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 the 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 co-crystallizing the enzyme with various combinations of the cofactor and substrate analogs, details of the active site structure have been established. Changes in subdomain orientation were observed by comparing structures in the presence and absence of substrate, suggesting that reorientation of the enzyme is involved in binding the substrate. In addition, residues important for substrate discrimination were predicted and probed through site directed mutagenesis and in vitro biochemical characterization.

Characterization of the Calicheamicin Orsellinate C2‐O‐Methyltransferase CalO6

“…Importantly, compared to typical kinetic values reported for aromatic O -MTs, [10c, 7b] the CalO6 K M values reflected in this study are >10-fold higher while the corresponding k cat values are ~1000-fold lower, suggesting the SNAC conjugates to be rather poor mimics of the putative native (CoA- or ACP-) substrate conjugate. However, this analysis revealed a ~3-fold kinetic bias toward P1 formation, consistent with C2-OH methylation as the preferred CalO6 regiospecificity.…”

“…These multifunctional enzymes assemble structurally diverse mono- or bicyclic- aromatic polyketides ( Scheme 1 ) [1] from acyl Co-enzyme A (CoA) precursors where subsequent modification/utilization of the aromatic core has been proposed or demonstrated to be dependent upon a dedicated acyltransferase (AT) (chlorothricin, [2] pactamycin, [3] avilamycin, [4] tiacumicin B, [5] and calicheamicin [6] ) or via adenylated intermediates (maduropeptin, [7] polyketomycin, [8] azinomycin B, [9] and neocarzinostatin, [10] ). A primary point of structural divergence of iPKS-derived aromatic polyketides derives from downstream ‘tailoring’ modifications (hydroxylation, epoxidation, acylation, glycosylation, transamination, halogenation and/or methylation) of the resulting polyketide scaffold.…”

“…Published work to date suggests the fundamental timing and/or biochemical basis of these subsequent modifications to also be widely variant and poorly understood. For example, the napthoic acid O -methyltransferase ( O -MT) NcsB1 in neocarzinostatin biosynthesis operates on the free napthoic acid moiety prior to adenylation [10c] while MdpB1-catalyzed C -methylation during the biosynthesis of maduropeptin utilizes a CoA-tethered 6-methylsalicylic acid substrate. [7b] In contrast, TiaM-catalyzed halogenation occurs as the last step in tiacumicin B maturation, after the iPKS-derived aromatic component has been attached to nearly mature natural product.…”

A Point Mutation inftmDBlocks the Fumitremorgin Biosynthetic Pathway inAspergillus fumigatusStrain Af293

“…These acidic residues are involved in the interaction with the ribosyl moiety of SAM. [19][20][21][22][23][24][25] The substitution presumably affects the substrate binding and the catalysis of FtmD. The genome of another strain of A. fumigatus, A1163, has also been sequenced.…”

Heterologous Expression and Manipulation of Three Tetracycline Biosynthetic Pathways

“…Indeed, the conserved histidine serving as general base in the catalytic pocket is substituted with Phe in SsfM3 (Figure S17). [30] …”