Molecular basis of substrate promiscuity for the SAM-dependent O-methyltransferase NcsB1, involved in the biosynthesis of the enediyne antitumor antibiotic neocarzinostatin

Abstract

The small molecule component of chromoprotein enediyne antitumor antibiotics is biosynthesized through a convergent route, incorporating amino acid, polyketide, and carbohydrate building blocks around a central enediyne hydrocarbon core. The naphthoic acid moiety of the enediyne neocarzinostatin plays key roles in the biological activity of the natural product by interacting with both the carrier protein and duplex DNA at the site of action. We have previously described the in vitro characterization of an S-adenosylmethionine-dependent O-methyltransferase (NcsB1) in the neocarzinostatin biosynthetic pathway [Luo, Y., Lin, S., Zhang, J., Cooke, H. A., Bruner, S. D., and Shen, B. (2008) J. Biol. Chem. 283, 14694-14702]. Here we provide a structural basis for NcsB1 activity, illustrating that the enzyme shares an overall architecture with a large family of S-adenosylmethionine-dependent proteins. In addition, NcsB1 represents the first enzyme to be structurally characterized in the biosynthetic pathway of neocarzinostatin. By cocrystallizing the enzyme with various combinations of the cofactor and substrate analogues, details of the active site structure have been established. Changes in subdomain orientation were observed via comparison of structures in the presence and absence of substrate, suggesting that reorientation of the enzyme is involved in binding of the substrate. In addition, residues important for substrate discrimination were predicted and probed through site-directed mutagenesis and in vitro biochemical characterization.

Figure 1

Biosynthesis of the naphthoic acid moiety and its attachment to the enediyne core, resulting in the natural product neocarzinostatin (1). The O-methyltransfer reaction catalyzed by NcsB1 is boxed (methyl group is highlighted in orange).

Figure 2

Cartoon representations of NcsB1. (A) Dimer of NcsB1 with the active site region indicated. Chain A is colored light blue and chain B yellow. Ligands are depicted in stick format with SAH in red and naphthoic acid 4 in cyan. B) Close up view of monomer with secondary structural elements and hinge regions labeled. C) Overlay of NcsB1/SAH/4 monomer (light blue) and NcsB1/SAH monomer (red).

Figure 3

Sequence alignment of NcsB1 structural homologs as determined by DALI structural alignment server. Abbreviations used with accession codes from the RCSB Protein Data Bank in parentheses: DnrK, S. peucetius O-MTase (Q06528); RdmB, S. purpurascens hydroxylase (Q54527); IOMT, alfalfa O-MTase (O24529); NPOMT, N. punctiforme putative O-MTase (ZP_00112478); PhzM, P. aeruginosa N-MTase (Q9HWH2); COMT, alfalfa O-MTase (P28002). Completely conserved residues are yellow, highly conserved residues are blue, and conserved residues are green. Catalytic residues are indicated by an asterisk and conserved glycine region is boxed.

Figure 4

Active site of NcsB1/SAM/2. (A) Substrate and SAM binding pocket. SAH is shown in green and naphthoic acid 2 in cyan; residues in the substrate and SAM binding pockets are shown in grey with pertinent hydrogen-bonds indicated. (B) Close up of active site in NcsB1/SAM/2 structure with 7-hydroxy group modeled onto 2, with the likely catalytic residues shown in grey. SAM is shown in green and the naphthoic acid 3 in orange. The methyl group on SAM is 2.0 Å from the hydroxyl group on 3.

Figure 5

Binding of alternate substrate, DHN (6). (A) Electron density map of NcsB1/SAH/6 active site, generated without 6. 2F_o – F_c map (blue, 1.5 σ) and F_o – F_c map (green, 3 σ) (B) Overlay of 2 and 6 in NcsB1 active site. Naphthoic acid 6 is hydrogen-bonded to Arg11 via a water and the 4-hydroxyl group is in sufficiently close proximity to SAM (2.1 Å, SAM from NcsB1/SAM/2 structure) for methyl transfer to occur.