UDP-glucose dehydrogenase catalyzes the NAD + -depend-ent twofold oxidation of UDP-glucose to give UDP-glucu-ronic acid. A sequestered aldehyde intermediate is produced in the first oxidation step and a covalently bound thioester is produced in the second oxidation step. This work demon-strates that theStreptococcus pyogenesenzyme incorporates a single solvent-derived oxygen atom during catalysis and probably does not generate an imine intermediate.