT on MU-NAG₂ substrate measured at 3 different substrate concentrations—0.5 μM, 5 μM or 50 μM. (C) NAG₂ inhibition of HCHT on ¹⁴C-CNW substrate (1.0 g/L). <i>v</i>_i and <i>v</i>_{i = 0} stand for the rates measured in the presence and absence of inhibitor, respectively. Error bars show standard deviations and are from three independent experiments.</p

SDS-PAGE analysis of purified chitinases.

<p>4–6 μg purified HCHT 50 kDa and 39 kDa isoforms and <i>S</i>. <i>marcescens</i> chitinases <i>Sm</i>ChiA, <i>Sm</i>ChiB and <i>Sm</i>ChiC were loaded and the gel was stained with Coomassie Brilliant Blue G-250.</p

Human Chitotriosidase-Catalyzed Hydrolysis of Chitosan

Chitotriosidase (HCHT) is one of two family 18 chitinases produced by humans, the other being acidic mammalian chitinase (AMCase). The enzyme is thought to be part of the human defense mechanism against fungal parasites, but its precise role and the details of its enzymatic properties have not yet been fully unraveled. We have studied the properties of HCHT by analyzing how the enzyme acts on high-molecular weight chitosans, soluble copolymers of β-1,4-linked <i>N</i>-acetylglucosamine (GlcNAc, A), and glucosamine (GlcN, D). Using methods for in-depth studies of the chitinolytic machinery of bacterial family 18 enzymes, we show that HCHT degrades chitosan primarily via an endoprocessive mechanism, as would be expected on the basis of the structural features of its substrate-binding cleft. The preferences of HCHT subsites for acetylated versus nonacetylated sugars were assessed by sequence analysis of obtained oligomeric products showing a very strong, absolute, and a relative weak preference for an acetylated unit in the −2, −1, and +1 subsites, respectively. The latter information is important for the design of inhibitors that are specific for the human chitinases and also provides insight into what kind of products may be formed in vivo upon administration of chitosan-containing medicines or food products

Thermodynamic Relationships with Processivity in <i>Serratia marcescens</i> Family 18 Chitinases

The enzymatic degradation of recalcitrant polysaccharides is accomplished by synergistic enzyme cocktails of glycoside hydrolases (GHs) and accessory enzymes. Many GHs are processive which means that they remain attached to the substrate in between subsequent hydrolytic reactions. Chitinases are GHs that catalyze the hydrolysis of chitin (β-1,4-linked <i>N</i>-acetylglucosamine). Previously, a relationship between active site topology and processivity has been suggested while recent computational efforts have suggested a link between the degree of processivity and ligand binding free energy. We have investigated these relationships by employing computational (molecular dynamics (MD)) and experimental (isothermal titration calorimetry (ITC)) approaches to gain insight into the thermodynamics of substrate binding to <i>Serratia marcescens</i> chitinases ChiA, ChiB, and ChiC. We show that increased processive ability indeed corresponds to more favorable binding free energy and that this likely is a general feature of GHs. Moreover, ligand binding in ChiB is entropically driven; in ChiC it is enthalpically driven, and the enthalpic and entropic contributions to ligand binding in ChiA are equal. Furthermore, water is shown to be especially important in ChiA-binding. This work provides new insight into oligosaccharide binding, getting us one step closer to understand how GHs efficiently degrade recalcitrant polysaccharides

Préface

<p>Effect of combinations of chitosan (DP_n 206) or CHOS (DP_n 30) and Signum on cumulative <i>Botrytis cinerea</i> infection of detached chickpea leaves.</p