Catalytic promiscuity in dihydroxy-acid dehydratase from the thermoacidophilic archaeon Sulfolobus solfataricus.

Dihydroxy-acid dehydratase (DHAD) is one of the key enzymes involved in the biosynthetic pathway of the branched chain amino acids. Although the enzyme has been purified and characterized in various mesophiles, including bacteria and eukarya, the biochemical properties of DHAD from hyperthermophilic archaea have not yet been reported. In this study we cloned, expressed in Escherichia coli, and purified a DHAD homologue from the thermoacidophilic archaeon Sulfolobus solfataricus, which grows optimally at 80 degrees C and pH 3. The recombinant S. solfataricus DHAD (rSso_DHAD) showed the highest activity on 2,3-dihydroxyisovalerate among 17 aldonic acids tested. Interestingly, this enzyme also displayed high activity toward d-gluconate and some other pentonic and hexonic sugar acids. The k(cat)/K(m) values were 140.3 mM(-1) s(-1) for 2,3-dihydroxyisovalerate and 20.0 mM(-1) s(-1) for d-gluconate, respectively. A possible evolutionary explanation for substrate promiscuity was provided through amino acid sequence alignments of DHADs and 6-phosphogluconate dehydratases from archaea, bacteria and eukarya.