Phosphoribosyl anthranilate isomerase from Thermotoga maritima is an extremely stable and active homodimer

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Abstract
The metabolism of hyperthermophilic microorganisms can function properly at temperatures close to 100 °C. It follows that they are equipped with both thermostable enzymes and mechanisms that handle labile metabolites. We wanted to understand how stable and active phosphoribosyl anthranilate isomerase (tPRAI) from the hyperthermophile Thermotoga maritima is at its optimum growth temperature of 80 °C, and how its thermolabile substrate, N‐(5′‐phosphoribosyl)‐anthranilate (PRA), is protected from rapid decomposition. To this end, the trpF gene of T. maritima was expressed heterologously in Escherichia coli and tPRAI was purified. In contrast to most PRAIs from mesophiles, which are monomers with the eightfold βα (or TIM) barrel fold, tPRAI is a homodimer. It is strongly resistant toward inactivation by temperatures up to 95 °C, by acidification to pH 3.2, and by proteases in the presence and absence of detergents. tPRAI is about 35‐fold more active at its physiologic temperature than is the enzyme from E. coli (ePRAI) at 37 °C. This high catalytic efficiency of tPRAI is likely to compete successfully with the rapid spontaneous hydrolysis of PRA at 80 °C. Thus, with respect to both stability and function, tPRAI appears well adapted to the extreme habitat of T. maritima. Single crystals of tPRAI have been obtained that are suitable for X‐ray analysis at high resolution.