AUTHOR=James Paul , Isupov Michail N. , De Rose Simone Antonio , Sayer Christopher , Cole Isobel S. , Littlechild Jennifer A. 

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

JOURNAL=Frontiers in Microbiology

VOLUME=Volume 11 - 2020

YEAR=2020

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2020.592353

DOI=10.3389/fmicb.2020.592353

ISSN=1664-302X

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 22 tetrameric enzyme has been biochemically characterised and its activity has been determined using a range of aldehydes including glycolaldehyde, phenylacetylaldehyde and cyclohexanecarboxaldehyde as the ketol acceptor and hydroxypyruvate as the donor. This reaction proceeds to 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 hour at 70 ºC and is stable after 1 hour incubation with 50 % of the organic solvents methanol, ethanol, isopropanol, DMSO, acetonitrile and acetone. The X-ray structure of the holo reconstituted 22 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 2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 rationalise 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 characterised allowing its potential for industrial biocatalysis to be evaluated.