-ManNAc) and uridine-diphospho-N
-mannosaminuronic acid (UDP-d
-ManNAcA) are ubiquitous and essential metabolites in cellular processes. They act as precursors for proteoglycans and glycoproteins as well as for the cell-wall components of bacteria (peptidoglycans and lipopolysaccharides). N
-mannosamine is a major component of the oligosaccharide chains of N- and O-glycans and of glycolipids (Schachter, 2000
; Herscovics & Orlean, 1993
). O-linked N
-acetylglucosamine modification of specific residues of intracellular proteins in higher eukaryotes has been shown to directly regulate important processes such as the cell cycle, transcription, translation, cell signalling and the stress response to carbohydrate metabolism (Wells et al.
; Zachara et al.
-ManNAcA is synthesized from UDP-N
-GlcNAc) by a two-step pathway. In the first step, UDP-N
-acetylglucosamine 2-epimerase catalyzes the C-2 epimerization of UDP-d
-GlcNAc to form UDP-d
-ManNAc; in the second step, UDP-d
-mannosamine dehydrogenase (UDPManNAcDH) catalyzes the C-6 dehydrogenation of UDP-d
-ManNAc to form UDP-d
-ManNAcA (Reid & Fewson, 1994
UDPManNAcDH is essential in the biosynthesis of UDP-N
-acetylglucosamine and belongs to an enzyme family known as the UDP-glucose/GDP-mannose dehydrogenase family. This family constitutes a small group of enzymes that catalyze the NAD+
-dependent twofold oxidation of a sugar nucleotide without the release of an aldehyde intermediate. Two known crystal structures of members of this family are those of UDP-glucose dehydrogenase (Campbell et al.
) and of GDP-mannose dehydrogenase (Snook et al.
), which show less than 30% sequence identity to UDPManNAcDH. These two enzymes belonging to the UDP-glucose/GDP-mannose dehydrogenase family were found to be dimers with a similar overall fold in their crystal structures. However, the biological role of the dimer formation of these enzymes is not fully understood. Some proteins belonging to this family of enzymes, such as UDPManNAcDH, are also of further interest because of their sugar nucleotide-modifying capabilities, involving a net four-electron oxidation of alcohol and aldehyde (Ge et al.
As the three-dimensional structure of UDPManNAcDH is unknown, our research on this protein may shed light on the UDPManNAc-biosynthesis pathway. Detailed analysis of the structure–function relationship of this enzyme will help in understanding the chemical principles of the biosynthesis of di-N-acetylated mannosamine sugar nucleotide. Here, we report the purification, crystallization and preliminary crystallographic analysis of the putative UDPManNAcDH from Pyrococcus horikoshii OT3 (PH1618), with the aim of determining its crystal structure and elucidating the mechanism of the enzymatic reactions. The PH1618 protein was assigned as a putative UDPManNAcDH, as it has a high sequence identity (40%) to the UDPManNAcDH from Escherichia coli.