The hyperthermophilic Archaea Sulfolobus solfataricus grows optimally above 80 degrees C and metabolizes
glucose by a non-phosphorylative variant of the Entner-Doudoroff pathway. In this pathway
glucose dehydrogenase and
gluconate dehydratase catalyze the oxidation of
glucose to
gluconate and the subsequent
dehydration of
gluconate to D-2-keto-3-deoxygluconate (KDG).
KDG aldolase (KDGA) then catalyzes the cleavage of KDG to D-
glyceraldehyde and
pyruvate. It has recently been shown that all the
enzymes of this pathway exhibit a catalytic promiscuity that also enables them to be used for the metabolism of
galactose. This phenomenon, known as metabolic pathway promiscuity, depends crucially on the ability of KDGA to cleave KDG and D-2-keto-3-deoxygalactonate (KDGal), in both cases producing
pyruvate and D-
glyceraldehyde. In turn, the
aldolase exhibits a remarkable lack of stereoselectivity in the condensation reaction of
pyruvate and D-
glyceraldehyde, forming a mixture of KDG and KDGal. We now report the structure of KDGA, determined by multiwavelength anomalous diffraction phasing, and confirm that it is a member of the tetrameric
N-acetylneuraminate lyase superfamily of
Schiff base-forming
aldolases. Furthermore, by soaking crystals of the
aldolase at more than 80 degrees C below its temperature activity optimum, we have been able to trap
Schiff base complexes of the natural substrates
pyruvate, KDG, KDGal, and
pyruvate plus D-
glyceraldehyde, which have allowed rationalization of the structural basis of promiscuous substrate recognition and catalysis. It is proposed that the active site of the
enzyme is rigid to keep its thermostability but incorporates extra functionality to be promiscuous.