Molecular architecture of KedS8, a sugar N-methyltransferase from Streptoalloteichus sp. ATCC 53650

Abstract

Kedarcidin, produced by Streptoalloteichus sp. ATCC 53650, is a fascinating chromoprotein of 114 amino acid residues that displays both antibiotic and anticancer activity. The chromophore responsible for its chemotherapeutic activity is an ansa-bridged enediyne with two attached sugars, l-mycarose, and l-kedarosamine. The biosynthesis of l-kedarosamine, a highly unusual trideoxysugar, is beginning to be revealed through bioinformatics approaches. One of the enzymes putatively involved in the production of this carbohydrate is referred to as KedS8. It has been proposed that KedS8 is an N-methyltransferase that utilizes S-adenosylmethionine as the methyl donor and a dTDP-linked C-4' amino sugar as the substrate. Here we describe the three-dimensional architecture of KedS8 in complex with S-adenosylhomocysteine. The structure was solved to 2.0 Å resolution and refined to an overall R-factor of 17.1%. Unlike that observed for other sugar N-methyltransferases, KedS8 adopts a novel tetrameric quaternary structure due to the swapping of β-strands at the N-termini of its subunits. The structure presented here represents the first example of an N-methyltransferase that functions on C-4' rather than C-3' amino sugars.

Keywords: N-methyltransferase; S-adenosylmethionine; kedarcidin; l-kedarosamine; trideoxysugar.

Figure 1. Scheme

Reaction catalyzed by KedS8.

Figure 2. Figure

Analysis of the quaternary structure of KedS8 by gel filtration chromatography. Shown is retention time on the HPLC versus milliabsorbance units. KedS8 and TylM1 migrate as tetrameric and dimeric species, respectively (retention times of 14.5 and 16.8 min). Standards used for comparison: alcohol dehydrogenase (retention time 12.5 min, MW=150,000), albumin (retention time 16.3 min, MW=66,000), and carbonic anhydrase (retention time 17.9 min, MW=29,000).

Figure 3. Figure

Structure of KedS8. Shown in (a) is a stereo ribbon representation of the KedS8 tetramer. A view of a single subunit is presented in (b) with the β-strands and α-helices highlighted in purple and green, respectively. The bound ligand, SAH, is shown in a stick representation. This figure and figures 3 and 4 were prepared using PyMOL.

Figure 4. Figure

Close-up view of the SAH binding pocket. The electron density corresponding to the bound SAH cofactor is shown. The map, contoured at 3σ, was calculated with coefficients of the form F_o–F_c, where F_o was the native structure factor amplitude and F_c was the calculated structure factor amplitude. The SAH ligand was not included in the coordinate file for the map calculation. Ordered water molecules are represented as red spheres. The dashed lines indicate possible hydrogen bonding interactions.

Figure 5. Figure

Quaternary structure of KedS8. One of the major subunit:subunit interfaces of the tetramer is shown in (a). This organization is also observed for DesVI and TylM1, which function as dimers. A superposition of the ribbon drawings for DesVI (yellow), TylM1 (purple), and KedS8 (white) is presented in (b). The overall folds are remarkably similar except for the positions of the N-terminal tails. The N-terminal tail in KedS8 is responsible for the change from a dimeric to tetrameric quaternary structure as shown in (c).