6. R.E. Campbell, S.C. Mosimann, M.E. Tanner*, and N.C.J. Strynadka*, “The Structure of UDP-N-Acetylglucosamine 2-Epimerase Reveals Homology to Phosphoglycosyl Transferases”, Biochemistry, 2000, 39: 14993-15001.

Bacterial UDP-N-acetylglucosamine 2-epimerase catalyzes the reversible epimerization at C-2 of UDP-N-acetylglucosamine (UDP-GlcNAc) and thereby provides bacteria with UDP-N-acetylmannosamine (UDP-ManNAc), the activated donor of ManNAc residues. ManNAc is critical for several processes in bacteria, including formation of the antiphagocytic capsular polysaccharide of pathogens such as Streptococcus pneumoniae types 19F and 19A. We have determined the X-ray structure (2.5 Å) of UDP-GlcNAc 2-epimerase with bound UDP and identified a previously unsuspected structural homology with the enzymes glycogen phosphorylase and T4 phage β-glucosyltransferase. The relationship to these phosphoglycosyl transferases is very intriguing in terms of possible similarities in the catalytic mechanisms. Specifically, this observation is consistent with the proposal that the UDP-GlcNAc 2-epimerase-catalyzed elimination and re-addition of UDP to the glycal intermediate may proceed through a transition state with significant oxocarbenium ion-like character. The homodimeric epimerase is composed of two similar α/β/α sandwich domains with the active site located in the deep cleft at the domain interface. Comparison of the multiple copies in the asymmetric unit has revealed that the epimerase can undergo a 10° interdomain rotation that is implicated in the regulatory mechanism. A structure-based sequence alignment has identified several basic residues in the active site that may be involved in the proton transfer at C-2 or stabilization of the proposed oxocarbenium ion-like transition state. This insight into the structure of the bacterial epimerase is applicable to the homologous N-terminal domain of the bifunctional mammalian UDP-GlcNAc “hydrolyzing” 2-epimerase/ManNAc kinase that catalyzes the rate-determining step in the sialic acid biosynthetic pathway.

5. R.E. Campbell, S.C. Mosimann, I. van de Rijn, M. E. Tanner, and N.C.J. Strynadka*, “The First Structure of UDP-Glucose Dehydrogenase Reveals the Catalytic Residues Necessary for the Two-fold Oxidation”, Biochemistry, 2000, 39: 7012-7023.

Bacterial UDP-glucose dehydrogenase (UDPGlcDH) is essential for formation of the antiphagocytic capsule that protects many virulent bacteria such as Streptococcus pyogenes and Streptococcus pneumoniae type 3 from the host's immune system. We have determined the X-ray structures of both native and Cys260Ser UDPGlcDH from S. pyogenes (74% similarity to S. pneumoniae) in ternary complexes with UDP-xylose/NAD+ and UDP-glucuronic acid/NAD(H), respectively. The 402 residue homodimeric UDPGlcDH is composed of an N-terminal NAD+ dinucleotide binding domain and a C-terminal UDP-sugar binding domain connected by a long (48 Å) central α-helix. The first 290 residues of UDPGlcDH share structural homology with 6-phosphogluconate dehydrogenase, including conservation of an active site lysine and asparagine that are implicated in the enzyme mechanism. Also proposed to participate in the catalytic mechanism are a threonine and a glutamate that hydrogen bond to a conserved active site water molecule suitably positioned for general acid/base catalysis.

4. R.E. Campbell and M.E. Tanner*, “UDP-Glucose Analogues as Inhibitors and Mechanistic Probes of UDP-Glucose Dehydrogenase”, J. Org. Chem., 1999, 64: 9487-9492.

UDP-glucose dehydrogenase catalyzes the NAD+-dependent 2-fold oxidation of UDP-glucose to give UDP-glucuronic acid. The putative aldehyde intermediate is not released from the active site and is presumably tightly bound. We have prepared UDP-7-deoxy-α-d-gluco-hept-6-ulopyranose, 5, that contains a methyl ketone at C-6 and cannot be further oxidized by the enzyme. Ketone 5 was found to be a competitive inhibitor of the dehydrogenase from Streptococcus pyogenes with a KI value of 6.7 μM. We have also prepared the secondary alcohols UDP-6S-6C-methylglucose, 4a, and UDP-6R-6C-methylglucose, 4b. Compound 4a, but not 4b, was found to be a slow substrate for the dehydrogenase and was converted into the ketone inhibitor 5. This is consistent with the notion that the pro-R hydride is transferred in the first oxidation step of the normal enzymatic reaction.

3. X. Ge, R. E. Campbell, I. van de Rijn, and M.E. Tanner*, “Covalent Adduct Formation with a Mutated Enzyme: Evidence for a Thioester Intermediate in the Reaction Catalyzed by UDP-Glucose Dehydrogenase”, J. Am. Chem. Soc., 1998, 120: 6613-6614.

The enzyme UDP-glucose dehydrogenase catalyzes the 2-fold oxidation of UDP-glucose to UDP-glucuronic acid in an NAD+-dependent process. In certain strains of pathogenic bacteria, such as group A streptococci or Streptococcus pneumoniae Type 3, UDP-glucuronic acid is used in the biosynthesis of a polysaccharide capsule that serves to protect the bacteria from the host's defense mechanisms. The capsule is known to be a necessary requirement for virulence since acapsular, mutant strains are rendered avirulent.

2. R.E. Campbell and M.E. Tanner*, “Uridine diphospho-alpha-D-gluco-hexodialdose: Synthesis and kinetic competence in the reaction catalyzed by UDP-glucose dehydrogenase”, Angew. Chem. Int. Ed. Eng. 1997, 36: 1520-1522.

The elusive aldehyde 1 has never been trapped nor detected during the enzymatic oxidation of UDP-glucose to UDP-glucuronic acid. Here the synthesis of 1 is reported; it proved to be kinetically competent to serve as an intermediate in the reaction catalyzed by UDP-glucose dehydrogenase.

1. R.E. Campbell, R.F. Sala, I. van de Rijn and M.E. Tanner*, “Properties and kinetic analysis of UDP-glucose dehydrogenase from group A streptococci. Irreversible inhibition by UDP-chloroacetol”, J. Biol. Chem., 1997, 272: 3416-22.

UDP-glucuronic acid is used by many pathogenic bacteria in the construction of an antiphagocytic capsule that is required for virulence. The enzyme UDP-glucose dehydrogenase catalyzes the NAD+-dependent 2-fold oxidation of UDP-glucose and provides a source of the acid. In the present study the recombinant dehydrogenase from group A streptococci has been purified and found to be active as a monomer. The enzyme contains no chromophoric cofactors, and its activity is unaffected by the presence of EDTA or carbonyl-trapping reagents. Initial velocity and product inhibition kinetic patterns are consistent with a bi-uni-uni-bi ping-pong mechanism in which UDP-glucose is bound first and UDP-glucuronate is released last. UDP-xylose was found to be a competitive inhibitor (Ki, 2.7 μM) of the enzyme. The enzyme is irreversibly inactivated by uridine 5′-diphosphate-chloroacetol due to the alkylation of an active site cysteine thiol. The apparent second order rate constant for the inhibition (ki/Ki) was found to be 2 × 103 mM−1 min−1. Incubation with the truncated compound, chloroacetol phosphate, resulted in no detectable inactivation when tested under comparable conditions. This supports the notion that uridine 5′-diphosphate-chloroacetol is bound in the place of UDP-glucose and is not simply acting as a nonspecific alkylating agent.