Properties of the recombinant glucose/galactose dehydrogenase from the extreme thermoacidophile, Picrophilus torridus

摘要

In Picrophilus torridus, a euryarchaeon that grows optimally at 60 degreesC and pH 0.7 and thus represents the most acidophilic thermophile known, glucose oxidation is the first proposed step of glucose catabolism via a nonphosphorylated variant of the Entner-Doudoroff pathway, as deduced from the recently completed genome sequence of this organism. The P. torridus gene for a glucose dehydrogenase was cloned and expressed in Escherichia coli, and the recombinant enzyme, GdhA, was purified and characterized. Based on its substrate and coenzyme specificity, physicochemical characteristics, and mobility during native PAGE, GdhA apparently resembles the main glucose dehydrogenase activity present in the crude extract of P. torridus DSM 9790 cells. The glucose dehydrogenase was partially purified from P. torridus cells and identified by MS to be identical with the recombinant GdhA. P. torridus GdhA preferred NADP(+) over NAD(+) as the coenzyme, but was nonspecific for the configuration at C-4 of the sugar substrate, oxidizing both glucose and its epimer galactose (K-m values 10.0 and 4.5 mm, respectively). Detection of a dual-specific glucose/galactose dehydrogenase points to the possibility that a 'promiscuous' Entner-Doudoroff pathway may operate in P. torridus, similar to the one recently postulated for the crenarchaeon Sulfolobus solfataricus. Based on Zn2+ supplementation and chelation experiments, the P. torridus GdhA appears to contain structurally important zinc, and conserved metal-binding residues suggest that the enzyme also contains a zinc ion near the catalytic site, similar to the glucose dehydrogenase enzymes from yeast and Thermoplasma acidophilum. Strikingly, NADPH, one of the products of the GdhA reaction, is unstable under the conditions thought to prevail in Picrophilus cells, which have been reported to maintain the lowest cytoplasmic pH known (pH 4.6). At the optimum growth temperature for P. torridus, 60 degreesC, the half-life of NADPH at pH 4.6 was merely 2.4 min, and only 1.7 min at 65 degreesC (maximum growth temperature). This finding suggests a rapid turnover of NADPH in Picrophilus.