[en] Adapting metabolic enzymes of microorganisms to low temperature environments may require a difficult compromise between velocity and affinity. We have investigated catalytic efficiency in a key metabolic enzyme (dihydrofolate reductase) of Moritella profunda sp. nov., a strictly psychrophilic bacterium with a maximal growth rate at 2degreesC or less. The enzyme is monomeric (M-r = 18,291), 55% identical to its Escherichia coli counterpart, and displays T-m and denaturation enthalpy changes much lower than E. coli and Thermotoga maritima homologues. Its stability curve indicates a maximum stability above the temperature range of the organism, and predicts cold denaturation below 0degreesC. At mesophilic temperatures the apparent K-m value for dihydrofolate is 50- to 80-fold higher than for E. coli, Lactobacillus casei, and T. maritima dihydrofolate reductases, whereas the apparent K-m value for NADPH, though higher, remains in the same order of magnitude. At 5degreesC these values are not significantly modified. The enzyme is also much less sensitive than its E. coli counterpart to the inhibitors methotrexate and trimethoprim. The catalytic efficiency (k(cat)/K-m) with respect to dihydrofolate is thus much lower than in the other three bacteria. The higher affinity for NADPH could have been maintained by selection since NADPH assists the release of the product tetrahydrofolate. Dihydrofolate reductase adaptation to low temperature thus appears to have entailed a pronounced trade-off between affinity and catalytic velocity. The kinetic features of this psychrophilic protein suggest that enzyme adaptation to low temperature may be constrained by natural limits to optimization of catalytic efficiency.
Disciplines :
Biochemistry, biophysics & molecular biology
Author, co-author :
Xu, Y.
Feller, Georges ; Université de Liège - ULiège > Département des sciences de la vie > Labo de biochimie
Gerday, Charles ; Université de Liège - ULiège > Services généraux (Faculté des sciences) > Relations académiques et scientifiques (Sciences)
Glansdorff, N.
Language :
English
Title :
Moritella cold-active dihydrofolate reductase: Are there natural limits to optimization of catalytic efficiency at low temperature?
Publication date :
September 2003
Journal title :
Journal of Bacteriology
ISSN :
0021-9193
eISSN :
1098-5530
Publisher :
Amer Soc Microbiology, Washington, United States - Washington
Baccanari, D. P., D. Stone, and L. Kuyper. 1981. Effect of a single amino acid substitution on Escherichia coli dihydrofolate reductase catalysis and ligand binding. J. Biol. Chem. 256:1738-1747.
Bentahir, M., G. Feller, M. Aittaleb, J. Lamotte-Brasseur, T. Himri, J. P. Chessa, and C. Gerday. 2000. Structural, kinetic and calorimetric characterization of the cold-active phosphoglycerate kinase from the antarctic Pseudomonas sp. TACII18. J. Biol. Chem. 275:11147-11153.
Bolin, J. T., D. J. Filman, D. A. Matthews, R. C. Hamlin, and J. Kraut. 1982. Crystal structures of Escherichia coli and Lactobacillus casei dihydrofolate reductase refined at 1.7 A resolution. I. General features and binding of methotrexate. J. Biol. Chem. 257:13650-13662.
Cavicchioli, R., K. S. Siddiqui, D. Andrews, and R. K. Sowers. 2002. Low-temperature extremophiles and their applications. Curr. Opin. Biotechnol. 13:253-261.
Clark, A. C., and C. Frieden. 1999. Native Escherichia coli and murine dihydrofolate reductases contain late-folding non-native structures. J. Mol. Biol. 285:1765-1776.
Dams, T., and R. Jaenicke. 1999. Stability and folding of dihydrofolate reductase from the hyperthermophilic bacterium Thermotoga maritima. Biochemistry 38:9169-9178.
Dams, T., G. Auerbach, G. Bader, U. Jacob, T. Ploom, R. Huber, and R. Jaenicke R. 2000. The crystal structure of dihydrofolate reductase of Thermotoga maritima: molecular features of thermostability. J. Mol. Biol. 297:659-672.
Dann, J. G., G. Ostler, R. A. Bjur, R. W. King, P. Scudder, P. C Turner, G. C. Roberts, and A. S. Burgen. 1976. Large-scale purification and characterization of dihydrofolate reductase from a methotrexate-resistant strain of Lactobacillus casei. Biochem. J. 157:559-571.
Feller, G., and C. Gerday. 1997. Psychrophilic enzymes: molecular basis of cold adaptation. Cell. Mol. Life Sci. 53:830-841.
Feller, G., D. d'Amico, and C. Gerday. 1999. Thermodynamic stability of a cold-active α-amylase from the Antarctic bacterium Alteromonas haloplanctis. Biochemistry 38:4613-4619.
Fields, P. A., and G. N. Somero. 1998. Hot spots in cold adaptation: localized increases in conformational flexibility in lactate dehydrogenase A (4) orthologs of Antarctic notothenioid fishes. Proc. Natl. Acad. Sci. USA 95:11476-11481.
Filman, D. J., J. T. Bolin, D. A. Matthews, and J. Kraut. 1982. Crystal structures of Escherichia coli and Lactobacillus casei dihydrofolate reductase refined at 1.7 Å resolution. II. Environment of bound NADPH and implications for catalysis. J. Biol. Chem. 257:13663-13672.
Gerday, C. Extremophiles. Basic concepts. In Unesco encyclopedia of life support systems, theme 6.73. extremophiles, in press.
Gerday, C., M. Aittaleb, J. L. Arpigny, E. Baise, J. P. Chessa, G. Garsoux, I. Petrescu, and G. Feller. 1997. Psychrophilic enzymes: a thermodynamic challenge. Biochim. Biophys. Acta 1342:119-131.
Glansdorff, N. 1965. Topography of cotransductible arginine mutations in Escherichia coli K12. Genetics 51:167-179.
Glansdorff, N., and Y. Xu. 2002. Microbial life at low temperatures: mechanisms of adaptation and extreme biotopes. Implications for exobiology and the origin of life. Recent Res. Dev. Microbiol. 6:1-21.
Hillcoat, B. L., P. F. Nixon, and R L. Blakley. 1967. Effect of substrate decomposition on the spectrophotometric assay of dihydrofolate reductase. Anal. Biochem. 21:178-189.
Hitchings, G. H., Jr. 1989. Nobel lecture in physiology or medicine, 1988: selective inhibitors of dihydrofolate reductase. In Vitro Cell Dev. Biol. 25:303-310.
Ionescu, R. M., V. F. Smith, J. C. O'Neill, Jr., and C. R. Matthews. 2000. Multistate equilibrium unfolding of Escherichia coli dihydrofolate reductase: thermodynamic and spectroscopic description of the native, intermediate, and unfolded ensembles. Biochemistry 39:9540-9550.
Kohlhoff, M., A. Dahm, and R. Hensel. 1996. Tetrameric triosephosphate isomerase from hyperthermophilic Archaea. FEBS Lett. 383:245-250.
Lonhienne, T., C. Gerday, and G. Feller. 2000. Psychrophilic enzymes: revisiting the thermodynamic parameters of activation may explain local flexibility. Biochim. Biophys. Acta. 1543:1-10.
Maes, D., J. P. Zeelen, N. Thanki, N. Beaucämp, M. Alvarez, M. H. Thi, J. Backmann, J. A. Martial, R. Jaenicke, R. K. Wierenga, and L. Wyns. 1999. The crystal structure of triosephosphate isomerase (TIM) from Thermotoga maritima: a comparative thermostability structural analysis of ten different TIM structures. Proteins 37:441-453.
Makhatadze, G. I., and P. L. Privalov. 1995. Energetics of protein structure. Adv. Protein Chem. 47:307-425.
Morita, R. Y. 1975. Psychrophilic bacteria. Bacteriol. Rev. 39:144-167.
Mozhaev, V. V., I. V. Berezin, and K. Martinek. 1988. Structure-stability relationship in proteins: fundamental tasks and strategy for the development of stabilized enzyme catalysts for biotechnology. Crit. Rev. Biochem. 23:235-281.
Pace, C. N. 1986. Determination and analysis of urea and guanidine hydrochloride denaturation curves. Methods Enzymol. 131:266-280.
Penner, M. H., and C. Frieden. 1987. Kinetic analysis of the mechanism of Escherichia coli dihydrofolate reductase. J. Biol. Chem. 262:15908-15914.
Perry, K. M., J. J. Onuffer, N. A. Touchette, C. S. Herndon, M. S. Gittelman, C. R. Matthews, J. T. Chen, R. J. Mayer, K. Taira, and S. J. Benkovic. 1987. Effect of single amino acid replacements on the folding and stability of dihydrofolate reductase from Escherichia coli. Biochemistry 26:2674-2682.
Poe, M., N. J. Greenfield, J. M. Hirshfield, M. N. Williams, and K. Hoogsteen. 1972. Dihydrofolate reductase. Purification and characterization of the enzyme from an amethopterin-resistant mutant of Escherichia coli. Biochemistry 11:1023-1030.
Privalov, P. L. 1979. Stability of proteins: small globular proteins. Adv. Protein Chem. 33:167-241.
Rood, J. I., A. J. Laird, and J. M. Williams. 1980. Cloning of the Escherichia coli K12 dihydrofolate reductase gene following Mu-mediated transposition. Gene 8:255-265.
Russell, N. J. 1992. Physiology and molecular biology of psychrophilic micro-organisms, p. 203-224. In R. A Herbert and R. J. Sharp (ed.), Molecular biology and bio/technology of extremophiles. Blackie & Son, Glasgow, Scotland.
Russell, N. J. 2000. Toward a molecular understanding of cold-activity of enzymes from psychrophiles. Extremophiles 4:83-90.
Sawaya, M. R., and J. Kraut. 1997. Loop and subdomain momvements in the mechanism of Escherichia coli dihydrofolate reductase: crystallographic evidence. Biochemistry 36:586-603.
Smalas, A. O., H. K. Leiros, V. Os, and N. P. Willassen. 2000. Cold adapted enzymes. Biotechnol. Annu. Rev. 6:1-57.
Stone, S. R., and J. F. Morrison. 1982. Kinetic mechanism of the reaction catalyzed by dihydrofolate reductase from Escherichia coli. Biochemistry 21:3757-3765.
Van de Casteele, M., P. Chen, M. Roovers, C. Legrain, and N. Glansdorff. 1997. Structure and expression of a pyrimidine gene cluster from the extreme thermophile Thermus strain ZO5. J. Bacteriol. 179:3470-3481.
Van de Casteele, M., C. Legrain, V. Wilquet, and N. Glansdorff. 1995. The dihydrofolate reductase-encoding gene dyrA of the hyperthermophilic bacterium Thermotoga maritima. Gene 158:101-105.
Villeret, V., B. Clantin, C. Tricot, C. Legrain, M. Roovers, V. Stalon, N. Glansdorff, and J. Van Beeumen. 1998. The crystal structure of Pyrococcus furiosus ornithine carbamoyltransferase reveals a key role for oligomerization in enzyme stability at extremely high temperatures. Proc. Natl. Acad. Sci. USA 95:2801-2806.
Wilquet, V. 2000. NAD(P)H-dependent enzymes from extremophilic micro-organisms: the eubacterial thermophilic dihydrofolate and dihydropteridine reductases and a cold-adapted glutamate dehydrogenase. Ph.D. thesis. Université Libre de Bruxelles, Brussels, Belgium.
Wilquet, V., J. A. Gaspar, M. Van de Lande, M. Van de Casteele, C. Legrain, E. M. Meiering, and N. Glansdorff. 1998. Purification and characterization of recombinant Thermotoga maritima dihydrofolate reductase Eur. J. Biochem. 255:628-637.
Xu, Y., G. Feller, C. Gerday, and N. Glansdorff. 2003. Metabolic enzymes from psychrophilic bacteria: challenge of adaptation to low temperatures in ornithine carbamoyltransferase from Moritella abyssi. J. Bacteriol. 185:2161-2168.
Xu, Y., Z. Liang, C. Legrain, H. J. Ruger, and N. Glansdorff. 2000. Evolution of arginine biosynthesis in the bacterial domain: novel gene-enzyme relationships from psychrophilic Moritella strains (Vibrionaceae) and evolutionary significance of N-α-acetyl ornithinase. J. Bacteriol. 182:1609-1615.
Xu, Y., Y. Nogi, C. Kato, Z. Liang, H. J. Rüger, D. De Kegel, and N. Glansdorff. 2003. Moritelia profunda sp. nov. and Moritella abyssi sp. nov. two psychropiezophilic organisms isolated from deep Atlantic sediments. Int. J. Syst. Evol. Microbiol. 53:533-538.
Zecchinon, L., P. Claverie, T. Collins, S. D'Amico, D. Delille, G. Feller, D. Georlette, E. Gratia, A. Hoyoux, M. A. Meuwis, G. Sonan, and C. Gerday. 2001. Did psychrophilic enzymes really win the challenge? Extremophiles 5:313-321.