The Unnatural Substrate Tolerance of the Enzymes Involved in Sialic Acid Biosynthesis

The possibility that sialic acid biosynthesis might be amenable to interception with unnatural substrates was first suggested by studies of the individual enzymes in the pathway. Several groups have analyzed the tolerance of various enzymes for unnatural substrates, revealing sites on metabolic intermediates at which functional group substitutions might be tolerated. Of the enzymes involved in sialic acid biosynthesis, the two that have attracted the most attention are CMP-Neu5Ac-synthetase and various sialyltransferases. Both bacterial and mammalian homologs from these two enzyme families have been characterized.

CMP-Neu5Ac-synthetase catalyzes the condensation of free Neu5Ac with CTP to make CMP-Neu5Ac. The enzymes from bovine brain and rat liver have been investigated with respect to their activity with unnatural sialic acids [118]. Sialic acids modified at C5 and C9 have been extensively studied; several of these are accepted by the enzyme and are successfully converted into the corresponding CMP-sialic acid analogs in yields of 40-90% [119] (Table 9). The efficiency of the reaction depended on the analog. While the enzyme tolerates rather large substituents at C9, structural perturbation at C5 must be conservative. Examples of acceptable modifications are shown in Table 9 [119-124]. The 4-deoxy derivative of Neu5Ac is also a substrate for CMP-Neu5Ac synthetase [125]. The large variety of compounds that have been successfully converted into their CMP analogs indicate that bovine CMP-Neu5Ac-synthetase might be useful for in vitro enzymatic synthesis of unnatural CMP-sialic acid analogs.

In eukaryotes, sialyltransferases are located in the Golgi compartments [126,127]. They transfer a sialic acid residue from CMP-sialic acid to a nonreducing Gal-, GalNAc-, GlcNAc-, or sialic acid residue via an a-glycosidic linkage. The possible linkages include a2,3, a2,6, or a2,8, and each is the product of a different sialyltransferase.

Sialyltransferases from rat and human liver have been studied extensively [128]. Similar to CMP-Neu5Ac-synthetase, the broad specificity of sialyltransferases has made them useful for the synthesis of neoglycoconjugates containing a variety of sialic acid analogs. Examples of CMP-sialic acid analogs that have been successfully converted into glycoconjugates by sialyltransferases are shown in Table 10 [118-120,125,128-131]. Even perturbations as large as a fluorescein group at C5 or C9 are tolerated by some sialyltransferases, allowing one to install fluorescent probes in cell surface glycoconjugates [132,133]. This approach has provided a method for kinetic analysis of the rates of different sialyltransferases within cells [134,135]. Furthermore, the C9 fluorescein derivative has been used to localize sia-lyltransferases to the Golgi compartments in rat liver [127]. Although alterations at C5 and C9 can reduce efficiency of sialyltransferases, the reactions occur at a reasonable rate and are therefore useful for studying cellular processes.

Figure 10 The sialoside biosynthetic pathway in eukaryotic cells: A, UDP-GlcNAc-2-epimerase; B, ManNAc-6-kinase; C, Neu5Ac-9-PO4~ synthase; D, Neu5Ac-9-PO;;~ phosphatase; E, CMP-Neu5Ac-synthetase; F, sialyltransferase. Enzymes A and B function together as a bifunctional enzyme.

Table 9 A Limited Number of Sialic Acid Derivatives Are Recognized as Substrates for CMP-Sialic Acid Synthetase from Bovine Brain and Rat Liver

Table 9 A Limited Number of Sialic Acid Derivatives Are Recognized as Substrates for CMP-Sialic Acid Synthetase from Bovine Brain and Rat Liver

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