Contribution of calcium ligands in substrate binding and product release in the Acetovibrio thermocellus glycoside hydrolase family 9 cellulase CelR

Kuch, Nathaniel J.; Kutschke, Mark E.; Parker, Alex; Bingman, Craig A.; Fox, Brian G.

doi:10.1016/j.jbc.2023.104655

Title: Contribution of calcium ligands in substrate binding and product release in the Acetovibrio thermocellus glycoside hydrolase family 9 cellulase CelR

Journal Article · 28 March 2023 · Journal of Biological Chemistry

DOI: https://doi.org/10.1016/j.jbc.2023.104655 · OSTI ID:1963726

^[1]; Kutschke, Mark E. ^[1]; Parker, Alex ^[2];

^[1];

^[1]

Univ. of Wisconsin, Madison, WI (United States); Great Lakes Bioenergy Research Center (GLBRC), Madison, WI (United States)
Univ. of Wisconsin, Madison, WI (United States); Great Lakes Bioenergy Research Center (GLBRC), Madison, WI (United States); Dane County School Consortium, Monona, WI (United States)

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_cat/K_M) 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.

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Research Organization:: Univ. of Wisconsin, Madison, WI (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Biological and Environmental Research (BER); USDOE

Grant/Contract Number:: SC0018409; FC02-07ER64494; AC02-05CH11231; AC02-06CH11357

OSTI ID:: 1963726

Alternate ID(s):: OSTI ID: 2472388

Journal Information:: Journal of Biological Chemistry, Vol. 299, Issue 5; ISSN 0021-9258

Publisher:: American Society for Biochemistry and Molecular BiologyCopyright Statement

Country of Publication:: United States

Language:: English

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59 BASIC BIOLOGICAL SCIENCES
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structure-function
enzyme structure
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enzyme mechanism

Title: Contribution of calcium ligands in substrate binding and product release in the Acetovibrio thermocellus glycoside hydrolase family 9 cellulase CelR

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