Characterization and mechanistic studies of DesII: a radical S-adenosyl-L-methionine enzyme involved in the biosynthesis of TDP-D-desosamine.
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.
Szu
Ping-Hui
PH
Division of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry and Biochemistry, University of Texas at Austin, Austin, Texas 78712, USA.
Ruszczycky
Mark W
MW
Choi
Sei-hyun
SH
Yan
Feng
F
Liu
Hung-wen
HW
D005418:Flavodoxin / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D053858:Metabolic Networks and Pathways; D009247:NADH, NADPH Oxidoreductases / Q000378:metabolism; D009702:Nucleoside Diphosphate Sugars / Q000096:biosynthesis*; D010088:Oxidoreductases / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism*
D005418:Flavodoxin; D007506:Iron-Sulfur Proteins; D009702:Nucleoside Diphosphate Sugars; C513911:TDP-D-desosamine; D012436:S-Adenosylmethionine; D010088:Oxidoreductases; D009247:NADH, NADPH Oxidoreductases; C116041:flavodoxin NADPH oxidoreductase