Characterization and Mechanistic Studies of DesII: A Radical S-Adenosyl-l-methionine Enzyme Involved in the Biosynthesis of TDP-d-Desosamine

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...

EPR-kinetic isotope effect study of the mechanism of radical-mediated dehydrogenation of an alcohol by the radical SAM enzyme DesII

“…Details regarding DesII expression, purification, and reconstitution have been previously reported (19,21). Initial rates were determined discontinuously using relative HPLC peak integrations.…”

“…In these cases (galactose oxidase, BtrN, and anSMEs), formation of a Lewis adduct between the hydroxyl to be oxidized and a Lewis acid (e.g., an active site metal ion) has been suggested. Although the observed DesII substrate radical does indeed appear to be protonated (23) and there is no reason to expect coordination of the C3 hydroxyl group to an active site metal ion (19), it nevertheless remains a possibility that the C3 hydroxyl group of 6 engages as a donor in a hydrogen-bonding interaction before hydrogen atom abstraction (e.g., 12 in Fig. 4).…”

“…Furthermore, direct coordination of the nascent substrate radical to the Cu II center or ½4Fe-4S 2+ aux cluster would permit rapid inner-sphere electron transfer to complete the dehydrogenation reaction. However, the results of iron-sulfur titration experiments (19), as well as the presence of only four cysteine residues (22) in the primary sequence of DesII, indicate that this enzyme possesses only a single [4Fe-4S] cluster. Thus, it is unlikely that the C3 hydroxyl of TDP-D-quinovose is coordinated to an active site metal ion.…”

“…The enzyme DesII from Streptomyces venezuelae is also a radical SAM enzyme and catalyzes the deamination of TDP-4-amino-4,6-dideoxy-D-glucose (4) to TDP-4,6-dideoxy-3-keto-D-glucose (5) in the biosynthetic pathway of TDP-D-desosamine (19,20) (Fig. 1A).…”

“…1A). DesII has also been shown to accept TDP-D-quinovose (6) as a substrate; however, rather than being dehydrated, the C3 hydroxyl group is oxidized to yield TDP-6-deoxy-3-keto-D-glucose (7) (19) (Fig. 1A).…”