Iiigalactosyltransferases A 14Galactosyltransferase

¡31,4-Galactosyltransferase (¡1,4-GalT, EC 2.4.1.22/38/90) has been commercially available for many years in unit quantities, where a unit is the amount of enzyme that converts one micromole of substrate to product per minute. It is the most widely studied glycosyltransferase with regard to substrate specificity and use in preparative synthesis and the first mammalian glycosyltransferase for which an X-ray crystal structure was determined [31]. Historically, ¡1,4-GalT was also the first enzyme used for the preparation of oligosaccharides on a large scale (>10 g) [12]. ¡1,4-GalT catalyzes the transfer of Gal from UDP-Gal to OH-4 of terminal ¡-linked GlcNAc to form V-acetyllactosamine (LacNAc) (Fig. 2). The acceptor specificity shifts to glucose in the presence of the protein modifier a-lactalbumin, giving lactose as the major product.

Since Berliner's pioneering work demonstrating that ¡1,4-GalT could transfer glucose, 4-deoxy-Gal and arabinose from their corresponding UDP donors [32], donors with modifications at every OH group have been employed for the synthesis of numerous LacNAc analogs (Fig. 3). While the enzyme transfers 2-deoxy-Gal at a rate comparable to that of UDP-Gal [33], 3-deoxy [34], 6-deoxy [35,36], and 6-deoxy-6-fluoro Gal [35,36] are transferred at reduced rates. The enzyme also utilizes UDP-GalNAc, UDP-GalNH2, and UDP-GlcNH donors [37,38] (Fig. 3). Replacement of the oxygen of Gal with sulfur in UDP-5'-thio-Gal is tolerated by ¡1,4-GalT [39], including transfer in the presence of a-lactalbumin [40].

The acceptor specificity of ¡1,4-GalT is equally relaxed, since numerous modifications in acceptors including the ring oxygen are possible as long as the 4-OH remains available for glycosylation (Fig. 3). The 4-OH is a "key polar group'' [41] where replacement or modification yields analogs that no longer bind to the enzyme.

The 2-NHAc group can be replaced with V-propanoyl [42,43], V-butanoyl [42], azido [38,44], allylcarbamate [45], and other amide derivatives, including bulky het-erocycles, charged groups, and glycuronamides [46]. Acceptors with the 3-OH group deoxygenated [47,48], alkylated with a methyl or allyl group, or oxidized to the ketone are active as acceptors with reduced reaction rates [48]. The 6-OH group of GlcNAc can be methylated [43,48], deoxygenated [47], fucosylated [43], or replaced with F or SH [47]. GlcNAc bearing a-linked sialic acid at 6-OH is not a substrate;

Figure 2 The reaction catalyzed by ¡1,4-galactosyltransferase.
Figure 3 Unnatural donors and acceptors for |61,4-GalT.

however, if the carboxylic acid of NeuAc is derivatized with a methyl ester, the resulting compound is an acceptor with 4% the rate of transfer compared to the parent acceptor [43].

¡1,4-GalT will utilize acceptors with the ring oxygen modified, including 5'-thio-Glc and 1-deoxy-nojirimycin [48]. Glucal with a flattened ring [48] and (±)-conduritol B are substrates; the latter mixture can be resolved because only a single galactosylated product (—)-conduritol B is produced [49]. In an exception to the

"one enzyme-one linkage'' hypothesis, the enzyme transfers galactose to the anomeric position of 3-acetamido-3-deoxy-d-glucose acceptors giving a ¡1—>1 trehalose type of linkage [29]. This atypical regiochemistry of galactosylation was also reported for N-acetyl-gentosamine, N-acetyl-5'-thiogentosamine, and xylose acceptors [50,51]. A variety of immobilized acceptors have been employed in solid phase or soluble polymer synthesis [52-58], and even UDP-GlcNAc is an acceptor substrate, yielding the nucleotide disaccharide UDP-N-acetyllactosamine [59]. Reactions at scales of 500 g or more with ¡1,4-GalT have been reported with in situ donor recycling [60]. Also ¡1,4-GalT enzymes have been found in bacterial sources, including Helicobacter pylori and Neisseria meningitidis [61-63] and utilized in 100 g reactions for N-acetyllactosamine production through bacterial coupling [64].

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