| Abstract |
Agmatine, produced from arginine by arginine decarboxylase (ADC), is an essential precursor for spermidine and agmatidine in hyperthermophilic archaea. In the hyperthermophilic archaeon Pyrobaculum calidifontis, native ADC activity was detectable but rapidly lost when extracts were kept on ice, suggesting cold sensitivity. To investigate this instability, the Pc-speA gene was cloned and expressed in Escherichia coli. The recombinant protein formed insoluble aggregates but could be solubilized and refolded at various temperatures. Enzymatic assays showed that activity was restored only after refolding at high temperature, with maximal activity at 90 °C. SDS-PAGE confirmed autocatalytic cleavage into α- and β-subunits, generating the pyruvoyl group required for catalysis, and substrate assays verified specificity for arginine but not ornithine. Notably, refolded Pc-SpeA was cold-labile: storage at ≤20 °C caused aggregation and loss of activity, whereas stability was maintained at 60-80 °C. These findings provide the first biochemical characterization of P. calidifontis ADC and reveal its unusual requirement for elevated temperature to achieve and preserve enzymatic function.
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