Kinetics of the Reductive Half-Reaction of the Iron−Sulfur Flavoenzyme CDP-6-deoxy-l-threo-d-glycero-4-hexulose-3-dehydrase Reductase

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Abstract
The conversion of CDP-4-keto-6-deoxy-d-glucose to CDP-4-keto-3,6-dideoxy-d-glucose is a key step in biosynthesis of ascarylose, the terminal dideoxyhexose of the O-antigen tetrasaccharide of the lipopolysaccharide from Yersinia pseudotuberculosis V. This transformation is catalyzed by two enzymes: CDP-6-deoxy-l-threo-d-glycero-4-hexulose-3-dehydrase (E1), which contains a pyridoxamine and a [2Fe-2S] center, and an NADH-dependent CDP-6-deoxy-l-threo-d-glycero-4-hexulose-3-dehydrase reductase (E3), which contains both an FAD and a [2Fe-2S] center. E1 reacts to form a Schiff base with CDP-4-keto-6-deoxy-d-glucose and catalyzes the elimination of the hydroxyl at position 3 of the glucose moiety, resulting in the formation of a covalently bound CDP-6-deoxy-Δ3,4-glucoseen intermediate. E3 transfers electrons from NADH to E1, which uses these to reduce the Δ3,4-glucoseen bond to produce CDP-4-keto-3,6-dideoxy-d-glucose. In this work, we have investigated the reductive half-reaction of E3 using both single wavelength and diode array stopped flow absorbance spectroscopy. We find that NADH binds to both oxidized (Kd = 52.5 ± 2 μM) and two-electron-reduced (Kd = 12.1 ± 1 μM) forms of E3. Hydride transfer from NADH to the FAD moiety occurs at 107.5 ± 3 s-1 and exhibits a 10-fold deuterium isotope effect when (4R)-[2H]NADH is substituted for NADH. Following the hydride transfer reaction, NAD+ is released at 42.5 ± 1 s-1 and electron transfer from the reduced FAD to the [2Fe-2S] center occurs rapidly. The extent of the intramolecular electron transfer reaction is pH-dependent with a pKa of 7.3 ± 0.1, which may represent the ionization state of the N-1 position of the FAD hydroquinone of E3. Finally, E3 is converted to the three-electron-reduced state in a slow disproportionation reaction that consumes NADH. The [2Fe-2S] center of E3 was selectively disassembled by titration with mersalyl to give E3(apoFeS). The properties of this form of the enzyme are compared to those of the holoenzyme. Similarities and differences of the reductive half-reactions of E3 and related iron−sulfur flavoenzymes are discussed.