Characterisation of a cold-adapted, thermostable glucokinase from psychrophilic Pseudoalteromonas sp. AS-131 reveals how the enzyme achieves high thermal stability without loss of cold adaptation.

Microorganisms living in cold environments such as the Antarctic and deep sea usually possess cold-adapted enzymes, which are known to have high catalytic efficiency and low stability owing to their flexible structures. Research on cold-adapted enzymes has not progressed much due to the challenge of these enzymes being less stable. However, several cold-adapted enzymes with high thermal stability have recently been reported. In this study, we investigated the biochemical properties of glucokinases from the psychrophilic Pseudoalteromonas sp. AS-131 (PsGK) isolated from the Antarctic Ocean and mesophilic Escherichia coli (EcGK). We demonstrated that PsGK is a cold-adapted enzyme with high thermal stability. A comparison of the crystal structures and spectroscopic studies revealed that PsGK has an additional disulfide bond connecting the N and C termini. To test whether this bond is important for stability, we prepared a PsGK variant by removing the bond and observed the significant reduction in thermal stability. In addition, the introduction of the artificial disulfide bonds in homologous positions in EcGK increased the thermal stability without the reduction in maximum activity. These results confirmed that the introduction of a disulfide bond at the proper position, such as the connection of the N and C termini, significantly improved stability without changing the nature of enzymes. Our findings propose a new strategy that will contribute to the industrial application of enzymes.