Phosphoribosyl anthranilate isomerase from Thermotoga maritima is an extremely stable and active homodimer.
The metabolism of hyperthermophilic microorganisms can function properly at temperatures close to 100 degrees 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 degrees 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 beta alpha (or TIM) barrel fold, tPRAI is a homodimer. It is strongly resistant toward inactivation by temperatures up to 95 degrees 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 degrees C. This high catalytic efficiency of tPRAI is likely to complete successfully with the rapid spontaneous hydrolysis of PRA at 80 degrees 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.
Sterner
R
R
Abteilung für Biophysikalische Chemie, Biozentrum der Universität Basel, Switzerland. sterner@ubaclu.unibas.ch
Kleemann
G R
GR
Szadkowski
H
H
Lustig
A
A
Hennig
M
M
Kirschner
K
K
D019747:Aldose-Ketose Isomerases; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002238:Carbohydrate Epimerases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D004269:DNA, Bacterial / Q000235:genetics; D019281:Dimerization; D004926:Escherichia coli / Q000201:enzymology; D006089:Gram-Negative Anaerobic Bacteria / Q000201:enzymology* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D012267:Ribosemonophosphates / Q000378:metabolism*; D013045:Species Specificity; D013696:Temperature; D062367:ortho-Aminobenzoates / Q000378:metabolism*
D001426:Bacterial Proteins; D004269:DNA, Bacterial; D011993:Recombinant Fusion Proteins; D012267:Ribosemonophosphates; D062367:ortho-Aminobenzoates; C063777:N-(5'-phosphoribosyl)anthranilate; D002238:Carbohydrate Epimerases; D019747:Aldose-Ketose Isomerases; C029865:phosphoribosylanthranilate isomerase