Using substrate analogues to probe the mechanisms of two biosynthetic enzymes : a thesis presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Chemistry at Massey University, Turitea, Palmerston North, New Zealand
3-Deoxy-D-arabino-heptulosonate 7-phosphate (DAH7P) synthase and 3-deoxy-Dmanno-
octulosonate 8-phosphate synthase (KDO8P) synthase are two enzymes that
catalyse very similar reactions. DAH7P synthase is the first enzyme of the shikimate
pathway and catalyses the condensation reaction between the four-carbon sugar
erythrose 4-phosphate (E4P) 1 and the three-carbon sugar phosphoenolpyruvate (PEP) 2
to give the seven-carbon sugar DAH7P 3. KDO8P synthase catalyses a similar
condensation reaction between the five-carbon sugar arabinose 5-phosphate (A5P) 8
and PEP 2 to give the eight-carbon sugar KDO8P 9. Early mechanistic studies have
shown the reaction mechanisms of these two enzymes to be very similar and structural
and phylogenic analysis has suggested that the two enzymes share a common ancestor.
However, there are differences between the two enzymes that have not been explained
by the current literature. Whereas all DAH7P synthases require a divalent metal ion for
activity, there exists both metallo and non-metallo KDO8P synthases. As well as this,
there is the difference in substrate specificity. The natural substrate of KDO8P
synthase, A5P, is one carbon longer and has the opposite C2 stereochemistry to E4P,
the natural substrate of DAH7P synthase.
This study investigates the role of the C2 and C3 hydroxyl groups of E4P and A5P in
the enzyme catalysed reactions. The E4P analogues 2-deoxyE4P 38 and 3-deoxyE4P
39 have been synthesised from [beta]-hydroxy-[gamma]-butyrolactone and malic acid respectively.
The two analogues were tested as substrates for DAH7P synthase from a variety of
organisms, including N. meningitidis, the purification and characterisation of which was carried
out during the course of these studies. It was found that both analogues were
substrates for DAH7P synthase. 2-DeoxyE4P was found to be the best alternative
substrate for DAH7P synthase to date.
The analogous study was carried out on KDO8P synthase from N. meningitidis with 2-
deoxyR5P 34 and 3-deoxyA5P 40. It was found that removal of the C2 and C3
hydroxyl groups of A5P was much more catastrophic for the KDO8P synthase catalysed
reaction. Commercially available 2-deoxyR5P was found to be a very poor substrate,
whereas 3-deoxyA5P, which was prepared according to a literature procedure was not a
The difference in substrate specificities of DAH7P synthase and KDO8P synthase is
consistent with the hypothesis that despite their similarities, these two related enzymes
have different mechanisms. The key step for DAH7P synthase appears to be
coordination of the E4P carbonyl to the divalent metal. The metal appears to play a less
important role in the KDO8P synthase reaction and the key step is the correct
orientation of A5P in the active site.