An extremely thermostable aldolase from Sulfolobus solfataricus with specificity for non-phosphorylated substrates

Sulfolobus solfataricus is a hyperthermophilic archaeon growing optimally a t 80-85 degrees C. It metabolizes glucose via a novel nonphosphorylated Ent ner-Doudoroff pathway, in which the reversible C-6 to C-3 aldol cleavage is catalysed by 2-keto-3-deoxygluconate aldolase (KDG-aldolase), generating p yruvate and glyceraldehyde. Given the ability of such a hyperstable enzyme to catalyse carbon-carbon-bond synthesis with nonphosphorylated metabolites , we report here the cloning and sequencing of the S. solfataricus gene enc oding KDG-aldolase, and its expression in Escherichia coli to give fully ac tive enzyme. The recombinant enzyme was purified in a simple two-step proce dure, and shown to possess kinetic properties indistinguishable from the en zyme purified from S. solfataricus cells. The KDG-aldolase is a thermostabl e tetrameric protein with a half-life at 100 degrees C of 2.5 h, and is equ ally active with both D- and L-glyceraldehyde. It exhibits sequence similar ity to the N-acetylneuraminate lyase superfamily of Schiff-base-dependent a ldolases, dehydratases and decarboxylases, and evidence is presented for a similar catalytic mechanism for the archaeal enzyme by substrate-dependent inactivation by reduction with NaBH4.