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

• Published in: Biochemical journal Vol. 343; no. 3; pp. 563 - 570
• Main Authors: BUCHANAN, Catriona L., CONNARIS, Helen, DANSON, Michael J., REEVE, Christopher D., HOUGH, David W.
• Format: Journal Article
• Language: English
• Published: 01.11.1999
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj3430563

Sulfolobus solfataricus is a hyperthermophilic archaeon growing optimally at 80-85 °C. It metabolizes glucose via a novel non-phosphorylated Entner-Doudoroff pathway, in which the reversible C6 to C3 aldol cleavage is catalysed by 2-keto-3-deoxygluconate aldolase (KDG-aldolase), generating pyruvate and glyceraldehyde. Given the ability of such a hyperstable enzyme to catalyse carbon-carbon-bond synthesis with non-phosphorylated metabolites, we report here the cloning and sequencing of the S. solfataricus gene encoding KDG-aldolase, and its expression in Escherichia coli to give fully active enzyme. The recombinant enzyme was purified in a simple two-step procedure, and shown to possess kinetic properties indistinguishable from the enzyme purified from S. solfataricuscells. The KDG-aldolase is a thermostable tetrameric protein with a half-life at 100 °C of 2.5 h, and is equally active with both D- and L-glyceraldehyde. It exhibits sequence similarity to the N-acetylneuraminate lyase superfamily of Schiff-base-dependent aldolases, dehydratases and decarboxylases, and evidence is presented for a similar catalytic mechanism for the archaeal enzyme by substrate-dependent inactivation by reduction with NaBH4.