No abstract

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), pikromycin (4), and narbomycin (5) produced by Streptomyces venezuelae. It plays an essential role in conferring biological activities to its parent aglycones. Previous genetic and biochemical studies of the biosynthesis of desosamine in S. venezuelae showed that the conversion of TDP-4-amino-4,6-dideoxy-D-glucose (8) to TDP-3-keto-4,6-dideoxy-D-glucose (9) is catalyzed by DesII, which is a member of the radical S-adenosyl-L-methionine (SAM) enzyme superfamily. Here, we report the purification and reconstitution of His 6 -tagged DesII, characterization of its [4Fe-4S] cluster using UV-Vis and EPR spectroscopies, and the capability of flavodoxin, flavodoxin reductase, and NADPH, to reduce the [4Fe-4S] 2+ cluster. Also included are a steady-state kinetic analysis of DesII-catalyzed reaction and an investigation of the substrate flexibility of DesII. Studies of deuterium incorporation into SAM using TDP-[3-2 H]-4-amino-4,6-dideoxy-D-glucose as the substrate provides strong evidence for direct hydrogen atom transfer to a 5′-deoxyadenosyl radical in the catalytic cycle. The fact that hydrogen atom abstraction occurs at C-3 also sheds light on the mechanism of this intriguing deamination reaction. 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 processes 2 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.*To whom correspondence should be addressed. Fax: 512-471-2746. h.w.liu@mail.utexas NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author ManuscriptThus far, more than 2800 proteins have been identified, mainly b...

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 non-physiological 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.Radical SAM enzymes are an important class of biological catalysts involved in radical mediated transformations in both primary and secondary metabolic pathways.1 -3 All of these enzymes contain a [4Fe-4S] cluster in the active-site and are S-adenosyl-L-methionine (1, SAM) dependent. The reactions are initiated by a single electron transfer from the reduced [4Fe-4S] 1+ cluster to SAM (1 → 2) to induce the homolytic cleavage of the C5'-S bond in SAM, which yields a reactive 5'-deoxyadenosyl radical (4) along with L-methionine (3), as shown in Scheme 1. 2,4-6 Utilization of a 5'-deoxyadenosyl radical (4) by the radical SAM enzymes is similar to the B 12 -dependent enzymes. In both cases, the 5'-deoxyadenosyl radical is used to generate a substrate radical intermediate that is subsequently converted to product. 1,2 However, unlike the radical SAM enzymes, the 5'-deoxyadenosyl radical (4) in the B 12 -dependent enzymes is derived from an adenosylcobalamin cofactor. 7-9 Moreover, the B 12 -dependent enzymes are primarily involved in redox neutral isomerization and lyase reactions, whereas the reactions catalyzed by radical SAM enzymes may or may not lead to a net change in the redox state of the substrate as it is converted to product. Thus, the fact that SAM can act as both a radical initiator as well as an oxidant highlights a clear difference between those enzymes utilizing adenosylcobalamin versus radical SAM chemistry.

[4 + 2]-Cycloadditions are increasingly being recognized in the biosynthetic pathways of many structurally complex natural products. A relatively small collection of enzymes from these pathways have been demonstrated to increase rates of cyclization and impose stereochemical constraints on the reactions. While mechanistic investigation of these enzymes is just beginning, recent studies have provided new insights with implications for understanding their biosynthetic roles, mechanisms of catalysis and evolutionary origin.

Background: UDP-galactopyranose mutase (UGM) requires the reduced FAD coenzyme to interconvert UDP-galactopyranose and UDP-galactofuranose. Results: Structural perturbations of the coenzyme inhibit bond cleavage in the substrate. Conclusion: Concerted bond breaking and formation between substrate and coenzyme occur during UGM catalysis. Significance: Mechanistic understanding of UGM offers new insight for clinically relevant inhibitor design.