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°C 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 0°C. 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 5°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 (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.The temperature range compatible with procaryotic life extends from well below 0°C up to at least 113°C. The comparative analysis of series of homologous enzymes spanning this range suggested the molecular strategies which achieve the necessary compromises between structural flexibility and stability. Thermophilic enzymes appear to owe their stability to a variety of extrinsic or intrinsic factors (for a recent review, see reference 13). Moreover, oligomerization is the dominant stabilizing factor in several proteins (20,22,39); a basically similar situation is obtained when an enzyme is stabilized by physical association with another one (37).At the other end of the temperature scale, psychrophilic (24) enzymes face the necessity of being efficient catalysts at a low energy cost (4, 9, 14). On the basis of a much more limited set of data than for thermophilic enzymes (see recent reviews in references 33, 35, and 45), two basic strategies for psychrophily were distinguished: either an overall increase of flexibility generally associated with a marked decrease of stability, or a more subtle strategy which keeps one domain rigid enough to provide enough affinity for the substrate while holding the catalytic domain in a more flexible state (2,21,45). In the former case thermodynamic and genetic data even suggest that some enzymes, such as...

Identification of a Novel Alkaliphilic Esterase Active at Low Temperatures by Screening a Metagenomic Library from Antarctic Desert Soil

“…values and lower k cat /K m ratios (27,28). Whether CHA3 fits with this theory is the subject of subsequent studies.…”

Catalysis by Dihydrofolate Reductase from the Psychropiezophile Moritella profunda

“…More recently, recombinant DHFR from Moritella profunda (MpDHFR), a psychropiezophilic bacterium isolated from Atlantic Ocean sediments at a depth of 2815 m, [2] has been examined with respect to its structural and kinetic adaptation to high pressure and low temperature. [3][4][5] In DHFR from E. coli (EcDHFR), as with many other enzymes, physical processes rather than the actual chemical step limit the reaction rate under steady-state conditions at pH 7. [6] EcDHFR cycles through five key intermediates during catalysis [6] that vary in the movement and coordination of various loops, subdomains and bound ligands [7][8][9] and solvent access.…”

Extreme Catalysts from Low-Temperature Environments

“…nov. (52) and the ornithine carbamoyltransferase from the psychrophilic Moritella abyssi (52) do not optimize the catalytic efficiency k cat /K m . Both heat-labile enzymes appear to be suboptimal in their physiological temperature range as far as the k cat /K m ratio is concerned, supporting the concept that complete metabolic adaptation to cold may remain elusive (52). However, it should be noted that the improvement of the catalytic efficiency k cat /K m could be related to the global or localized decrease in the stability of psychrophilic enzymes.…”

“…The hydrophobic core and ion-pair network at the subunit interface are the major stabilizing factors which stabilize the intersubunit interface (50). Moreover, oligomerization can be a significant stabilization mechanism for hyperthermophilic enzymes (50)(51)(52)(53). On the basis of stability studies of dimeric globular proteins, it was calculated that quaternary interactions could provide 25% to 100% of the conformational stability in protein dimers (54).…”