Metabolic Enzymes from Psychrophilic Bacteria: Challenge of Adaptation to Low Temperatures in Ornithine Carbamoyltransferase from Moritella abyssi

Abstract

The enzyme ornithine carbamoyltransferase (OTCase) of Moritella abyssi (OTCase Mab ), a new, strictly psychrophilic and piezophilic bacterial species, was purified. OTCase Mab displays maximal activity at rather low temperatures (23 to 25°C) compared to other cold-active enzymes and is much less thermoresistant than its homologues from Escherichia coli or thermophilic procaryotes. In vitro the enzyme is in equilibrium between a trimeric state and a dodecameric, more stable state. The melting point and denaturation enthalpy changes for the two forms are considerably lower than the corresponding values for the dodecameric Pyrococcus furiosus OTCase and for a thermolabile trimeric mutant thereof. OTCase Mab displays higher K m values for ornithine and carbamoyl phosphate than mesophilic and thermophilic OTCases and is only weakly inhibited by the bisubstrate analogue ␦-N-phosphonoacetyl-L-ornithine (PALO). OTCase Mab differs from other, nonpsychrophilic OTCases by substitutions in the most conserved motifs, which probably contribute to the comparatively high K m values and the lower sensitivity to PALO. The K m for ornithine, however, is substantially lower at low temperatures. A survey of the catalytic efficiencies (k cat /K m ) of OTCases adapted to different temperatures showed that OTCase Mab activity remains suboptimal at low temperature despite the 4.5-fold decrease in the K m value for ornithine observed when the temperature is brought from 20 to 5°C. OTCase Mab adaptation to cold indicates a trade-off between affinity and catalytic velocity, suggesting that optimization of key metabolic enzymes at low temperatures may be constrained by natural limits.Intracellular biosynthetic enzymes are usually exposed to low substrate concentrations, in contrast to extracellular enzymes. Optimizing their catalytic efficiency (k cat /K m ) in psychrophilic (cold-adapted) organisms may thus be challenging, since improving k cat at low temperatures by decreasing the activation enthalpy may have a cost in terms of affinity for the substrate(s) of the reaction (18,35). The study of cold-active enzymes is thus an important topic in terms of physiology and metabolic evolution (for recent reviews, see references 6, 21, 25, 44, and 60).No cold-active ornithine carbamoyltransferase (OTCase; EC 2.1.3.3) had been characterized until now. However, the presence of this enzyme in microorganisms adapted to the full range of environments compatible with life makes it an excellent candidate for investigations of protein evolution and of molecular adaptations to extreme conditions. OTCase catalyzes the conversion of ornithine and carbamoyl phosphate (CP) into citrulline and inorganic phosphate in the de novo pathway for arginine biosynthesis and in the detoxifying urea cycle. Biosynthetic and urea cycle OTCases are usually homotrimers of 33-to 40-kDa subunits (7, 9), except in the hyperthermophilic archaeon Pyrococcus furiosus, where OTCase is a dodecamer (51). In Pseudomonas aeruginosa, a dodecameric catabolic OTCase catalyzes the revers...

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