Abstract
Studies of cold adaptation in lactate dehydrogenase (LDH) implicated structural rearrangements of the molecule associated with substrate and cofactor binding. Analysis of these regions suggests that these movements are much smaller than previously thought and unlikely to play a key role in adapting LDH to function at low temperatures. Recent homology mod- eling of LDH from Antarctic notothenioid fish based on the spiny dogfish LDH structure, identified flexibility in a key loop, βJ-α1G as implicated in cold-adaptation in notothenioid fish (Fields and Somero, 1998).
The gene sequence of LDH from the spiny dogfish appeared in 1995 and contained numerous deviations from the sequence used by Abad-Zapatero et al. (1987) to build the spiny dogfish LDH structure, and these devi- ations are concentrated in a few key areas including the βJ-α1G region (Stock and Power, 1995). Further, the sequence alignment of LDH from a number of Antarctic fish reveals significant variability in βJ-α1G loop re- gion. Taken together the variability in this region, which combined with the knowledge that the Abad-Zapatero LDH structure was built with the incorrect sequence in this loop, suggested the need to re-examine the three- dimensional structure of spiny dogfish LDH and the proposed movements in βJ-α1G loop. Further, whether its movements, considered to play a role in cold-adaptation are real or an artifact of an incomplete structure.
We present the crystal structure of LDH from spiny dogfish in apo and ternary form with the corrected amino acid sequence. The structure re- vealed no change in the βJ-α1G loop region in the transition from apo to holo form. This suggests that the loop area may not play a crucial role in cold adaptation. The dissociation constant (KD) determined by surface plasmon resonance suggests weak binding of NADH to cold-adapted LDH at low temperatures compared to LDH from temperate fish. Similarly, the catalytic rate constant (kcat) for cold adapted LDH showed a reduced cat- alytic rate at low temperature. However, at physiological temperatures, cold and temperate LDH exhibit similar Michaelis constant. In summary, we found kinetic evidence for cold adaptation in LDH from cold adapted fish but a structural basis for the cold adaptation is yet to be established.