N-Terminal 112 amino acid residues are not required for the sialyltransferase activity of Photobacterium damsela alpha2,6-sialyltransferase.

Photobacterium damsela alpha2,6-sialyltransferase was cloned as N- and C- His-tagged fusion proteins with different lengths (16-497 aa or 113-497 aa). Expression and activity assays indicated that the N-terminal 112 amino acid residues of the protein were not required for its alpha2,6-sialyltransferase activity. Among four truncated forms tested, N-His-tagged Delta15Pd2,6ST(N) containing 16-497 amino acid residues had the highest expression level. Similar to the Delta15Pd2,6ST(N), the shorter Delta112Pd2,6ST(N) was active in a wide pH range of 7.5-10.0. A divalent metal ion was not required for the sialyltransferase activity, and the addition of EDTA and dithiothreitol did not affect the activity significantly.

The Hd0053 gene of Haemophilus ducreyi encodes an alpha2,3-sialyltransferase.

Haemophilus ducreyi is a Gram-negative bacterium that causes chancroid, a sexually transmitted genital ulcer disease. Different lipooligosaccharide (LOS) structures have been identified from H. ducreyi strain 35000, including those sialylated glycoforms. Surface LOS of H. ducreyi is considered an important virulence factor that is involved in ulcer formation, cell adhesion, and invasion of host tissue. Gene Hd0686 of H. ducreyi, designated lst (for lipooligosaccharide sialyltransferase), was identified to encode an alpha2,3-sialyltransferase that is important for the formation of sialylated LOS. Here, we show that Hd0053 of H. ducreyi genomic strain 35000HP, the third member of the glycosyltransferase family 80 (GT80), also encodes an alpha2,3-sialyltransferase that may be important for LOS sialylation.

Cytidine 5'-monophosphate (CMP)-induced structural changes in a multifunctional sialyltransferase from Pasteurella multocida.

Sialyltransferases catalyze reactions that transfer a sialic acid from CMP-sialic acid to an acceptor (a structure terminated with galactose, N-acetylgalactosamine, or sialic acid). They are key enzymes that catalyze the synthesis of sialic acid-containing oligosaccharides, polysaccharides, and glycoconjugates that play pivotal roles in many critical physiological and pathological processes. The structures of a truncated multifunctional Pasteurella multocida sialyltransferase (Delta24PmST1), in the absence and presence of CMP, have been determined by X-ray crystallography at 1.65 and 2.0 A resolutions, respectively. The Delta24PmST1 exists as a monomer in solution and in crystals. Different from the reported crystal structure of a bifunctional sialyltransferase CstII that has only one Rossmann domain, the overall structure of the Delta24PmST1 consists of two separate Rossmann nucleotide-binding domains. The Delta24PmST1 structure, thus, represents the first sialyltransferase structure that belongs to the glycosyltransferase-B (GT-B) structural group. Unlike all other known GT-B structures, however, there is no C-terminal extension that interacts with the N-terminal domain in the Delta24PmST1 structure. The CMP binding site is located in the deep cleft between the two Rossmann domains. Nevertheless, the CMP only forms interactions with residues in the C-terminal domain. The binding of CMP to the protein causes a large closure movement of the N-terminal Rossmann domain toward the C-terminal nucleotide-binding domain. Ser 143 of the N-terminal domain moves up to hydrogen-bond to Tyr 388 of the C-terminal domain. Both Ser 143 and Tyr 388 form hydrogen bonds to a water molecule, which in turn hydrogen-bonds to the terminal phosphate oxygen of CMP. These interactions may trigger the closure between the two domains. Additionally, a short helix near the active site seen in the apo structure becomes disordered upon binding to CMP. This helix may swing down upon binding to donor CMP-sialic acid to form the binding pocket for an acceptor.