4.8 Article

Active-Site Engineering Switches Carbohydrate Regiospecificity in a Fungal Copper Radical Oxidase

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ACS CATALYSIS
卷 12, 期 16, 页码 10264-10275

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AMER CHEMICAL SOC
DOI: 10.1021/acscatal.2c0195610264

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copper radical oxidase; protein engineering; aryl alcohol oxidase; galactose oxidase; Auxiliary Activity Family 5 (AA5)

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Scientists have discovered a unique enzyme that can selectively oxidize alcohols to aldehydes and adapt to different carbohydrates. This finding has important implications for the design of improved biocatalysts.
Family 5, Subfamily 2 (AA5_2), are organic cofactor-free biocatalysts for the selective oxidation of alcohols to the corresponding aldehydes. AA5_2 CROs comprise canonical galactose-6-oxidases as well as the more recently discovered general alcohol oxidases and aryl alcohol oxidases. Guided by primary and tertiary protein structural analyses, we targeted a distinct extended loop in the active site of a Colletotrichum graminicola aryl alcohol oxidase (CgrAAO) to explore its effect on catalysis in the broader context of AA5_2. Deletion of this loop, which is bracketed by a conserved disulfide bridge, significantly reduced the inherent activity of the enzyme toward extended galacto-oligosaccharides, as anticipated from molecular modeling. Unexpectedly, kinetic and product analysis on a range of monosaccharides and disaccharides revealed that an altered carbohydrate specificity in CgrAAO-Delta loop was accompanied by a complete change in regiospecificity from C-6 to C-1 oxidation, thereby generating aldonic acids. C-1 regiospecificity is unprecedented in AA5 enzymes and is classically associated with flavin-dependent carbohydrate oxidases of Auxiliary Activity Family 3. Thus, this work further highlights the catalytic adaptability of the unique mononuclear copper radical active site and provides a basis for the design of improved biocatalysts for diverse potential applications.

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