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Chapter 9 : Contribution of Domain Interactions and Calcium Binding to the Stability of Carbohydrate-Active Enzymes

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Abstract:

This chapter focuses on the contributions that domain interactions and calcium have on the properties of carbohydrate-active enzymes. It also focuses on the interactions between domains in one of the largest cellulosomal catalytic components, cellobiohydrolase A (CbhA). CbhA is the only cellulosomal enzyme whose domain interactions and role of calcium have been studied in detail by different techniques including genetic manipulations, crystallography, circular dichroism (CD) spectroscopy, and differential scanning calorimetry (DSC). In the presence of calcium, the stabilities of the domains are relatively independent, while in the absence of Ca, domain interactions play a stabilizing role. Thermal unfolding in buffer assumes coexistence of protein molecules (i) with calcium bound to all binding sites, (ii) with partially lost calcium, and (iii) without calcium. This chapter describes modular architectures of carbohydrate-active enzymes and analyzes the role of interdomain interactions in the structure, stability, and functionality of the interesting and important proteins. Even linkers between domains are crucial for the functionality of carbohydrate-active enzyme in that they serve as “molecular springs” allowing catalytic sites to reach and hydrolyze new glycosidic bonds, while the CBM is still bound to the substrate surface. As a general conclusion one may say that domain interactions in modular carbohydrate hydrolytic enzymes enhance the activity of the catalytic domains of the enzymes.

Citation: Ljungdahl L, Kataeva I, Uversky V. 2008. Contribution of Domain Interactions and Calcium Binding to the Stability of Carbohydrate-Active Enzymes, p 115-127. In Wall J, Harwood C, Demain A (ed), Bioenergy. ASM Press, Washington, DC. doi: 10.1128/9781555815547.ch9

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Figures

Image of Figure 1.
Figure 1.

Domain structure of CbhA and its truncated variants. Abbreviations: CBD4 and CBD3, carbohydrate-binding domains of family 4 and 3, respectively; Ig, immunoglobulin-like domain; GH9, catalytic domain of family 9 glycoside hydrolases; X1 and X1, X domains of family 1; DD, duplicated dockerin domain. The content of calcium is also shown. (From .)

Citation: Ljungdahl L, Kataeva I, Uversky V. 2008. Contribution of Domain Interactions and Calcium Binding to the Stability of Carbohydrate-Active Enzymes, p 115-127. In Wall J, Harwood C, Demain A (ed), Bioenergy. ASM Press, Washington, DC. doi: 10.1128/9781555815547.ch9
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Image of Figure 2.
Figure 2.

Comparison of near-UV CD spectra recorded at 25°C of Fn3 in the presence (A) and absence (C) of calcium and of Fn3-CBM3 in the presence (B) and absence (D) of calcium to the spectra calculated as simple weighted sums based on spectra recorded for the individual domains: (Fn3 Fn3)/2 (A and C, dotted lines) and (Fn3 Fn3 CBM3)/3 (B and D, dotted lines) or (Fn3 CBM3)/2 (B and D, dashed lines). Experimental spectra of the domain combinations Fn3 and Fn3-CBM3 are shown with solid lines. (From .)

Citation: Ljungdahl L, Kataeva I, Uversky V. 2008. Contribution of Domain Interactions and Calcium Binding to the Stability of Carbohydrate-Active Enzymes, p 115-127. In Wall J, Harwood C, Demain A (ed), Bioenergy. ASM Press, Washington, DC. doi: 10.1128/9781555815547.ch9
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Image of Figure 3.
Figure 3.

Phase diagrams based on [θ] versus [θ] (see the text for details) characterize heat-induced denaturation of different domains of CbhA, based on the temperature-induced changes in the near-UV CD spectra of individual domains Fn3 (A), Fn3 (B), and CBM3 (C) and of domain combinations Fn3 (D) and Fn3-CBM3 (E). Data for holo- and apoproteins are given with closed symbols and solid lines, and open symbols and dashed lines, respectively. (From .)

Citation: Ljungdahl L, Kataeva I, Uversky V. 2008. Contribution of Domain Interactions and Calcium Binding to the Stability of Carbohydrate-Active Enzymes, p 115-127. In Wall J, Harwood C, Demain A (ed), Bioenergy. ASM Press, Washington, DC. doi: 10.1128/9781555815547.ch9
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Image of Figure 4.
Figure 4.

Denaturation peaks obtained for different constructs of CbhA in 20 mM sodium-phosphate buffer, pH 6.0 (A through D) and in the presence of 2 mM Ca as well (E through H) or in the presence of 2 mM EDTA (I). (From Kataeva et al., 2005.)

Citation: Ljungdahl L, Kataeva I, Uversky V. 2008. Contribution of Domain Interactions and Calcium Binding to the Stability of Carbohydrate-Active Enzymes, p 115-127. In Wall J, Harwood C, Demain A (ed), Bioenergy. ASM Press, Washington, DC. doi: 10.1128/9781555815547.ch9
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Image of Figure 5.
Figure 5.

DSC thermograms of Ig-GH9 module pair (1), its D264A (2) and T230/D262A (3) mutants, and individual GH9 module (4) and Ig-like module (5). The protein concentrations and scan rate were 6 mg/ml and 60°C/h, respectively. All thermal transitions were completely irreversible, so that second scans of the proteins were used as baselines. (From .)

Citation: Ljungdahl L, Kataeva I, Uversky V. 2008. Contribution of Domain Interactions and Calcium Binding to the Stability of Carbohydrate-Active Enzymes, p 115-127. In Wall J, Harwood C, Demain A (ed), Bioenergy. ASM Press, Washington, DC. doi: 10.1128/9781555815547.ch9
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Tables

Generic image for table
Table 1.

Denaturation parameters of CbhA individual domains and their combinations

Citation: Ljungdahl L, Kataeva I, Uversky V. 2008. Contribution of Domain Interactions and Calcium Binding to the Stability of Carbohydrate-Active Enzymes, p 115-127. In Wall J, Harwood C, Demain A (ed), Bioenergy. ASM Press, Washington, DC. doi: 10.1128/9781555815547.ch9

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