Stoichiometry of the redox neutral deamination and oxidative dehydrogenation reactions catalyzed by the radical SAM enzyme DesII

Abstract

DesII from Streptomyces venezuelae is a radical SAM (S-adenosyl-l-methionine) enzyme that catalyzes the deamination of TDP-4-amino-4,6-dideoxy-d-glucose to form TDP-3-keto-4,6-dideoxy-d-glucose in the biosynthesis of TDP-d-desosamine. DesII also catalyzes the dehydrogenation of the nonphysiological substrate TDP-D-quinovose to TDP-3-keto-6-deoxy-d-glucose. These properties prompted an investigation of how DesII handles SAM in the redox neutral deamination versus the oxidative dehydrogenation reactions. This work was facilitated by the development of an enzymatic synthesis of TDP-4-amino-4,6-dideoxy-d-glucose that couples a transamination equilibrium to the thermodynamically favorable oxidation of formate. In this study, DesII is found to consume SAM versus TDP-sugar with stoichiometries of 0.96 +/- 0.05 and 1.01 +/- 0.05 in the deamination and dehydrogenation reactions, respectively, using Na(2)S(2)O(4) as the reductant. Importantly, no significant change in stoichiometry is observed when the flavodoxin/flavodoxin NADP(+) oxidoreductase/NADPH reducing system is used in place of Na(2)S(2)O(4). Moreover, there is no evidence of an uncoupled or abortive process in the deamination reaction, as indicated by the observation that dehydrogenation can take place in the absence of an external source of reductant whereas deamination cannot. Mechanistic and biochemical implications of these results are discussed.

Figure 1

Representative HPLC chromatograms for stoichiometry experiments with Na₂S₂O₄ as the reductant. (A) Deamination of TDP-4-amino-4,6-dideoxy-d-glucose (15). (B) Dehydrogenation of TDP-d-quinovose (18). The bottom chromatogram in both (A) and (B) corresponds to the reaction before adding DesII. Subsequent traces are at increasing incubation times not exceeding 90 and 130 min, respectively. Peaks 14, 16 and 19 correspond to reaction products 5'-deoxyadenosine, TDP-3-keto-4,6-dideoxy-d-glucose and TDP-3-keto-6-deoxy-d-glucose, respectively. Peaks a, b and c are contaminants in the commercial SAM. Peak d is a contaminant in the TDP-d-quinovose (18) and corresponds to deoxythymidine monophosphate. Peaks a and 14 overlap, and the correction for f_SAM is described in the Results and further detailed in the Supporting Information.

Figure 2

Representative fits of the fraction of reaction in terms of SAM, f_SAM, versus TDP-4-amino-4,6-dideoxy-d-glucose (15) or TDP-d-quinovose (18), f_sub. Trend lines are fit under the assumption of equal variance in both f_SAM and f_sub. The stoichiometry is equal to the slope divided by the correction factor, γ, for the given experiment. (A) Substrate 15 with Na₂S₂O₄ (γ = 1.23). (B) Substrate 15 with FLD/FNR/NADPH (γ = 1.05). (C) Substrate 18 with Na₂S₂O₄ (γ = 1.16). (D) Substrate 18 with FLD/FNR/NADPH (γ = 2.04).

Figure 3

HPLC experiments demonstrating a requirement for a constant source of reducing equivalents in the deamination of TDP-4-amino-4,6-dideoxy-d-glucose (15) to TDP-3-keto-4,6-dideoxy-d-glucose (16) by DesII. (A) Incubation of reduced DesII with 15 at 5 and 85 min demonstrating less than 2% turnover at each time point. (B) Incubation of reduced DesII with 15 in the presence of fresh Na₂S₂O₄ at 5 and 85 min demonstrating 23% and 94% turnover in terms of 15, respectively. Peak 14 corresponds to 5'-deoxyadenosine generated as SAM is consumed. Peaks a and b correspond to contaminants in the commercial SAM. See text for details and Figure 4 for a comparison with the dehydrogenation reaction.

Figure 4

HPLC experiments demonstrating that a constant external source of reducing equivalents is not required for the dehydrogenation of TDP-d-quinovose (18) to TDP-3-keto-6-deoxy-d-glucose (19) by DesII. (A) Incubation of reduced DesII with 18 at 5 and 85 min demonstrating 26% and 55% reaction in terms of 18, respectively (SAM is substoichiometric versus 18 in this experiment). (B) Incubation of nonreduced DesII with 18 in the presence of deproteinized reduced DesII filtrate at 5 and 85 min demonstrating less than 1% reaction at each time point. Peak 14 corresponds to 5'-deoxyadenosine generated as SAM is consumed. Peaks a and b correspond to contaminants in the commercial SAM. See text for details and Figure 3 for a comparison with the deamination reaction.

Scheme 1

Scheme 2

Scheme 3

Scheme 4

Scheme 5

Scheme 6

Scheme 7