Adaptations for Pressure and Temperature in Dihydrofolate Reductases.

Enzymes from extremophilic microbes that live in extreme conditions are generally adapted so that they function under those conditions, although adaptations for extreme temperatures and pressures can be difficult to unravel. Previous studies have shown mutation of Asp27 in Escherichia coli dihydrofolate reductase (DHFR) to Glu27 in Moritella profunda (Mp). DHFR enhances activity at higher pressures, although this may be an adaptation for cold. Interestingly, MpDHFR unfolds at ~70 MPa, while Moritella yayanosii (My) was isolated at depths corresponding to ~110 MPa, indicating that MyDHFR might be adapted for higher pressures. Here, these adaptations are examined using molecular dynamics simulations of DHFR from different microbes in the context of not only experimental studies of activity and stability of the protein but also the evolutionary history of the microbe. Results suggest Tyr103 of MyDHFR may be an adaptation for high pressure since Cys103 in helix F of MpDHFR forms an intra-helix hydrogen bond with Ile99 while Tyr103 in helix F of MyDHFR forms a hydrogen bond with Leu78 in helix E. This suggests the hydrogen bond between helices F and E in MyDHFR might prevent distortion at higher pressures.

How adding a single methylene to dihydrofolate reductase can change its conformational dynamics.

Studies of the effects of pressure on proteins from piezophilic (pressure-loving) microbes compared with homologous proteins from mesophilic microbes have been relatively rare. Interestingly, such studies of dihydrofolate reductase show that a single-site mutation from an aspartic acid to a glutamic acid can reverse the pressure-dependent monotonic decrease in activity to that in a monotonic pressure-dependent activation. This residue is near the active site but is not thought to directly participate in the catalytic mechanism. Here, the ways that addition of one carbon to the entire protein could lead to such a profound difference in pressure effects are explored using molecular dynamics simulations. The results indicate that the glutamate changes the coupling between a helix and the ß-sheet due to the extra flexibility of the side chain, which further changes correlated motions of other regions of the protein.