Cyclase: Intramolecular Aldol Condensation in the Biosynthesis of Tetracenomycin C in Streptomyces gZaucescens7

Tetracenomycin (Tcm) F2 cyclase, which catalyzes the cyclization of the anthrone Tcm F2 to the naphthacenone Tcm F1 in the biosynthesis of the anthracycline antibiotic Tcm C in Streptomyces glaucescens, has been purified to homogeneity and characterized. The N-terminal sequence of the enzyme establishes that it is encoded by the tcml gene, whose deduced product has a molecular weight of 12 728. SDS-PAGE analysis gave a single band with a molecular weight of 12 500, whereas gel-filtration chromatography yielded a molecular weight of 37 500, indicating that the Tcm F2 cyclase is a homotrimer in solution. Under pH 1 8.0, the enzyme catalyzes the cyclization of Tcm F2 to Tcm F1 and has a Km of 121 * 18.2 p M and V, , , of 704 * 62.3 nmol.min-'-mg'. In contrast, under pH I 6.5, it catalyzes the cyclization of Tcm F2 to 9-decarboxy Tcm F1, a known shunt metabolite of the Tcm C biosynthetic pathway. Tcm F2 cyclase represents the first discrete enzyme for carbon-carbon bond formation via an intramolecular aldol condensation-dehydration mechanism, a key biochemical operation proposed in the early steps of the biosynthesis of all aromatic polyketides. Polyketides encompass natural compounds with diverse structures including macrolides, polyenes, polyethers, anthracyclines, and tetracyclines, yet apparently they share a common mechanism of biosynthesis: the carbon skeleton of a polyketide is synthesized by sequential condensation of the CoA esters of small fatty acids. This process is catalyzed by a polyketide synthase in the manner that is conceptually similar to the biosynthesis of long-chain fatty acids catalyzed by a fatty acid synthase (Hopwood & Sherman, 1990; Katz & Donadio, 1993). Unlike the latter substances, many polyketides are cyclic compounds whose biosynthesis often involves the formation of one or more six-membered rings. This process is mechanistically the result of one or more intramolecular aldol or Claisen condensations of the oligoketide intermediate, which presumably are catalyzed by the polyketide cyclase as shown in Figure 1. Little is known about the role that polyketide cyclases play in the cyclization of the oligoketides in vivo, although a number of synthetic oligoketides have been studied in vitro to examine their reactivity and regioselectivity for intramolecular aldol or Claisen condensations (Harris & Harris, 1986). Since different kinds of cyclized compounds such as aklaviketone in Streptomyces peucetius (Connors et al., 1990), dehydrorabelomycin in Streptomyces murayamaensis (Gould et al., 1992), and tetracenomycin (Tcm)' F2 in Streptomyces glaucescens (Shen et al., 1993; Yue et al., 1986) formally can be derived from the same class of oligoketide intermediate (Figure 1A-C), a polyketide cyclase could be an important determinant of the structural variation among such cyclized, This work was supported by National Institutes of Health Grant * Corresponding author: School of Pharmacy, University of Wisconsin, * School of Pharmacy. 5 Department of Bacteriology. Abstract published in Advonce ACSAbsrr~crs, September 15,1993. * Abbreviations: DTT, dithiothreitol; 6 , molar absorbance index; EDTA, ethylenediaminetetraacetic acid; FPLC, fast protein liquid chromatography; HPLC, high-performance liquid chromatography; SDSPAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; Tcm, tetracenomycin; TLC, thin-layer chromatography. CA3 538 1. 425 N . Charter St., Madison, WI 53706. 0006-2960/93/0432-11149%04.00/0 aromatic polyketides. However, elucidation of the mechanism of these enzymatic aldol or Claisen condensations is challenging because they have no precedents among the well-studied enzymes of primary metabolism. Molecular genetic analysis of several streptomycetes has recently led to the characterization of genes encoding a number of putative polyketide cyclases. For example, the act VZZgene for the production of actinorhodin in Streptomyces coelicolor (Figure 1D) (Sherman et al., 1991), thegra-rf4genefor the production of granaticin in Streptomyces violaceoruber (Shermanet al., 1991), the whiE-orjVZgenefor the production of a spore pigment in S. coelicolor (Davis & Chater, 1990) and the tcmZJN genes for the production of tetracenomycins in S. glaucescens (Figure 1A) (Summers et al., 1992; R. G. Summers, E. Wendt-Pienkowski, H. Motamedi, and C. R. Hutchinson, 1993, unpublished data) have been sequenced and analyzed. These studies have uncovered a highly conserved gene organization but, unfortunately, do not reveal any putative active sites by sequence comparisons; therefore, essentially nothing is known about the reaction mechanism of these enzymatic aldol or Claisen condensations other than the presumption that carbon-carbon bond formation is a separate event from the dehydration (Sherman et al., 1991). Tcm C, 1, is an antitumor antibiotic produced by S . glaucescens GLA.0 (Weber et al., 1976). We have previously established the biosynthetic pathway of 1 from acetate and malonate with all the biosynthetic intermediates identified (Shen et al., 1993; Yue et al., 1986) and cloned the gene cluster for the biosynthesis of 1 (Motamedi & Hutchinson, 1987). Sequence analysis of the biosynthetic genes (Bibb et al., 1989; Decker & Hutchinson, 1993; Guilfoile & Hutchinson, 1992; Summers et al., 1992; R. G. Summers, E. WendtPienkowski, H. Motamedi, and C. R. Hutchinson, 1993, unpublished data) has provided further support for the proposed biosynthetic pathway to 1 (Figure 1A). From this information, we believe that the naphthacenone Tcm F1, 2, is biosynthesized by an intramolecular aldol condensation catalyzed by a polyketide cyclase from the corresponding anthrone precursor Tcm F2,3. To provide some mechanistic