(a) SDS-PAGE of purified DesII isolated from E. coli BL21 Star (DE3): lane 1, the molecular mass standards: MBP-β-galactosidase (175 kDa), MBP-paramyosin (83 kDa), glutamic dehydrogenase (62 kDa), aldolase (47.5 kDa), triosephosphate isomerase (32.5 kDa), β-lactoglobulin A (25 kDa), lysozyme (16.5 kDa), and aprotinin (6.5 kDa), lanes 2–4, fractions of DesII purified using a Ni-NTA column followed by FPLC on a MonoQ HR (16/10) column. (b) UV-vis absorption spectrum of aerobically purified DesII in 50 mM Tris HCl buffer, pH 7.5. The protein concentration is 0.35 mM.

(a) EPR spectrum of as-purified DesII containing a [3Fe-4S]¹⁺ cluster with a g value centered at 2.010. The protein concentration is 0.2 mM in 100 mM Tris HCl buffer, pH 8.0. The spectrum was recoded at 10 K with a modulation amplitude of 10 G, a microwave power of 1 mW, and a microwave frequency of 9.60 GHz. (b) EPR spectrum of reconstituted DesII containing a [4Fe-4S]¹⁺ cluster with g values at 2.01, 1.96, and 1.87 (g_av = 1.95). The protein concentration is 0.2 mM in 100 mM Tris HCl buffer, pH 8.0. The spectrum was recoded at 10 K with a modulation amplitude of 10 G, a microwave power of 10 mW, and a microwave frequency of 9.60 GHz.

Plot of v_o versus [S] (8) determined by an HPLC assay from which the steady state kinetic constants for the DesII-catalyzed reaction were determined. See Experimental Procedures for details.

TDP-D-quinovose (14) and TDP-D-fucose (15) were prepared following a literature procedure26 (Scheme 2). First, TDP-4-keto-6-deoxy-D-glucose (7) was prepared by incubating TDP-D-glucose (6, 46.2 mg, 8.2 mM) and RfbB (28 μM) in 10 mL of 50 mM potassium phosphate buffer, pH 7.5, at 37 oC for 1 h. The enzyme was removed by filtration through an Amicon ultrafiltration unit (YM10 membrane). To the filtrate was added NaBH₄ (10 μM) and the reaction was allowed to proceed at room temperature for 1 h. The reaction mixture was desalted on a Sephadex G-10 column equilibrated with water. After lyophilization, the reaction mixture was analyzed by HPLC using a Dionex CarboPac PA1 column (4 × 250 mm). The flow rate was 1.0 mL/min and the detector was set at 267 nm. A linear gradient from 2.5% to 10% 1 M ammonium acetate, pH 7.0, versus ddH₂O over 20 min, followed by a second linear gradient from 10% to 30% 1 M ammonium acetate, pH 7.0, versus ddH₂O over 20 min gave satisfactory separation between TDP-D- quinovose (14) and TDP-D-fucose (15) (the ratio of 14 : 15 = 3:1). The retention times are 18.9 min and 13.5 min for TDP-D-quinovose (14) and TDP-D-fucose (15), respectively. Both compounds were collected, lyophilized to dryness, and confirmed by NMR spectroscopy.

DesII, which also contains the CXXXCXXC iron-sulfur cluster binding motif, has been identified as a member of the radical SAM enzyme superfamily.5b Previous studies have established the dependence on a [4Fe-4S] cluster and SAM for DesII activity.3 In this paper, we report a full account of the characterization of the [4Fe-4S] cluster in DesII, a steady-state kinetic analysis of the DesII-catalyzed reaction, an investigation of the substrate flexibility of DesII, and the demonstration of a biological reducing system, flavodoxin, flavodoxin reductase, and NADPH, capable of reducing the [4Fe-4S]²⁺ cluster. Moreover, studies of deuterium incorporation into SAM using TDP-[3-²H]-4-amino-4,6-dideoxy-D-glucose (18, see Scheme 4) provide insight into the mechanism of this intriguing deamination reaction.

Thus far, more than 2800 proteins have been identified, mainly based on sequence alignment, as members of the radical SAM enzyme superfamily.5 These enzymes catalyze a variety of reactions, such as isomerization, protein radical formation, sulfur insertion, ring formation, anaerobic oxidation, and unusual methylations, and are involved in the biosynthesis of DNA precursors, cofactors, vitamins, many types of secondary metabolites, and various biodegradation pathways.6 Enzymes of this superfamily possess a characteristic conserved sequence motif, CXXXCXXC, which coordinates a [4Fe-4S] cluster.7 Four different oxidation states of the iron-sulfur cluster have been observed. The intact iron-sulfur cluster exists mainly as [4Fe-4S]²⁺ in the purified enzyme, but [4Fe-4S]³⁺ is found as a minor component and readily converts to [3Fe-4S]¹⁺ via air oxidation. Upon treatment with dithionite in the presence of SAM, the iron-sulfur cluster is reduced to [4Fe-4S]¹⁺, which is the catalytically active form of all radical SAM enzymes.8 Catalysis by this class of enzymes is always initiated by one electron transfer from the [4Fe-4S]¹⁺ cluster to SAM. This induces the homolytic cleavage of the C_5′–S bond of SAM to generate methionine and a 5′-deoxyadenosyl radical (22, see Schemes 5 and 6).6 The subsequent abstraction of a hydrogen atom from the substrate by the reactive 5′-deoxyadenosyl radical triggers the chemical transformations during turnover.6,9

UV-vis absorption spectra of apo-DesII (solid line), reconstituted DesII before (dotted line) and after (dashed line) reduction with 1.14 mM sodium dithionite. The protein concentration is 0.19 mM in 100 mM Tris HCl buffer, pH 8.0.

(a) HPLC traces demonstrating DesII activity using dithionite as the reducing agent. (b) HPLC traces demonstrating DesII substrate specificity using TDP-D-quinovose (14) and TDP-D-fucose (15) in the incubation. (c) HPLC traces demonstrating DesII substrate specificity using TylB product (17) in the incubation. See Experimental Procedures for details.

D-Desosamine (1) is a 3-(N,N-dimethylamino)-3,4,6-trideoxyhexose found in a number of macrolide antibiotics including methymycin (2), neomethymycin (3), narbomycin (4), and pikromycin (5) produced by Streptomyces venezuelae. Both biochemical and structural studies have shown that desosamine is an essential structural component crucial to the biological activity of the parent aglycones (e.g., 12, 13).1 Desosamine is biosynthetically derived from TDP-D-glucose (6), and a key step in its formation is the removal of the C-4 hydroxyl group of the hexose ring. Early studies of desosamine biosynthesis in S. venezuelae revealed that the C-4 deoxygenation is unique among biological deoxygenation processes2 and the reaction proceeds in two stages: the conversion of TDP-4-keto-6-deoxy-D-glucose (7) to the corresponding 4-amino sugar intermediate (8), and the deamination of 8 to afford TDP-3-keto-4,6-dideoxy-D-glucose as the final product (9, Scheme 1). The former reaction is catalyzed by DesI, a pyridoxal-5′-phosphate (PLP)-dependent aminotransferase, whereas the latter reaction is catalyzed by DesII, a radical S-adenosyl-L-methionine (SAM)-dependent enzyme.3 Subsequent C-3 aminotransfer (9→ 10) by DesV followed by N,N-dimethylation (10→ 11) by DesVI complete the desosamine biosynthesis.4 All enzymes in the pathway have been biochemically characterized, but the details of the catalytic properties of DesII and the mechanism of its catalysis remain obscure.

TDP-3-amino-3,6-dideoxy-D-glucose (17), which is the product of the TylB reaction,27 was prepared according to a published procedure28 (Scheme 3). The assay mixture contained 0.5 mM of 17, 50 μM dithionite-reduced DesII, and 50 μM SAM in 1 mL of 100 mM Tris HCl buffer (pH 8 with 2 mM DTT). The reaction was incubated at 25 °C for 3 h, and quenched by removing the enzyme (YM10 membrane, Amicon). The reaction was subjected to HPLC analysis as described above using a CarboPac PA1 column and a two-stage linear gradient: first from 2.5% to 10% of 1 M ammonium acetate, pH 7.0, versus ddH₂O over 20 min, followed by a second gradient from 10% to 30% 1 M ammonium acetate, pH 7.0, versus ddH₂O over 20 min (1 mL/min). The retention times are 18.1 min and 37.2 min for 17 and the resulting product, respectively. The product was verified via HPLC coinjection with the DesII products from both TDP-4-amino-4,6-dideoxy-D-glucose (8) and TDP-D-quinovose (14).

Thus far, more than 2800 proteins have been identified, mainly based on sequence alignment, as members of the radical SAM enzyme superfamily.5 These enzymes catalyze a variety of reactions, such as isomerization, protein radical formation, sulfur insertion, ring formation, anaerobic oxidation, and unusual methylations, and are involved in the biosynthesis of DNA precursors, cofactors, vitamins, many types of secondary metabolites, and various biodegradation pathways.6 Enzymes of this superfamily possess a characteristic conserved sequence motif, CXXXCXXC, which coordinates a [4Fe-4S] cluster.7 Four different oxidation states of the iron-sulfur cluster have been observed. The intact iron-sulfur cluster exists mainly as [4Fe-4S]²⁺ in the purified enzyme, but [4Fe-4S]³⁺ is found as a minor component and readily converts to [3Fe-4S]¹⁺ via air oxidation. Upon treatment with dithionite in the presence of SAM, the iron-sulfur cluster is reduced to [4Fe-4S]¹⁺, which is the catalytically active form of all radical SAM enzymes.8 Catalysis by this class of enzymes is always initiated by one electron transfer from the [4Fe-4S]¹⁺ cluster to SAM. This induces the homolytic cleavage of the C_5′–S bond of SAM to generate methionine and a 5′-deoxyadenosyl radical (22, see Schemes 5 and 6).6 The subsequent abstraction of a hydrogen atom from the substrate by the reactive 5′-deoxyadenosyl radical triggers the chemical transformations during turnover.6,9

However, these results do not rule out stoichiometric turnover of SAM suggested by the observation of the formation of [5′-²H]-5′-deoxyadenosine when 18 was used in the reaction. Under this scenario, the consumption of one molecule of SAM should be accompanied by the production of one molecule of [5′-²H]-5′-deoxyadenosine after each catalytic cycle. The formation of [5′-²H₂]SAM in this case may be ascribed to the reversible formation of the substrate radical (23) via direct H-atom interchange with 5′-deoxyadenosine (27). As depicted in Scheme 7, H-atom return from [5′-²H]-27 to the substrate radical (23) could eventually lead to a Michaelis complex with [5′-²H₂]SAM, which will then be released.