7V0J

Crystal structure of a CelR catalytic domain active site mutant with bound cellobiose product

7V0J の概要

• エントリーDOI: 10.2210/pdb7v0j/pdb
• 分子名称: Glucanase, beta-D-glucopyranose-(1-4)-beta-D-glucopyranose, CALCIUM ION, ... (4 entities in total)
• 機能のキーワード: biomass deconstruction, glycoside hydrolase family 9, gh9, cellulose binding domain 3c, cbm3c, hydrolase, hydrolase-product complex, hydrolase/product
• 由来する生物種: Acetivibrio thermocellus
• タンパク質・核酸の鎖数: 3
• 化学式量合計: 161281.97

構造登録者

Bingman, C.A.,Kuch, N.,Kutsche, M.E.,Parker, A.,Smith, R.W.,Fox, B.G. (登録日: 2022-05-10, 公開日: 2023-04-05, 最終更新日: 2023-10-25)

主引用文献

Kuch, N.J.,Kutschke, M.E.,Parker, A.,Bingman, C.A.,Fox, B.G.
Contribution of calcium ligands in substrate binding and product release in the Acetovibrio thermocellus glycoside hydrolase family 9 cellulase CelR.
J.Biol.Chem., 299:104655-104655, 2023

PubMed Abstract: Enzymatic deconstruction of lignocellulosic biomass is crucial to establishment of the renewable biofuel and bioproduct economy. Better understanding of these enzymes, including their catalytic and binding domains, and other features offer potential avenues for improvement. Glycoside hydrolase family 9 (GH9) enzymes are attractive targets because they have members that exhibit exo- and endo-cellulolytic activity, processivity of reaction, and thermostability. This study examines a GH9 from Acetovibrio thermocellus ATCC 27405, AtCelR containing a catalytic domain and a carbohydrate binding module (CBM3c). Crystal structures of the enzyme without substrate, bound to cellohexaose (substrate) or cellobiose (product), show the positioning of ligands to calcium and adjacent residues in the catalytic domain that may contribute to substrate binding and facilitate product release. We also investigated the properties of the enzyme engineered to contain an additional carbohydrate binding module (CBM3a). Relative to the catalytic domain alone, CBM3a gave improved binding for Avicel (a crystalline form of cellulose), and catalytic efficiency (k/K) was improved 40× with both CBM3c and CBM3a present. However, because of the molecular weight added by CBM3a, the specific activity of the engineered enzyme was not increased relative to the native construct consisting of only the catalytic and CBM3c domains. This work provides new insight into a potential role of the conserved calcium in the catalytic domain and identifies contributions and limitations of domain engineering for AtCelR and perhaps other GH9 enzymes.

PubMed: 36990218
DOI: 10.1016/j.jbc.2023.104655

実験手法

X-RAY DIFFRACTION (2.4 Å)