Evolution of Lactate Dehydrogenase-A Homologs of Barracuda Fishes (Genus Sphyraena) from Different Thermal Environments:  Differences in Kinetic Properties and Thermal Stability Are Due to Amino Acid Substitutions Outside the Active Site^,

Abstract

Orthologous homologs of lactate dehydrogenase-A (LDH-A) (EC 1.1.1.27; NAD+:lactate oxidoreductase) of six barracuda species (genus Sphyraena) display differences in Michaelis-Menten constants (apparent Km) for substrate (pyruvate) and cofactor (NADH) that reflect evolution at different habitat temperatures. Significant increases in Km with increasing measurement temperature occur for all homologs, yet Km at normal body temperatures is similar among species because of the inverse relationship between adaptation temperature and Km. Thermal stabilities of the homologs also differ. To determine the amino acid substitutions responsible for differences in Km and thermal stability, peptide mapping of the LDH-As of all six species was first performed. Then, the amino acid sequences of the three homologs having the most similar peptide maps, those of the north temperate species, S. argentea, the subtropical species, S. lucasana, and the south temperate species, S. idiastes, were deduced from the respective cDNA sequences. At most, there were four amino acid substitutions between any pair of species, none of which occurred in the loop or substrate binding sites of the enzymes. The sequence of LDH-A from S. lucasana differs from that of S. idiastes only at position 8. The homolog of S. argentea differs from the other two sequences at positions 8, 61, 68, and 223. We used a full-length cDNA clone of LDH-A of S. lucasana to test, by site-directed mutagenesis, the importance of these sequence changes in establishing the observed differences in kinetics and thermal stability. Differences in sequence at sites 61 and/or 68 appear to account for the differences in Km between the LDH-As of S. argentea and S. lucasana. Differences at position 8 appear to account for the difference in thermal stability between the homologs of S. argentea and S. lucasana. Evolutionary adaptation of proteins to temperature thus may be achieved by minor changes in sequence at locations outside of active sites, and these changes may independently affect kinetic properties and thermal stabilities.

Adaptations of protein structure and function to temperature: there is more than one way to ‘skin a cat’

“…A subsequent study, however, extended the work of Graves and Somero on barracuda A 4 -LDHs, and for the first time was able to associate a specific amino acid substitution with a measurable, significant difference in a temperature-associated functional attribute, K m,pyr (Holland et al, 1997). In this study, A 4 -LDHs of five Sphyraena congeners from differing thermal habitats were examined, but two orthologs produced especially noteworthy results.…”

“…Some, but not all of these conclusions are supported in a retrospective examination of the location of the amino acid substitution found between A 4 -LDH orthologs of S. idiastes and S. lucasana described above (Holland et al, 1997). Position 8 is close to the N-terminus, on a structure that seems to extend away from the molecule and shows no intra-molecular interactions when the LDH-A monomer is examined (Fig.…”

Resurrecting prehistoric parvalbumins to explore the evolution of thermal compensation in extant Antarctic fish parvalbumins

“…It has been shown in several groups of enzyme orthologs that protein function correlates tightly with environmental temperature, which is thought to be due to adaptive amino acid substitutions that maintain protein function at an optimal level at physiological temperatures (Holland et al, 1997;Fields and Somero, 1998;Dong and Somero, 2009). The corresponding states theory (Somero, 1978;Somero, 1983;Somero, 1995;Jaenicke, 2000;Fields, 2001;Hochachka and Somero, 2002) predicts that differentially adapted orthologs (homologous protein isoforms separated by speciation) existing as populations of a series of related conformational states will sample a similar subset of states at their physiological temperature.…”

“…Typically, studies of thermal adaptation in enzymes have been conducted on proteins from closely related, often congeneric species that occupy different thermal niches (Holland et al, 1997;Fields and Somero, 1998). The polar and temperate species studied by Erickson et al (Erickson et al, 2005) do not fall into the same study paradigm as the enzyme work.…”

The protective role of aquaporins in the freeze-tolerant insect Eurosta solidaginis: functional characterization and tissue abundance of EsAQP1

“…Although this particular substitution does not explain the reduction in permeability of EsAQP1, which contains two NPA motifs, an amino acid substitution(s) elsewhere in the protein could alter its water transport abilities. Changes to residues outside the active site of proteins can still impact their activity, as demonstrated by a single amino acid substitution altering the kinetic properties of lactate dehydrogenase A in barracudas (Holland et al, 1997). Another possible cause of the relatively lower permeability of EsAQP1 is the non-native environment in which it was tested.…”