A ‘Split-Gene’ Transketolase From the Hyper-Thermophilic Bacterium Carboxydothermus hydrogenoformans: Structure and Biochemical Characterization

• Published in: Frontiers in microbiology 2020-10, Vol.11, p.592353-592353
• Main Authors: James, Paul, Isupov, Michail N., De Rose, Simone Antonio, Sayer, Christopher, Cole, Isobel S., Littlechild, Jennifer A.
• Format: Article
• Language: English
• Subjects: hyperthermophilic; industrial applications; Microbiology; split-gene; thermal stability; transketolase
• ISSN: 1664-302X; 1664-302X
• DOI: 10.3389/fmicb.2020.592353

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 α 2 β 2 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 α 2 β 2 tetrameric transketolase has been determined to 1.4 Å resolution. In addition, the structure of an inactive tetrameric β 4 protein has been determined to 1.9 Å resolution. The structure of the active reconstituted α 2 β 2 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.