Uracil DNA glycosylase (UDG) is a DNA repair enzyme in the base excision repair pathway and removes uracil from the DNA strand. Atlantic cod UDG (cUDG), which is a cold-adapted enzyme, has been found to be up to 10 times more catalytically active in the temperature range 15-37 degrees C as compared with the warm-active human counterpart. The increased catalytic activity of cold-adapted enzymes as compared with their mesophilic homologues are partly believed to be caused by an increase in the structural flexibility. However, no direct experimental evidence supports the proposal of increased flexibility of cold-adapted enzymes. We have used molecular dynamics simulations to gain insight into the structural flexibility of UDG. The results from these simulations show that an important loop involved in DNA recognition (the Leu(272) loop) is the most flexible part of the cUDG structure and that the human counterpart has much lower flexibility in the Leu(272) loop. The flexibility in this loop correlates well with the experimental k(cat)/K(m) values. Thus, the data presented here add strong support to the idea that flexibility plays a central role in adaptation to cold environments.

The characteristic of cold-adapted enzymes, high catalytic efficiency at low temperatures, is often associated with low thermostability and high flexibility. In this context, we analyzed the catalytic properties and solved the crystal structure of phenylalanine hydroxylase from the psychrophilic bacterium Colwellia psychrerythraea 34H (CpPAH). CpPAH displays highest activity with tetrahydrobiopterin (BH(4)) as cofactor and at 25 degrees C (15 degrees C above the optimal growth temperature). Although the enzyme is monomeric with a single L-Phe-binding site, the substrate binds cooperatively. In comparison with PAH from mesophilic bacteria and mammalian organisms, CpPAH shows elevated [S(0.5)](L-Phe) (= 1.1 +/- 0.1 mm) and K(m)(BH(4))(= 0.3 +/- 0.1 mm), as well as high catalytic efficiency at 10 degrees C. However, the half-inactivation and denaturation temperature is only slightly lowered (T(m) approximately 52 degrees C; where T(m) is half-denaturation temperature), in contrast to other cold-adapted enzymes. The crystal structure shows regions of local flexibility close to the highly solvent accessible binding sites for BH(4) (Gly(87)/Phe(88)/Gly(89)) and l-Phe (Tyr(114)-Pro(118)). Normal mode and COREX analysis also detect these and other areas with high flexibility. Greater mobility around the active site and disrupted hydrogen bonding abilities for the cofactor appear to represent cold-adaptive properties that do not markedly affect the thermostability of CpPAH.