Abstract
The cDNAs of lactate dehydrogenase b (LDH-b) from both deep-sea and shallow living fish species, Corphaenoides armatus and Gadus morhua respectively, have been isolated, sequenced and their encoded products overproduced as recombinant enzymes in E. coli. The proteins were characterised in terms of their kinetic and physical properties and their ability to withstand high pressures. Although the two proteins are very similar in terms of their primary structure, only 21 differences at the amino acid level exist between them, the enzyme from the deep-sea species has a significantly increased tolerance to pressure and a higher thermostability. It was possible to investigate whether the changes in the N-terminal or C-terminal regions played a greater role in barophilic adaptation by the construction of two chimeric enzymes by use of a common restriction site within the cDNAs. One of these hybrids was found to have even greater pressure stability than the recombinant enzyme from the deep-living fish species. It was possible to conclude that the major adaptive changes to pressure tolerance must be located in the N-terminal region of the protein. The types of changes that are found and their spatial location within the protein structure are discussed. An analysis of the kinetic parameters of the enzymes suggests that there is clearly a trade off between Km and kcat values, which likely reflects the necessity of the deep-sea enzyme to operate at low temperatures.
Highlights
The physical properties of the deep-sea create a unique environment, which is characterised by high pressures and low temperatures
In this study we compared the kinetic and stability properties of recombinantly produced lactate dehydrogenase b (LDH-b) from two species of fish: the abyssal grenadier Coryphaenoides armatus and the Atlantic cod Gadus morhua. These represent two related (Gadiform) teleost species from entirely distinct pressure habitats: C. armatus typically living below 2000 m depth, though ranging between 300 and 5000 m (1 – 4uC); and G. morhua between 50–200 metres (0 – 20uC) [12]
The cDNA sequences have been deposited in the EMBL nucleotide database; accession numbers are C. armatus lactate dehydrogenase (LDH)-b AJ 609232, G. morhua LDH-b AJ 609233
Summary
The physical properties of the deep-sea create a unique environment, which is characterised by high pressures and low temperatures. Temperature in the deep-sea is generally low, typically in the range 2 to 4uC, necessitating low temperature adaptations, in enzyme systems [1,2] These environmental extremes of the deep-sea have required suitable adjustments within living organisms, especially at the molecular level, to allow normal biological processes to operate [1,2,3]. In one of the few studies undertaken in this research area, proteins of deep-sea fish have been shown to have an increased resistance to thermal denaturation compared to homologous proteins from shallow-water relatives [1,2,6,9] This increase in thermal stability is thought to be due to the evolution of especially rigid proteins that are able to resist disruption of tertiary and quaternary structure under high pressure [1,2,6,9]. This cannot always be the case since some barophilic enzymes still show a rate dependency upon pressure [9]
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
