Tm. Hallis et al., New insights into the mechanism of CDP-D-tyvelose 2-epimerase: An enzyme-catalyzing epimerization at an unactivated stereocenter, J AM CHEM S, 122(43), 2000, pp. 10493-10503
Tyvelose is a 3,6-dideoxyhexose found in the Q-antigen of Yersinia pseudotu
berculosis IVA and is the only member of this class of sugars to be produce
d directly from another 3,6-dideoxyhexose, paratose. The C-2 epimerization
required for this conversion has been proposed to be catalyzed by CDP-D-tyv
elose 2-epimerase. This enzyme is intriguing since it belongs to a group of
epimerases, including the well-studied UDP-D-galactose 4-epimerase, thar c
an invert unactivated stereocenters. To study the mechanism of this enzyme,
we have cloned and expressed the tyv gene that encodes CDP-D-tyvelose 2-ep
imerase. The purified tetrameric protein contains approximately one equival
ent of bound NAD(+) per monomer and a small fraction of NADH. Four possible
mechanisms involving NAD(+) can be proposed for this enzyme; two involve o
xidation at C-2 of the substrate, while the other two require oxidation at
C-4. In a previous contribution, we presented preliminary data that support
ed a retro-aldol-type mechanism initiated by C-4 oxidation. However, this m
echanism was refuted by further investigations, which revealed that the 4-f
luoro analogue of DP-D-paratose could be turned over by the enzyme. More im
portantly, the direct transfer of a deuterium from C-2 of the labeled subst
rate to the enzyme-bound NAD(+) was observed by mass spectrometry. These re
sults suggest that epimerization is in fact initiated by oxidation at C-2,
followed by the transfer of the hydride from the transiently formed NADH to
the opposite side of the 2-hexulose intermediate.