A2,6-sialyltransferase

E. Formation of the Sialyl a2,3-Linkage Using a2,3-Sialyltransferase with Regeneration of CMP-NeuAc [24]... 

The regeneration system for CMP-NeuAc can be employed both for a2,3-sialyltransferase-catalyzed reactions and for reactions mediated by a2,6-sialyltransferase. The system starts with NeuAc, the glycosyl acceptor, PEP, and catalytic amounts of ATP and CMP. CMP is converted to CDP by nucleoside monophosphate kinase (EC 2.7.4.4 NMK) in the presence of ATP, which is regenerated from the by-product ADP, catalyzed by PK in the presence ol PEP, then to CTP with PEP by PK. The CTP thus formed reacts with NeuAc, catalyzed b>... 

CMP-NeuAc synthetase (EC 2.7.7.43) to produce CMP-NeuAc. The by-product pyrophosphate (PPi) is hydrolyzed to phosphate (Pi) by inorganic pyrophosphatase (PPase). Sialyla-tion is accomplished with a2,3-sialyltransferase (< 2,3NeuAcT) or a2,6-sialyltransferase (a2,3NeuAcT), respectively. The released CMP is again converted to CDP, to CTP, and finally to CMP-NeuAc. The UDP-Gal and CMP-NeuAc regeneration schemes have been combined in a one-pot reaction and applied to the synthesis of sialyl Lewis X. 

CMP-neuraminic acid can be used in oligosaccharide synthesis as a substrate for various a2-6-sialyltransferases isolated from rat liver [50] or bovine colostrum [51] as well as a2-3-sialyltransferases isolated from porcine liver [52] and porcine submaxillary glands [53]. Recently, the enzyme has been overexpressed in a baculovirus system [54]. 

D. Formation of the sialyl a2,3-linkage using a2,3-sialyltransferase without regeneration of CMP-NeuAc 497... 

Lee YC, Kurosawa N, Hamamoto T, Nakaoka T, Tsuji S (1993) Molecular cloning and expression of Gal/il,3GalNAc a2,3-sialyltransferase from mouse brain. Eur J Biochem 216 377-385... 

Kono M, Takashima S, Liu H, Inoue M, Kojima N, Lee YC, Hamamoto T, Tsuji S (1998) Molecular cloning and functional expression of a fifth-type a2,3-sialyltransferase (mST3Gal V GM3 synthase). Biochem Biophys Res Commun 253 170-175... 

Fukumoto S, Miyazaki H, Goto G, Urano T, Furukawa K, Furukawa K (1999) Expression cloning of mouse cDNA of CMP-NeuAc Lactosylceramide a2,3-sialyltransferase, an enzyme that initiates the synthesis of ganglio-sides. J Biol Chem 274 9271-9276... 

Our group has reported a novel multifunctional bacterial enzyme, Pasteurella multocida sialyltransferase (PmSTl). PmSTl is an easy to express extremely soluble, and highly active sialyltransferase. Routinely, about 100 mg of PmSTl can be purified from one liter E. coli cell culture. PmSTl has four different activities including a2,3-sialyltransferase (optimal activity at pH 7.5-9.0), a2,6-sialyltransferase (optimal activity at pH 4.5-7.0), a2,3-sialidase (optimal activity at pH 5.0-5.5), and trans-sialidase (optimal activity at pH 5.5-6.5) activities. Among them, a2,3-sialyltransferase activity is the major function of the PmSTl with a specific activity of 60 U/mg protein (24),... 

Sialyltransferase (SiaT). a2,6- and a2,3-sialyltransferase have been used for oligosaccharide synthesis [166 1681. Sialyltransferases generally transfer N-acetylneura-minic acid (NeuAc) to either the 3- or 6-position of terminal Gal or GalNAc residues... 

Aldolases, CMP-Sia synthetases, and/or sialyltransferases have been combined in a number of one-pot, chemoenzymatic syntheses of naturally occurring sialosides (e.g., see Ichikawa et /. ). The use of permissive enzymes broadens the scope of these one-pot methods and has facilitated the synthesis of a number of unnatural sialosides. The Chen group reported a Pasteurella multocida sialyltransferase that can function as an a2-3 sialyltransferase, o 2-6 sialyltransferase, o 2-3 sialidase, or a2-3 rr j-sialidase, depending on the pH of the reaction mixture. Using this enzyme in a one-pot reaction in combination with E. coli aldolase and... 

Sialyltransferase P-Galactoside a2-3-sialyltransferase CMP-Neu5Ac Neu5Aca2-3Gaipi-3GalNac-R... 

For the synthesis of sialylated oligosaccharides, a fusion enzyme consisting of CMP-NeuAc synthetase and a2,3-sialyltransferase was created (29). The... 

Four of the six different a2,3-sialyltransferases that catalyze the transfer of sialic acid residues in a2,3-linkage onto the terminal Galp residue are involved in mucin glycan chains biosynthesis (Table 3). 

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