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

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-delta(3,4)-glucoseen intermediate. E3 transfers electrons from NADH to E1, which uses these to reduce the delta(3,4)-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 microM) and two-electron-reduced (Kd = 12.1 +/- 1 microM) 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.

Coenzyme B6 dependent novel bond cleavage reactions

“…On the basis of results gathered from stopped-flow spectrophotometry and freeze-quench EPR spectroscopy experiments, a mechanism of electron transfer in the E1-E3 coupled reaction can be proposed (Scheme 2) (12,14). First, NADH binds to E3 and forms a charge-transfer complex with the oxidized FAD (5, FADox).…”

“…Removal of the [2Fe-2S] center impairs the ability of E3 to catalyze final product formation in the presence of El and substrate. By stopped-flow spectroscopic experiments, the kinetic mechanism of this E3 mediated electron transfer was fully characterized (12). Interestingly, after the FAD is reduced to the hydroquinone form, the subsequent electron transfer to the [2Fe-2S] center was found to be pH-dependent.…”

“…Subsequently, electrons are shuttled one at a time from the hydroquinone through the [2Fe-2S] centers of both E3 and El to reduce the PMP-glucoseen intermediate bound in the El active-site as a Schiff base (7 to 10). Such a one-electron transfer process is expected to produce transient radicals, and two kinetically competent organic radicals formed during this process have been observed using both stopped-flow spectrophotometry and freeze-quench EPR spectroscopy (12,14). While the first radical is clearly the flavin semiquinone, the nature of the second radical is less wellcharacterized.…”

“…Overall, the proposed electron transport sequence is consistent with the role of E3 as a 2e-/le-switch, and the evidence provides compelling support for a radical mechanism in the C-3 deoxygenation of 1. In light of the fact that a PMP-glucoseen adduct (4) is the ultimate acceptor receiving electrons from E3, the catalytic role of E3 in the biosynthesis of 3d-dideoxy sugars clearly constitutes a novel example of biological deoxygenation.On the basis of results gathered from stopped-flow spectrophotometry and freeze-quench EPR spectroscopy experiments, a mechanism of electron transfer in the E1-E3 coupled reaction can be proposed (Scheme 2) (12,14). First, NADH binds to E3 and forms a charge-transfer complex with the oxidized FAD (5, FADox).…”

PLP and PMP Radicals: A New Paradigm in Coenzyme B6 Chemistry

“…In fact, two kinetically competent radicals formed during this reaction have been observed by using stopped-flow spectrophotometry and freeze-quench EPR spectroscopy (75,76). The first radical was clearly identified as the flavin semiquinone radical.…”