Structural and biochemical characterizations of Thermus thermophilus HB8 transketolase producing a heptulose

Transketolase is a key enzyme in the pentose phosphate pathway in all organisms, recognizing sugar phosphates as substrates. Transketolase with a cofactor of thiamine pyrophosphate catalyzes the transfer of a 2-carbon unit from D-xylulose-5-phosphate to D-ribose-5-phosphate (5-carbon aldose), giving D-sedoheptulose-7-phosphate (7-carbon ketose). Transketolases can also recognize non-phosphorylated monosaccharides as substrates, and catalyze the formation of non-phosphorylated 7-carbon ketose (heptulose), which has attracted pharmaceutical attention as an inhibitor of sugar metabolism. Here, we report the structural and biochemical characterizations of transketolase from Thermus thermophilus HB8 (TtTK), a well-characterized thermophilic Gram-negative bacterium. TtTK showed marked thermostability with maximum enzyme activity at 85 degrees C, and efficiently catalyzed the formation of heptuloses from lithium hydroxypyruvate and four aldopentoses: D-ribose, L-lyxose, L-arabinose, and D-xylose. The X-ray structure showed that TtTK tightly forms a homodimer with more interactions between subunits compared with transketolase from other organisms, contributing to its thermal stability. A modeling study based on X-ray structures suggested that D-ribose and L-lyxose could bind to the catalytic site of TtTK to form favorable hydrogen bonds with the enzyme, explaining the high conversion rates of 41% (D-ribose) and 43% (L-lyxose) to heptulose. These results demonstrate the potential of TtTK as an enzyme producing a rare sugar of heptulose. Key points Transketolase catalyzes the formation of a 7-carbon sugar phosphate Structural and biochemical characterizations of thermophilic transketolase were done The enzyme could produce non-phosphorylated 7-carbon ketoses from sugars.

Automated summary

In this study, the structural and biochemical characteristics of transketolase from the thermophilic bacterium Thermus thermophilus HB8 (TtTK) were investigated. TtTK showed remarkable thermostability and efficiently catalyzed the formation of 7-carbon ketoses from various sugars. The results demonstrate the potential of TtTK as an enzyme producing a rare sugar, heptulose.