Lactate dehydrogenase from the hyperthermophilic bacterium thermotoga maritima: the crystal structure at 2.1 A resolution reveals strategies for intrinsic protein stabilization.
BACKGROUND
L(+)-Lactate dehydrogenase (LDH) catalyzes the last step in anaerobic glycolysis, the conversion of pyruvate to lactate, with the concomitant oxidation of NADH. Extensive physicochemical and structural investigations of LDHs from both mesophilic and thermophilic organisms have been undertaken in order to study the temperature adaptation of proteins. In this study we aimed to determine the high-resolution structure of LDH from the hyperthermophilic bacterium Thermotoga maritima (TmLDH), the most thermostable LDH to be isolated so far. It was hoped that the structure of TmLDH would serve as a model system to reveal strategies of protein stabilization at temperatures near the boiling point of water.


RESULTS
The crystal structure of the extremely thermostable TmLDH has been determined at 2.1 A resolution as a quaternary complex with the cofactor NADH, the allosteric activator fructose-1,6-bisphosphate, and the substrate analog oxamate. The structure of TmLDH was solved by Patterson search methods using a homology-based model as a search probe. The native tetramer shows perfect 222 symmetry. Structural comparisons with five LDHs from mesophilic and moderately thermophilic organisms and with other ultrastable enzymes from T. maritima reveal possible strategies of protein thermostabilization.


CONCLUSIONS
Structural analysis of TmLDH and comparison of the enzyme to moderately thermophilic and mesophilic homologs reveals a strong conservation of both the three-dimensional fold and the catalytic mechanism. Going from lower to higher physiological temperatures a variety of structural differences can be observed: an increased number of intrasubunit ion pairs; a decrease of the ratio of hydrophobic to charged surface area, mainly caused by an increased number of arginine and glutamate sidechains on the protein surface; an increased secondary structure content including an additional unique 'thermohelix' (alphaT) in TmLDH; more tightly bound intersubunit contacts mainly based on hydrophobic interactions; and a decrease in both the number and the total volume of internal cavities. Similar strategies for thermal adaptation can be observed in other enzymes from T. maritima.
Auerbach
G
G
Max-Planck-Institut für Biochemie Abt. Strukturforschung, 82152, Martinsried, Germany. auerbach@biochem.mpg.de
Ostendorp
R
R
Prade
L
L
Korndörfer
I
I
Dams
T
T
Huber
R
R
Jaenicke
R
R
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites; D018360:Crystallography, X-Ray; D003545:Cysteine / Q000737:chemistry; D019281:Dimerization; D004795:Enzyme Stability; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology*; D006358:Hot Temperature; D006860:Hydrogen Bonding; D007770:L-Lactate Dehydrogenase / Q000737:chemistry*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017386:Sequence Homology, Amino Acid; D013499:Surface Properties
D001120:Arginine; D007770:L-Lactate Dehydrogenase; D003545:Cysteine