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A ‘split-gene’ transketolase from the hyper-thermophilic bacterium Carboxydothermus hydrogenoformans: Structure and biochemical characterization

DOI: 10.3389/fmicb.2020.592353 DOI Help

Authors: Paul James (University of Exeter) , Michail N. Isupov (University of Exeter) , Simone Antonio De Rose (University of Exeter) , Christopher Sayer (University of Exeter) , Isobel S. Cole (University of Exeter) , Jennifer A. Littlechild (University of Exeter)
Co-authored by industrial partner: No

Type: Journal Paper
Journal: Frontiers In Microbiology , VOL 11

State: Published (Approved)
Published: October 2020
Diamond Proposal Number(s): 8889

Open Access Open Access

Abstract: A novel transketolase has been reconstituted from two separate polypeptide chains encoded by a ‘split-gene’ identified in the genome of the hyperthermophilic bacterium, Carboxydothermus hydrogenoformans. The reconstituted active α2β2 tetrameric enzyme has been biochemically characterized and its activity has been determined using a range of aldehydes including glycolaldehyde, phenylacetaldehyde and cyclohexanecarboxaldehyde as the ketol acceptor and hydroxypyruvate as the donor. This reaction proceeds to near 100% completion due to the release of the product carbon dioxide and can be used for the synthesis of a range of sugars of interest to the pharmaceutical industry. This novel reconstituted transketolase is thermally stable with no loss of activity after incubation for 1 h at 70°C and is stable after 1 h incubation with 50% of the organic solvents methanol, ethanol, isopropanol, DMSO, acetonitrile and acetone. The X-ray structure of the holo reconstituted α2β2 tetrameric transketolase has been determined to 1.4 Å resolution. In addition, the structure of an inactive tetrameric β4 protein has been determined to 1.9 Å resolution. The structure of the active reconstituted α2β2 enzyme has been compared to the structures of related enzymes; the E1 component of the pyruvate dehydrogenase complex and D-xylulose-5-phosphate synthase, in an attempt to rationalize differences in structure and substrate specificity between these enzymes. This is the first example of a reconstituted ‘split-gene’ transketolase to be biochemically and structurally characterized allowing its potential for industrial biocatalysis to be evaluated.

Journal Keywords: hyperthermophilic; ‘split-gene’; transketolase; thermal stability; industrial applications

Subject Areas: Biology and Bio-materials, Chemistry

Instruments: I04-1-Macromolecular Crystallography (fixed wavelength) , I04-Macromolecular Crystallography


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