title: Moritella cold-active dihydrofolate reductase: are there natural limits to optimization of catalytic efficiency at low temperature?
abstract: 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 2 degrees C or less. The enzyme is monomeric (Mr=18,291), 55% identical to its Escherichia coli counterpart, and displays Tm 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 0 degrees C. At mesophilic temperatures the apparent Km value for dihydrofolate is 50- to 80-fold higher than for E. coli, Lactobacillus casei, and T. maritima dihydrofolate reductases, whereas the apparent Km value for NADPH, though higher, remains in the same order of magnitude. At 5 degrees C 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 (kcat/Km) 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.
authors.affiliation: J. M. Wiame Research Institute, Microbiology, Free University of Brussels (VUB), B-1070 Brussels, Belgium. xuying@vub.ac.be
authors.forename: Ying
authors.initials: Y
authors.lastname: Xu
authors.forename: Georges
authors.initials: G
authors.lastname: Feller
authors.forename: Charles
authors.initials: C
authors.lastname: Gerday
authors.forename: Nicolas
authors.initials: N
authors.lastname: Glansdorff
mesh_terms: D000595:Amino Acid Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D003080:Cold Temperature; D004789:Enzyme Activation / Q000502:physiology; D004795:Enzyme Stability; D005493:Folic Acid Antagonists / Q000494:pharmacology; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D007700:Kinetics; D008727:Methotrexate / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D034221:Moritella / Q000201:enzymology* / Q000235:genetics; D009249:NADP / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017386:Sequence Homology, Amino Acid; D013696:Temperature; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D014295:Trimethoprim / Q000494:pharmacology
chemical_list: D005493:Folic Acid Antagonists; D009249:NADP; D014295:Trimethoprim; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate