9590 Biochemistry 2009, 48, 9590–9598 DOI: 10.1021/bi901257q Molecular Basis of Substrate Promiscuity for the SAM-Dependent O-Methyltransferase NcsB1, Involved in the Biosynthesis of the Enediyne Antitumor Antibiotic Neocarzinostatin† ^ Heather A. Cooke,§ Elizabeth L. Guenther,§ Yinggang Luo, ) Ben Shen, ,) and Steven D. Bruner*,§ §Department of Chemistry, Merkert Chemistry Center, Boston College, Chestnut Hill, Massachusetts 02467, Division) of Pharmaceutical Sciences and ^Department of Chemistry, University of Wisconsin, Madison, Wisconsin 53705 Received July 22, 2009; Revised Manuscript Received August 20, 2009 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-adenosyl- methionine-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. Enediyne antitumor antibiotics are structurally complex nat- may be a way to control or mediate the reactivity and specificity ural products possessing remarkable cytotoxicity (1). Members of NCS (1). of the nine-member subfamily of enediynes are composed of a The recent cloning and sequencing of the NCS biosynthetic small molecule chromophore and an apoprotein, which seques- pathway from Streptomyces carzinostaticus revealed a conver- ters and stabilizes the reactive enediyne and aids in delivery to gent path with two polyketide synthases playing central roles in target cells, leading to single- and double-stranded DNA clea- building the carbon framework (4, 12, 16). The scaffold for the vage (2). Biosynthetic gene clusters of multiple enediynes from naphthoate moiety in NCS is constructed by an iterative type I actinomycetes have recently been sequenced and annotated, polyketide synthase via a mechanism common to the biosynthesis allowing detailed investigation into their complex biosynthesis of diverse aromatic products (17-19). Four additional gene (3-8). The pathways utilize diverse enzymology to construct the products decorate and couple the naphthoic acid to the enediyne natural products, including the use of polyketide and nonribo- core (Figure 1). The product of NcsB (2) is first hydroxylated at somal peptide machinery. Neocarzinostatin (NCS,1 1)isan the C-7 position by a cytochrome P450 hydroxylase (NcsB3) to archetypal example of the nine-membered enediynes, the studies produce 3. The resulting hydroxyl group is methylated by NcsB1, of which have contributed to the general understanding of producing the methyl ether 4 present in the natural product. This enediyne biosynthesis and mode of action (4, 9-12). The functionalized naphthoic acid is activated via adenylation, con- naphthoate moiety of NCS functions by both binding to the verted to the aryl-CoA 5 by the CoA-ligase NcsB2, and finally apoprotein and intercalating target DNA, thereby positioning attached to the enediyne core by NcsB4 (a putative acyl- the enediyne in the minor groove (9, 10, 13-15). Alterations of transferase) to produce the natural product (1). The assignment the peripheral moieties of NCS surrounding the enediyne core of the naphthoic acid biosynthetic pathway is based on bioinfor- matic analysis (4) and biochemical characterization of NcsB2 as a CoA-ligase (20) and NcsB1 as an S-adenosylmethionine (SAM)- †This work was supported in part by funds from the Damon Runyon Cancer Research Fund (to S.D.B.) and National Institutes of Health dependent O-methyltransferase (O-MTase) (21). NcsB1 was Grants CA78747 and CA113297 (to B.S.). shown to catalyze the third step in the biosynthesis of the *To whom correspondence should be addressed. Telephone: (617) naphthoate moiety of the NCS chromophore (3 to 4). Bio- 552-2931. Fax: (617) 552-2705. E-mail: [email protected]. 1 Abbreviations: NCS, neocarzinostatin; SAH, S-adenosyl-L-homo- chemical analysis also revealed a relaxed substrate flexibility cysteine; SAM, S-adenosyl-L-methionine; CoA, coenzyme A; PPi, pyro- toward substituted naphthoic acids, methylating a variety of phosphate; MTase, methyltransferase; DHN, 1,4-dihydroxy-2-naph- 3-hydroxy-2-naphthoic acids with the reactive hydroxyl group thoic acid; rmsdCR, root-mean-square difference at R-carbons; PDB, Protein Data Bank; SDS-PAGE, sodium dodecyl sulfate-polyacry- located at either the 5 or 7 position on the ring. NcsB1 also lamide gel electrophoresis. was demonstrated to methylate 1,4-dihydroxy-2-naphthoic acid pubs.acs.org/Biochemistry Published on Web 08/25/2009 r 2009 American Chemical Society Article Biochemistry, Vol. 48, No. 40, 2009 9591 FIGURE 1: Biosynthesis of the naphthoic acid moiety and its attachment to the enediyne core, resulting in the natural product neocarzinostatin (1). The O-methyl transfer reaction catalyzed by NcsB1 is boxed (methyl group highlighted in orange). (see Figure 5, 6) at the 4-hydroxyl group, which was surprising including examples requiring a cation for catalysis. Cation- because of the large difference in the location of the reactive dependent O-MTases include CmcI from cephamycin biosynth- hydroxyl group from that on the natural substrate, necessitating esis (29), LiOMT from the pathogenic bacterium Leptospira a reorientation of the substrate in the active site. This observation interrogans (30), and BcOMT2 from Bacillus cereus (31). A led to the prediction of a model by which naphthoic acids flexibly limited number of cation-independent bacterial O-MTase struc- bind the active site and in which the 1,2-hydroxy acid function- tures have been structurally characterized, including DnrK and ality was required for substrate binding and SAM-dependent RebM from rebeccamycin biosynthesis (32). methylation (21). Here we report the structural basis for NcsB1 activity in a There are ∼160 classes of SAM-dependent MTases comprised variety of cocomplexes with SAM or S-adenosyl-L-homocysteine of thousands of identified members. These functionally diverse (SAH) with or without 5-methyl-2-hydroxynaphthoic acid (2), enzymes methylate a wide range of substrates, including nucleic product (4), or 1,4-dihydroxy-2-naphthoic acid (6), an alternate acids for regulation of gene expression, DNA repair or protection substrate. The structures reveal that NcsB1 shares an overall against restriction enzymes, proteins for repair or control of architecture common to the large MTase family. The active site signal transduction pathways, hormones and neurotransmitters, binding pocket is able to accommodate the natural substrate and and biosynthetic intermediates to produce secondary metabo- structurally diverse analogues, allowing efficient methylation lites. Structurally characterized MTases can be segregated into with distinct regiospecificity. The specificity determinants of five general classes on the basis of specific structural features (22). the naphthoate binding pocket were probed using site-directed Among the several examples of MTases that act on small mutagenesis and alternate substrates. On the basis of the results, molecules, catechol O-MTase isolated from rat liver was the first residues that affect the substrate specificity of the enzyme were to be structurally characterized (23). This structure exemplifies identified. the basic SAM-MTasecorefold:amixedR, β secondary structure with a β-sheet flanked on each side by three MATERIALS AND METHODS R-helices (24). A common feature of the active site of MTases is a glycine-rich region within the SAM binding pocket. Despite a Protein Expression and Purification. NcsB1 was over- low degree of overall sequence homology among family mem- produced as an N-terminal His6-tagged fusion protein using bers, structurally characterized SAM-MTase complexes share expression plasmid pBS5039 in Escherichia coli BL21(DE3) cells this common fold and the biologically active unit is a homodimer. as reported previously (21). Cells were grown in 1 L of Luria- The role of NcsB1 was assigned in the enediyne neocarzinos- Bertani medium at 37 °C and 150 rpm to an OD600 of 0.5-0.8. tatin biosynthetic pathway on the basis of sequence homology to Overexpression was induced with 50 μMisopropylβ-D-thioga- small molecule SAM-dependent O-MTases, including DnrK, an lactopyranoside at 18 °C for 16 h. Cells were collected by enzyme involved in the biosynthesis of