An extremely thermostable aldolase from Sulfolobus solfataricus with specificity for non-phosphorylated substrates.
Sulfolobus solfataricus is a hyperthermophilic archaeon growing optimally at 80-85 degrees C. It metabolizes glucose via a novel non-phosphorylated Entner-Doudoroff pathway, in which the reversible C(6) to C(3) 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. solfataricus cells. The KDG-aldolase is a thermostable tetrameric protein with a half-life at 100 degrees 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 NaBH(4).
Buchanan
C L
CL
Centre for Extremophile Research, Department of Biology and Biochemistry, University of Bath, Bath BA2 7AY, UK.
Connaris
H
H
Danson
M J
MJ
Reeve
C D
CD
Hough
D W
DW
D000446:Aldehyde-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000201:enzymology; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D006193:Haemophilus influenzae / Q000201:enzymology; D006358:Hot Temperature; D007700:Kinetics; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D016944:Sulfolobus / Q000201:enzymology* / Q000235:genetics; D013816:Thermodynamics
D011994:Recombinant Proteins; D000446:Aldehyde-Lyases; C021468:2-keto-3-deoxy-D-glucarate aldolase