L-Colitose (
1) is a 3,6-dideoxyhexose found in the
O-antigen of gram-negative lipopoly-saccharides. While the biosynthesis of many deoxysugars have previously been investigated,
L-colitose is distinct in that it originates from GDP-
D-mannose. In contrast, other 3,6-dideoxyhexoses arise from CDP-
D-glucose. Therefore, the enzymes involved in the
L-colitose biosynthetic pathway must be specifically tailored to utilize such a modified substrate. The mode for deoxygenation at C-3 of colitose is of particular interest because this conversion in other naturally occurring 3,6-dideoxyhexoses requires a pair of enzymes, E
1 and E
3, acting in concert. Interestingly, no E
3 equivalent was identified in the five open reading frames of the
col biosynthetic gene cluster from
Yersinia pseudotuberculosis IVA. However, the gene product of
colD showed moderate similarity with the E
1 gene (
ddhC/
ascC) of the ascarylose pathway (27% identity and 42% similarity). Because E
1 is a pyridoxamine 5'-phosphate (PMP)-dependent enzyme, it was thought that ColD might also utilize PMP. Indeed, turnover was observed during incubation of ColD with substrate in the presence of excess PMP, but not with pyridoxal 5'-phosphate (PLP). However, the rate of product formation increased by more than 40-fold when
L-glutamate was included in the PLP incubation. The formation of
-ketoglutarate as a byproduct under these conditions clearly indicated that ColD functions as a transaminase, recognizing both PMP and PLP. In this paper, we propose a novel biosynthetic route for colitose, including the unprecedented C-3 deoxygenation performed solely by ColD. The utilization of PMP in a dehydration reaction is rare, but the combined deoxygenation-transamination activity makes ColD a unique enzyme.