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

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 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-5phosphate synthase, in an attempt to rationalize differences in structure and substrate specificity between these enzymes. This is the first example of a reconstituted 'splitgene' transketolase to be biochemically and structurally characterized allowing its potential for industrial biocatalysis to be evaluated.

Characterisation of a hyperthermophilic transketolase from Thermotoga maritima DSM3109 as a biocatalyst for 7-keto-octuronic acid synthesis

“…The pH optimum was found between 6 and 6.4 which is below the optima pH reported for other thermophilic TKs ( pH 7 and 8); 15,25,26 in fact, TK tmar lost 57% of its activity at pH 7.4 compared to pH 6.4 (Fig. 2B).…”

“…32,33 TK tmar has a very low Ala content (7% compared to the 11% average) and 5 Cys residues (compared to only 1 for G. stearothermophilus and C. hydrogenoformans) that could potentially be forming disulfide bonds as a mechanism for protein stabilisation (ESI Table 1 †). 34 It has been suggested that Pro residues in the loop regions play an important role in enzyme thermostability; 25,35 however, TK tmar contains only 26 Pro residues (4.1%), which together with B. anthracis has the lowest number of Pro residues, amongst the analysed TKs.…”

“…TK tmar showed better organic solvent stability properties compared to TK chy whose loss of activity was around 50% after 1 h incubation in the same organic solvents. 25…”

“…Recently a "split-gene" TK from Carboxydothermus hydrogenoformans (TK chy ) has been reported showing an optimum temperature of 70°C and good stability in the presence of different organic solvents. 25 Also, Bawn et al 15 isolated and characterised TKs from Deinococcus geothermalis (TK dge ) and Deinococcus radiodurans (TK dra ) which were used to produce L-glucoheptulose from L-arabinose; and both TKs have optimum temperatures of 50°C. Notably, TK from Geobacillus stearothermophilus (TK gst ) was the first thermostable TK reported also with an optimum temperature of 50°C.…”

Complete Biosynthetic Pathway of the Phosphonate Phosphonothrixin: Two Distinct Thiamine Diphosphate-Dependent Enzymes Divide the Work to Form a C–C Bond

“…During the condensation reaction, TPP-dependent decarboxylation of the first substrate, pyruvate, occurs, resulting in elongation from the two carbons. , PtxB5/6 shows sequence similarity to the first example of the “split-gene” transketolase that catalyzes the transfer of a two-carbon unit from hydroxypyruvate to an acceptor aldehyde to yield a ketose product and carbon dioxide in the hyperthermophilic bacterium Carboxydothermus hydrogenoformans (Figure S7). Ptx5/6 was indeed reconstituted from two separate polypeptide chains as a heterodimer (Figure S6). As both enzymes require a keto substrate as an acceptor of a two-carbon unit, we incubated PtxB5/6 and/or PtxB7 with 6 , possessing a keto group and pyruvate, as the donor of a two-carbon unit in the presence of TPP for 1 h. 31 P NMR analysis of the reaction mixture clearly revealed the formation of PTX with dependence on both PtxB5/6 and PtxB7 (Figure A).…”