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The structure and mechanism of UDP-glucose dehydrogenase Campbell, Robert E.

Abstract

UDP-glucose dehydrogenase (UDPGlcDH) catalyses the two-fold oxidation of UDPglucose to UDP-glucuronic acid with sequential hydride transfers to two equivalents of NAD ⁺. Bacterial 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 carried out a variety of biochemical and X-ray crystallographic studies of UDPGlcDH and have proposed the first detailed and consistent mechanism for this enzyme. UDPGlcDH operates by a Bi Uni Uni Bi ping pong kinetic mechanism in which UDPglucose is bound first by the enzyme and UDP-glucuronic acid is released last. In the first step of the enzyme mechanism, the pro-R hydride of UDP-glucose is transferred to NAD ⁺ to produce the aldehyde intermediate and NADH. The oxidation of the aldehyde intermediate to the carboxylic acid product is initiated by nucleophilic attack of the thiol of Cys 260 to yield a thiohemiacetal. Collapse of the thiohemiacetal intermediate with a second hydride transfer to NAD ⁺ produces a thioester intermediate that is hydrolyzed in the final step of the mechanism. The putative aldehyde intermediate has been chemically synthesized and demonstrated to be kinetically competent to serve as an intermediate in the enzymatic reaction. Evidence for transfer of the pro-R hydride occurring in the first oxidation comes from the observation that the UDPglucose analogue, UDP-6S-6C-methylglucose, is oxidized by UDPGlcDH to the corresponding ketone product but the 6R epimer is not. The involvement of a nucleophilic cysteine thiol is supported by covalent labeling studies with the affinity label, UDP-chloroacetol phosphate. The X-ray structures of wild-type UDPGlcDH and Cys260Ser UDPGlcDH have been determined 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 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.

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