Mechanistic Studies of the Radical S‐Adenosylmethionine Enzyme DesII with TDP‐D‐Fucose

Abstract

DesII is a radical SAM enzyme that catalyzes the C4-deamination of TDP-4-amino-4,6-dideoxyglucose via a C3 radical intermediate. However, if the C4 amino group is replaced with a hydroxyl (TDP-quinovose), the hydroxyl at C3 is oxidized to a ketone with no C4-dehydration. It is hypothesized that hyperconjugation between the C4 C-N/O bond and the partially filled porbital at C3 of the radical intermediate modulates the degree to which elimination competes with dehydrogenation. To investigate this hypothesis, the reaction of DesII with the C4-epimer of TDPquinovose (TDP-fucose) was examined. The majority of the reaction results in the formation of TDP-6-deoxygulose and likely regeneration of TDP-fucose. The remainder of the substrate radical partitions roughly equally between C3-dehydrogenation and C4-dehydration. Thus, changing the stereochemistry at C4 permits a more balanced competition between elimination and dehydrogenation. KeywordsEnzyme catalysis; Radical SAM; Alcohols; Biosynthesis The radical S-adenosylmethionine (SAM) enzyme DesII from Streptomyces venezuelae catalyzes the redox-neutral deamination of TDP-4-amino-4,6-dideoxy-D-glucose (1) to generate TDP-4,6-dideoxy-3-keto-D-glucose (2, see Scheme 1). 1,2 In its biological context, the deamination of 1 is the key reaction in the biosynthesis of TDP-desosamine (3), which is an essential component of many macrolide antibiotics. [2][3][4] This deamination reaction is radical-mediated and is initiated via hydrogen atom abstraction from the substrate by a 5′-deoxyadenosyl radical. The latter is derived from reductive homolysis of SAM by an active site [4Fe-4S] 1+ cluster and represents the hallmark of radical SAM enzymology. 5 Two general mechanisms have been proposed for the DesII-catalyzed deamination as shown in Figure 1. 1,4 In both cases, the p-orbital harboring the unpaired electron at C3 of the radical intermediate 6 must overlap productively with the C-N σ-system at C4 in order to ** This work was supported by grants from the National Institutes of Health (GM035906) and the Welch Foundation (F-1511). NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript facilitate either 1,2-migration (6→7→9/10→2) or direct elimination (6→8→9→2) of the adjacent amino group. In this regard, DesII is highly reminiscent of ethanolamine ammonia lyase (EAL), which catalyzes the deamination of ethanolamine albeit using a 5′-deoxyadenosyl radical produced from the homolysis of adenosylcobalamin rather than SAM. 6 Although the chemistry of DesII is also very similar to the dehydration of 1,2-diols by the B 12 -dependent dioldehydratases, 7 no elimination of the C2 hydroxyl from 1 is observed during the catalytic cycle of DesII.DesII can also accept TDP-D-quinovose (4) as a substrate where the C4 amino group of 1 is replaced with a hydroxyl group. However, DesII does not catalyze elimination of the C4 hydroxyl from 4 to produce 2, but rather oxidation of the C3 hydroxyl group to yield 5 (see Scheme 1). 1 This second, dehydrogenase activity of D...

C3′‐Deoxygenation of Paromamine Catalyzed by a Radical S‐Adenosylmethionine Enzyme: Characterization of the Enzyme AprD4 and Its Reductase Partner AprD3

“…When 10 μM AprD4 was incubated with 1 mM [4′- 2 H]-paromamine, 2 mM SAM, and 2 mM dithionite in 50 mM NH 4 HCO 3 (pH 7.8) for 8 hr at room temperature, formation of 5′-dAdo was again observed and this time with single-deuterium incorporation beyond natural abundance as determined by ESI MS (see Figure S5). A complimentary assay with unlabeled paromamine ( 11 ) and AprD4 using the dideuterated [5′- 2 H 2 ]-SAM isotopolog 40 in the incubation revealed hydrogen transfer from 11 to [5′- 2 H 2 ]-5′-dAdo• to yield [5′- 2 H 2 ]-5′-dAdo as the product (Figure S6). These observations further substantiate the proposed early steps of AprD4 catalysis shown Scheme 2, whereby AprD4 acts as a radical SAM enzyme that initiates a radical-mediated dehydration of paromamine via hydrogen atom abstraction from the C4′ position.…”

“…The dehydration reaction catalyzed by AprD4 bears a close resemblance to the deamination catalyzed of DesII, which is also a radical SAM enzyme catalyzing the conversion of TDP-4,6-dideoxy-4-amino-D-glucose to TDP-4,6-dideoxy-3-keto-D-glucose. 40,43–49 However, the latter is unable to catalyze dehydration upon direct substitution of the eliminated amine with a hydroxyl and instead acts as a dehydrogenase. 45 This makes the comparative study of the AprD4 and DesII of particular interest for understanding how enzymes are able to control the fate of their radical intermediates during turnover.…”

Mechanistic Investigation of the Radical S-Adenosyl-l-methionine Enzyme DesII Using Fluorinated Analogues

“…The epimerization likely results from reduction of the C3-radical intermediate ( 7 ) by a solvent-derived H-atom equivalent as has been previously reported in the case of TDP- d -fucose. 21 …”

“…However, these latter pathways are relatively minor in the case of DesII unless the substrate exhibits significant structural perturbations. 21 …”

C3′‐Deoxygenation of Paromamine Catalyzed by a Radical S‐Adenosylmethionine Enzyme: Characterization of the Enzyme AprD4 and Its Reductase Partner AprD3

“…When AprD4 (10 mm)w as incubated with [4'-2 H]-paromamine (1 mm), SAM (2 mm), and dithionite (2 mm)i nN H 4 HCO 3 (50 mm ;pH7.8) for 8hat room temperature,formation of 5'-dAdo was again detected and this time with single-deuterium incorporation beyond natural abundance as determined by ESIMS ( Figure S5). Ac omplimentary assay with unlabeled paromamine (11)and AprD4 using the dideuterated [5'-2 H 2 ]-SAM isotopologue [40] in the incubation revealed hydrogen transfer from 11 to [5'-2 H 2 ]-5'-dAdoC to yield [5'-2 H 2 ]-5'-dAdo as the product ( Figure S6). These results further substantiate the proposed early steps of AprD4 catalysis shown Scheme 2, whereby AprD4 acts as ar adical SAM enzyme that initiates ar adical-mediated dehydration of paromamine through hydrogen-atom abstraction from the C4' position.…”