Regiospecific O-Methylation of Naphthoic Acids Catalyzed by NcsB1, an O-Methyltransferase Involved in the Biosynthesis of the Enediyne Antitumor Antibiotic Neocarzinostatin

Abstract

Neocarzinostatin, a clinical anticancer drug, is the archetypal member of the chromoprotein family of enediyne antitumor antibiotics that are composed of a nonprotein chromophore and an apoprotein. The neocarzinostatin chromophore consists of a nine-membered enediyne core, a deoxyaminosugar, and a naphthoic acid moiety. We have previously cloned and sequenced the neocarzinostatin biosynthetic gene cluster and proposed that the biosynthesis of the naphthoic acid moiety and its incorporation into the neocarzinostatin chromophore are catalyzed by five enzymes NcsB, NcsB1, NcsB2, NcsB3, and NcsB4. Here we report the biochemical characterization of NcsB1, unveiling that: (i) NcsB1 is an S-adenosyl-L-methionine-dependent O-methyltransferase; (ii) NcsB1 catalyzes regiospecific methylation at the 7-hydroxy group of its native substrate, 2,7-dihydroxy-5-methyl-1-naphthoic acid; (iii) NcsB1 also recognizes other dihydroxynaphthoic acids as substrates and catalyzes regiospecific O-methylation; and (iv) the carboxylate and its ortho-hydroxy groups of the substrate appear to be crucial for NcsB1 substrate recognition and binding, and O-methylation takes place only at the free hydroxy group of these dihydroxynaphthoic acids. These findings establish that NcsB1 catalyzes the third step in the biosynthesis of the naphthoic acid moiety of the neocarzinostatin chromophore and further support the early proposal for the biosynthesis of the naphthoic acid and its incorporation into the neocarzinostatin chromophore with free naphthoic acids serving as intermediates. NcsB1 represents another opportunity that can now be exploited to produce novel neocarzinostatin analogs by engineering neocarzinostatin biosynthesis or applying directed biosynthesis strategies. Neocarzinostatin (NCS),3 a clinical anticancer drug used to treat leukemia and cancers of the bladder, stomach, pancreas, liver, and brain, is an archetypal member of the chromoprotein family of enediyene antitumor antibiotics that are composed of a nonprotein chromophore and an apoprotein (1-3). NCS was originally isolated from Streptomyces carzinostaticus in 1965 (1-3). Its apoprotein NcsA is encoded by the ncsA gene and protects, carries, and delivers the reactive NCS chromophore (4). The NCS chromophore (1, Fig. 1B) is composed of a ninemembered enediyne core, a deoxyaminosugar, and a naphthoic acid moiety (2, 5). As a member of the enediyne family, the biological activity of NCS is derived from its ability to cleave DNA (6). The NCS chromophore undergoes Myers-Saito cycloaromatization to form a 2,6-indacene diradical species that subsequently abstracts hydrogen atoms from the deoxyribose of DNA, leading to single-and double-stranded DNA breaks (2, 7). For NCS, a thiol is often required to trigger the cycloaromatization, although a few enediynes have been reported to form diradicals via the cycloaromatization in a thiol-independent manner, likely the result of the intrinsic reactivity of the nine-membered ring enediyne core (1, 2, 7). The mechanism of action for NCS also invo...

Cloning and sequencing of the kedarcidin biosynthetic gene cluster from Streptoalloteichus sp. ATCC 53650 revealing new insights into biosynthesis of the enediyne family of antitumor antibiotics

“…Similar aromatic polyketide moieties have been found in other enediyne natural products, as exemplified by the benzoic acid moiety in MDP and the 1-naphthoic acid moiety in NCS, and the biosynthesis of both moieties are catalyzed by the iterative type I PKSs, MdpB 26,52 and NcsB, 25,49–51 respectively (Fig. S7).…”

“…S7). 25,26,49–51 These findings lend additional support to the intermediacy of 3,6,8-trihydroxy-2-naphthoic acid in KED biosynthesis. Thus, as depicted in Fig.…”

Molecular Basis of Substrate Promiscuity for the SAM-Dependent O-Methyltransferase NcsB1, Involved in the Biosynthesis of the Enediyne Antitumor Antibiotic Neocarzinostatin

“…The LDXGXGXG motif indicative of SAM-utilizing proteins is found in NcsB1 as VDVGGGSG and is located between β3 and α14. We previously reported that NcsB1 alkylates the substrate 3 with a variety of SAM analogs, including S -ethyl and S -n-propyl, to the corresponding 7-alkyl ether with reasonable efficiency (21). The nearest side chain to the methyl-group on SAM is the sterically small Ala243 (3.5 Å distance).…”

“…NcsB1 also was demonstrated to methylate 1,4-dihydroxy-2-naphthoic acid (see Figure 5, 6) at the 4-hydroxyl group, which was surprising because of the large difference in the location of the reactive hydroxyl group from that on the natural substrate, necessitating a reorientation of the substrate in the active site. This observation led to the prediction of a model by which naphthoic acids flexibly bind the active site, and that the 1,2-hydroxy acid functionality was required for substrate binding and SAM-dependant methylation (21). …”

The Crystal Structure of the Novobiocin Biosynthetic Enzyme NovP: The First Representative Structure for the TylF O-Methyltransferase Superfamily

“…Particular interest surrounds the mammalian O -methyltransferases (OMTs) as they are the targets of therapeutic agents designed to treat Parkinson's disease 2. More recently, OMTs have attracted considerable interest on account of their roles in decorating antibiotics3; 4;5 and other therapeutic agents6 and their potential application for creating chemical diversity in a range of compound classes.…”