In this report, we present evidence that NeuC catalyzes the epimerization of UDP-GlcNAc to ManNAc, the first committed step in sialic acid biosynthesis in E. coli
K1. These data are in good agreement with both the protein homologies and the indirect results reported by other investigators (29
). Two radiolabeled reaction products that comigrated in paper chromatography with ManNAc and 2-acetamidoglucal resulted from the in vitro incubation of purified NeuC protein with UDP-[14
C]GlcNAc. However, no products were observed when the enzyme fraction was boiled or when an excess of unlabeled UDP-GlcNAc was added (Fig. ). This result indicated that neither product was the result of nonenzymatic hydrolysis or epimerization of the substrate. In their studies of UDP-GlcNAc 2-epimerase purified from rat liver, Sommar and Ellis (30
) proposed a reaction mechanism that could explain these data (Fig. ). They posited that the enzymatic mechanism was an ordered one in which the first product released was UDP, followed by the irreversible formation of ManNAc. This mechanism included a 2-acetamidoglucal intermediate, which was enzyme bound. This mechanism does not require a cofactor and is consistent with the requirement for a nucleotide sugar in the formation of ManNAc. It is also consistent with the observation that purified NeuC did not catalyze the epimerization of GlcNAc to ManNAc.
Proposed mechanism of the reaction catalyzed by the mammalian UDP-GlcNAc 2-epimerase.
The proposed role for NeuC as a UDP-GlcNAc 2-epimerase is supported by the observed sequence homology between this enzyme and the well-characterized mammalian and E. coli
) UDP-GlcNAc 2-epimerases (27 and 21% identities, respectively). This enzyme clearly is required for sialic acid biosynthesis. It is therefore expected that this enzyme converts UDP-GlcNAc to ManNAc and UDP. Both the chromatographic and the NMR spectroscopic assays indicate, however, that the primary reaction observed is the conversion of UDP-GlcNAc to 2-acetamidoglucal and UDP. The chromatographic assay indicated that a very low level of ManNAc is also formed, but this was an extremely slow process. Thus, two scenarios emerge: either a second enzyme, such as a glycosidase (glycosidases are known to hydrate glycals [18
]), is required to complete the conversion to ManNAc, or the purified enzyme is missing an important regulator or coenzyme that is required to complete the catalytic cycle. The second scenario is consistent with the observation that even the formation of 2-acetamidoglucal seems to be quite slow. One attempt was made to determine whether CMP-N
-acetylneuraminic acid is an allosteric activator required for full activity; however, no effect was observed.
The formation of 2-acetamidoglucal nevertheless strengthens the link between NeuC and other UDP-GlcNAc 2-epimerases (27
). The E. coli
UDP-GlcNAc 2-epimerase, RffE, is a true epimerase that interconverts UDP-GlcNAc and UDP-ManNAc. Several lines of evidence support a mechanism involving the anti
-elimination of UDP to form 2-acetamidoglucal, followed by the syn
-addition of UDP to form a product (20
). In fact, the enzyme is known to release 2-acetamidoglucal and UDP into solution once every 1,000 turnovers.
The mammalian UDP-GlcNAc 2-epimerase converts UDP-GlcNAc to UDP and free ManNAc (this action essentially is irreversible and technically is not epimerization) and plays a key role in sialic acid biosynthesis. This enzyme also is thought to use a mechanism involving the anti
-elimination of UDP to give 2-acetamidoglucal, followed by the syn
-addition of water to give ManNAc (7
) (Fig. ). It has been shown that this enzyme will accept the intermediate 2-acetamidoglucal from solution and hydrate it to form ManNAc. Given that NeuC also catalyzes the anti
-elimination of UDP to form 2-acetamidoglucal, it seems reasonable to assume that it also functions as a UDP-GlcNAc 2-epimerase in vivo. During the in vitro studies reported in this article, it is possible that a key protein or regulatory molecule that is required for NeuC to complete the reaction was lacking. Therefore, we simply might have been seeing the products of a “crippled” enzyme that was unable to complete its normal reaction under the specific conditions of the assay.
While the previous information argues strongly for the assignment of NeuC as a UDP-GlcNAc 2-epimerase, it is conceivable that NeuC actually catalyzes a different reaction in vivo. One possibility is that the true substrate is UDP-ManNAc and that the role of NeuC simply is to catalyze hydrolysis of the glycosyl-UDP bond. This possibility seems unlikely, however, since 2-acetamidoglucal and UDP would be the expected intermediates in this process and the former should be converted readily to ManNAc. An alternate possibility is that NeuC is actually a glycosyl transferase that utilizes UDP-GlcNAc as a substrate. This possibility is not unreasonable, since the UDP-GlcNAc 2-epimerases share structural similarities with a family of glycosyltransferases (26
). In the absence of an acceptor molecule, the glycal could be formed as an unnatural product. This possibility also seems unlikely, since one would not expect to observe the formation of any ManNAc in this process, and it does not help to explain the role of NeuC in sialic acid biosynthesis.
Surface-displayed sialic acid is an important virulence determinant in a number of bacterial pathogens besides E. coli K1. These include the N. meningitidis polysialic acid capsule, terminal sialic acid residues on the S. agalactiae capsule, and the sialylated flagella of C. coli. The synthesis of sialic acid differs in prokaryotes and eukaryotes.
GBS synthesize a branched-chain polysaccharide capsule, and the only similarity with the E. coli
K1 capsule is a single terminal sialic acid residue. The NeuIII
C gene of GBS, however, complements ΔneuC
. It has also been shown that the NeuIII
A gene (previously designated cpsF
) of GBS can complement a mutation in the E. coli
gene, which encodes the CMP-N
-acetylneuraminic acid synthetase (13
). Indeed, a plasmid containing the four GBS genes involved in sialic acid synthesis, pDC128, successfully complemented strains with mutations in neuD
, or neuA
, the E. coli
K1 region 2 sialic acid synthesis genes (D. Daines, unpublished data). This result indicates that the sialic acid biosynthetic pathways for incorporation into capsular polysaccharides are probably identical in these two pathogens.
The epimerases encoded by rffE
catalyze the conversion of UDP-GlcNAc to UDP-ManNAc. That rfbC
does not complement the neuC
deletion is not surprising, since NeuC cleaves UDP-GlcNAc during catalysis. During the preparation of this article, Ringenberg et al. reported (25
) that rffE
is not necessary for polysialic acid synthesis in E. coli
K1. These authors also reported that ManNAc-6-phosphate is not involved as an intermediate in the formation of sialic acid. The observations of these authors support our suggestion that NeuC converts UDP-GlcNAc to ManNAc and fit well with the observed homology to the epimerase domain of the mammalian UDP-GlcNAc 2-epimerase.