The analysis presented allows the specificity of A domains of unknown function (e.g. from polymerase chain reaction amplification or genome sequencing) to be predicted. Furthermore, it provides a rational framework for altering of A domain specificity by site-directed mutagenesis, which has significant potential for engineering the biosynthesis of novel natural products.

With the escalation of obesity-related disease, there is great interest in defining the mechanisms that control appetite and body weight. We have identified a link between anabolic energy metabolism and appetite control. Both systemic and intracerebroventricular treatment of mice with fatty acid synthase (FAS) inhibitors (cerulenin and a synthetic compound C75) led to inhibition of feeding and dramatic weight loss. C75 inhibited expression of the prophagic signal neuropeptide Y in the hypothalamus and acted in a leptin-independent manner that appears to be mediated by malonyl-coenzyme A. Thus, FAS may represent an important link in feeding regulation and may be a potential therapeutic target.

PksA, which initiates biosynthesis of the environmental carcinogen aflatoxin B1, is one of the multidomain iterative polyketide synthases (IPKSs), a large, poorly understood family of biosynthetic enzymes. We found that dissection of PksA and its reconstitution from selected sets of domains allows the accumulation and characterization of advanced octaketide intermediates bound to the enzyme, permitting the reactions controlled by individual catalytic domains to be identified. A product template (PT) domain unites with the ketosynthase and thioesterase in this IPKS system to assemble precisely seven malonyl-derived building blocks to a hexanoyl starter unit and mediate a specific cyclization cascade. Because the PT domain is common among nonreducing IPKSs, these mechanistic features should prove to be general for IPKS-catalyzed production of aromatic polyketides.Tens of thousands of natural products are known from microorganisms, plants, and animals that provide hormones, toxins, flavors and fragrances, pigments, drugs, and other materials of commercial value. A handful of biosynthetic pathways give rise to this rich diversity of useful structures. Among these, polyketides are synthesized from simple acyl-coenzyme A (acylCoA) substrates by polyketide synthases (PKSs) (1). We understand a great deal about the function of giant modular PKSs that synthesize complex products, for example, the antibiotic erythromycin and the immunosup-pressant rapamycin (2). Each catalytic domain in these multidomain (type I) megaproteins is used once in an overall "assembly-line" process as a growing intermediate is advanced along the enzyme to yield a product. In contrast to these bacterial systems, in eukaryotes iterative PKSs (IPKSs) are generally the rule where a markedly smaller number of catalytic domains are similarly fused but individually reused in multiple catalytic cycles (iteration) that are "programmed" to yield specific products. How programming is achieved is a central unanswered question of iterative catalysis.We undertook a "deconstruction" approach by domain dissection and reassembly of PksA, the nonreducing IPKS of aflatoxin biosynthesis, to reveal the global division of labor among the domains in these macromolecular machines to control polyketide chain length, cyclization of an extended poly-β-keto intermediate, and product release. A domain hypothesized to be a "product template" (PT) has been discovered to play a central role in product formation. These studies are combined with high-resolution mass spectrometry (MS) to monitor the highly †To whom correspondence should be addressed.

Clavaminate synthase (CS) is one member of a large class of non-heme iron enzymes that require α-ketoglutarate (α-KG) as a cosubstrate. While the majority of this class catalyzes the hydroxylation of unactivated C−H bonds, CS is unusual in that in addition to performing hydroxylation chemistry, it also catalyzes the key oxidative ring closure and desaturation steps in the biosynthetic pathway to the potent β-lactamase inhibitor clavulanic acid. A single non-heme Fe2+ site is responsible for all three of these reactions (hydroxylation, oxidative ring closure, and desaturation), during which 1 equiv of α-KG per reaction is decarboxylated into succinate and CO2. We have applied circular dichroism (CD), magnetic circular dichroism (MCD), and variable-temperature, variable-field (VTVH) MCD spectroscopies to probe the geometric and electronic structure of the ferrous active site in the isozyme CS2 and its interaction with α-KG. CD titration experiments show stoichiometric binding of Fe2+ to the apoenzyme, either with or without α-KG, as well as stoichiometric binding of α-KG to the iron-containing enzyme. However, in the absence of the metal, the α-KG binding constant is reduced, indicating that Fe2+ facilitates cosubstrate binding at the active site. Ligand field CD and MCD data show that resting CS2 contains a six-coordinate ferrous center (10Dq = 10 050 cm-1, Δ5Eg = 1690 cm-1) and that addition of α-KG perturbs the site to produce a different six-coordinate center (10Dq = 9500 cm-1, Δ5Eg = 1630 cm-1). VTVH MCD analysis finds a ground-state splitting for resting CS2 (Δ5T2g ≈ −400 cm-1) that is fairly typical of six-coordinate ferrous sites, but a much larger splitting for CS2 + α-KG (Δ5T2g ≈ −1000 cm-1), indicative of Fe2+−α-KG π interactions. UV/vis absorption, CD, and MCD spectroscopies have been applied to further probe the interaction of the cosubstrate with the metalloenzyme. These data show the appearance of low-lying metal-to-ligand charge-transfer transitions which demonstrate that α-KG binds directly to the iron. Furthermore, analysis and comparison to model complex data support a bidentate binding mode of α-KG, indicating that cosubstrate displaces two ligands from the six-coordinate resting active site to form a new six-coordinate α-KG-bound Fe2+ site. These results provide the first direct spectroscopic information about the nature of the CS2 ferrous active site and its interaction with α-KG and lend insight into the mechanism of this multifunctional enzyme.