Hot spots in cold adaptation: Localized increases in conformational flexibility in lactate dehydrogenase A₄orthologs of Antarctic notothenioid fishes

Abstract

To elucidate mechanisms of enzymatic adaptation to extreme cold, we determined kinetic properties, thermal stabilities, and deduced amino acid sequences of lactate dehydrogenase A 4 (A 4 -LDH) from nine Antarctic (؊1.86 to 1°C) and three South American (4 to 10°C) notothenioid teleosts. Higher Michaelis-Menten constants (K m ) and catalytic rate constants (k cat ) distinguish orthologs of Antarctic from those of South American species, but no relationship exists between adaptation temperature and the rate at which activity is lost because of heat denaturation. In all species, active site residues are conserved fully, and differences in k cat and K m are caused by substitutions elsewhere in the molecule. Within geographic groups, identical kinetic properties are generated by different substitutions. By combining our data with A 4 -LDH sequences for other vertebrates and information on roles played by localized conformational changes in setting k cat , we conclude that notothenioid A 4 -LDHs have adapted to cold temperatures by increases in f lexibility in small areas of the molecule that affect the mobility of adjacent active-site structures. Using these findings, we propose a model that explains linked temperatureadaptive variation in K m and k cat . Changes in sequence that increase f lexibility of regions of the enzyme involved in catalytic conformational changes may reduce energy (enthalpy) barriers to these rate-governing shifts in conformation and, thereby, increase k cat . However, at a common temperature of measurement, the higher configurational entropy of a cold-adapted enzyme may foster conformations that bind ligands poorly, leading to high K m values relative to warmadapted orthologs.

Structure and dynamics of Antarctic fish neuroglobin assessed by computer simulations

“…These results support the general hypothesis that alterations in protein conformational mobility can happen through one/few substitutions, providing insights into the evolutionary rates at which adaptive change may occur (43,44). They also indicate that a small change in the primary structure, namely a shortterm response, may be very efficient as such for generating an adaptive response to a challenge.…”

Urea-Dependent Adenylate Kinase Activation following Redistribution of Structural States

“…Similar covariation of k cat and K M has been observed previously in cold-adaptation of lactate-dehydrogenase A 4 (A 4 -LDH). It was shown that A 4 -LDH isolated from organisms living in colder habitats have increased K cat and K M values compared to A 4 -LDH isolated from organisms living in warmer habitats (42). Further K cat -versus-K M compensation has been observed for the oxidative DNA repair enzyme AlkB (43).…”

Adaptation of model proteins from cold to hot environments involves continuous and small adjustments of average parameters related to amino acid composition

“…However, hyperthermophilic proteins are endowed with an extraordinary heat stability, as a consequence of a more tight compactness of the protein structure (Vieille and Zeikus, 2001). Vice versa, the psychrophilic counterparts possess an increased protein flexibility, which in most cases leads to a decreased stability compared to mesophilic proteins (D'Amico et al, 2006); in some psychrophilic enzymes the protein flexibility is enhanced in localized regions of the protein structure (Fields and Somero, 1998). Temperature adaptation of proteins is mostly relevant for the catalytic properties of the enzymes, as they must adapt the rate of the catalyzed reaction to the growth temperature of the organism.…”