The authors wish to note the following: ''We wish to add direct references to a stochastic model of DNA replication previously applied to the Xenopus laevis early embryonic divisions. That model was applied to molecular combing experiments on cellfree extracts from Xenopus laevis embryos.'' The additional references appear below. www.pnas.org/cgi

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Polyketides are a class of natural products with diverse structures and biological activities. The structural variability of aromatic products of fungal nonreducing, multidomain iterative polyketide synthases (NR-PKS group of IPKSs) results from regiospecific cyclizations of reactive poly-β-keto intermediates [1][2][3] . How poly-β-keto species are synthesized and stabilized, how their chain lengths are determined, and, in particular, how specific cyclization patterns are controlled have been largely inaccessible and functionally unknown until recently 4 . A product template (PT) domain is responsible for controlling specific aldol cyclization and aromatization of these mature polyketide precursors, but the mechanistic basis is unknown. Here we present the 1.8 Å crystal structure and mutational studies of a dissected PT monodomain from PksA, the NR-PKS that initiates the biosynthesis of the potent hepatocarcinogen aflatoxin B 1 in Aspergillus parasiticus. Despite having minimal sequence similarity to known enzymes, the structure displays a distinct 'double hot dog' (DHD) fold. Co-crystal structures with palmitate or a bicyclic substrate mimic illustrate that PT can bind both linear and bicyclic polyketides. Docking and mutagenesis studies reveal residues important for substrate binding and catalysis, and identify a phosphopantetheine localization channel and a deep two-part interior binding pocket and reaction chamber. Sequence similarity and extensive conservation of active site residues in PT domains suggest that the mechanistic insights gleaned from these studies will prove general for this class of IPKSs, and lay a foundation for defining the molecular rules controlling NR-PKS cyclization specificity.Aflatoxin B 1 (Fig. 1a, 3) biosynthesis is initiated by the NR-PKS PksA, which accepts a hexanoyl starter unit from a dedicated fungal fatty acid synthase (FAS) and extends it through seven iterative, malonyl-derived ketide extensions to norsolorinic acid anthrone ((1), noranthrone) (Fig. 1a)5. Application of the Udwary-Merski algorithm (UMA) afforded the unanticipated insight that PksA was composed of two unrecognized domains, in additionCorrespondence and requests for materials should be addressed to C.A.T. (ctownsend@jhu.edu) or S.-C.T. (sctsai@uci.edu). * These authors contributed equally to this work.Full Methods and any associated references are available in the online version of the paper at www.nature.com/nature.Supplementary Information is linked to the online version of the paper at www.nature.com/nature. (Fig. 1a, c) 4 . When the TE domain was absent, naphthopyrone (4) was produced by self-condensation of the stalled bicyclic PT product 7 (Fig. 1b). Author ContributionsWe crystallized PT in P2 1 2 1 2 1 and P4 1 2 1 2 space groups (Supplementary Table 1) and solved the PT crystal structure by multi-wavelength anomalous dispersion (MAD) using selenomethionine-derivatized PT followed by phase extension with the native data to 1.8 Å.The crystal structures from both crystal forms showed distinct ...

Polyketides are a class of natural products that exhibit a wide range of functional and structural diversity. They include antibiotics, immunosuppressants, antifungals, antihypercholesterolemics, and cytotoxins. Polyketide synthases (PKSs) use chemistry similar to fatty acid synthases (FASs), although building block variation and differing extents of reduction of the growing polyketide chain underlie their biosynthetic versatility. In contrast to the well studied sequential modular type I PKSs, less is known about how the iterative type I PKSs carry out and control chain initiation, elongation, folding, and cyclization during polyketide processing. Domain structure analysis of a group of related fungal, nonreducing PKSs has revealed well defined N-terminal domains longer than commonly seen for FASs and modular PKSs. Predicted structure of this domain disclosed a region similar to malonyl-CoA:acyl-carrier protein (ACP) transacylases (MATs). MATs play a key role transferring precursor CoA thioesters from solution onto FASs and PKSs for chain elongation. On the basis of site-directed mutagenesis, radiolabeling, and kinetics experiments carried out with individual domains of the norsolorinic acid PKS, we propose that the Nterminal domain is a starter unit:ACP transacylase (SAT domain) that selects a C6 fatty acid from a dedicated yeast-like FAS and transfers this unit onto the PKS ACP, leading to the production of the aflatoxin precursor, norsolorinic acid. These findings could indicate a much broader role for SAT domains in starter unit selection among nonreducing iterative, fungal PKSs, and they provide a biochemical rationale for the classical acetyl ''starter unit effect.'' aflatoxin biosynthesis ͉ fatty acid synthase

Shewanellae are gram-negative facultatively anaerobic metal-reducing bacteria commonly found in chemically (i.e., redox) stratified environments. Occupying such niches requires the ability to rapidly acclimate to changes in electron donor/acceptor type and availability; hence, the ability to compete and thrive in such environments must ultimately be reflected in the organization and utilization of electron transfer networks, as well as central and peripheral carbon metabolism. To understand how Shewanella oneidensis MR-1 utilizes its resources, the metabolic network was reconstructed. The resulting network consists of 774 reactions, 783 genes, and 634 unique metabolites and contains biosynthesis pathways for all cell constituents. Using constraint-based modeling, we investigated aerobic growth of S. oneidensis MR-1 on numerous carbon sources. To achieve this, we (i) used experimental data to formulate a biomass equation and estimate cellular ATP requirements, (ii) developed an approach to identify cycles (such as futile cycles and circulations), (iii) classified how reaction usage affects cellular growth, (iv) predicted cellular biomass yields on different carbon sources and compared model predictions to experimental measurements, and (v) used experimental results to refine metabolic fluxes for growth on lactate. The results revealed that aerobic lactate-grown cells of S. oneidensis MR-1 used less efficient enzymes to couple electron transport to proton motive force generation, and possibly operated at least one futile cycle involving malic enzymes. Several examples are provided whereby model predictions were validated by experimental data, in particular the role of serine hydroxymethyltransferase and glycine cleavage system in the metabolism of one-carbon units, and growth on different sources of carbon and energy. This work illustrates how integration of computational and experimental efforts facilitates the understanding of microbial metabolism at a systems level.