Comparing independent high-throughput gene-expression experiments can generate hypotheses about which gene-expression programs are shared between particular biological processes. Current techniques to compare expression profiles typically involve choosing a fixed differential expression threshold to summarize results, potentially reducing sensitivity to small but concordant changes. We present a threshold-free algorithm called Rank–rank Hypergeometric Overlap (RRHO). This algorithm steps through two gene lists ranked by the degree of differential expression observed in two profiling experiments, successively measuring the statistical significance of the number of overlapping genes. The output is a graphical map that shows the strength, pattern and bounds of correlation between two expression profiles. To demonstrate RRHO sensitivity and dynamic range, we identified shared expression networks in cancer microarray profiles driving tumor progression, stem cell properties and response to targeted kinase inhibition. We demonstrate how RRHO can be used to determine which model system or drug treatment best reflects a particular biological or disease response. The threshold-free and graphical aspects of RRHO complement other rank-based approaches such as Gene Set Enrichment Analysis (GSEA), for which RRHO is a 2D analog. Rank–rank overlap analysis is a sensitive, robust and web-accessible method for detecting and visualizing overlap trends between two complete, continuous gene-expression profiles. A web-based implementation of RRHO can be accessed at http://systems.crump.ucla.edu/rankrank/.

Type II polyketides are a class of natural products that include pharmaceutically important aromatic compounds such as the antibiotic tetracycline and antitumor compound doxorubicin. The type II polyketide synthase (PKS) is a complex consisting of 5-10 standalone domains homologous to fatty acid synthase (FAS). Polyketide ketoreductase (KR) provides regio-and stereochemical diversity during the reduction. How the type II polyketide KR specifically reduces only the C9 carbonyl group is not well understood. The cocrystal structures of actinorhodin polyketide ketoreductase (actKR) bound with NADPH or NADP + and the inhibitor emodin were solved with the wild type and P94L mutant of actKR, revealing the first observation of a bent p-quinone in an enzyme active site. Molecular dynamics simulation help explain the origin of the bent geometry. Extensive screening for in vitro substrates shows that unlike FAS KR, the actKR prefers bicyclic substrates. Inhibition kinetics indicate that actKR follows an ordered Bi Bi mechanism. Together with docking simulations that identified a potential phosphopantetheine binding groove, the structural and functional studies reveal that the C9 specificity is a result of active site geometry and substrate ring constraints. The results lay the foundation for the design of novel aromatic polyketide natural products with different reduction patterns.The pharmaceutical potential of bacterial or fungal natural products is illustrated by the large number of compounds that are clinically applied as therapeutics. Many pharmaceutically relevant natural products are derived from polyketides and are used as antibiotic (tetracyclines, actinorhodin), anticancer (doxorubicin), antiviral (rebeccamycin derivatives), and cholesterollowering (statins) compounds (1). The antibiotics such as tetracycline and actinorhodin are biosynthesized from acyl-CoA thiosters by type II polyketide synthases (PKSs 1 ), which are structurally and functionally related to the type II fatty acid synthase (FAS) (2). Compared to the type I FAS and PKS, which have enzyme domains covalently linked together, the type II FAS and PKS consist of 5-10 standalone enzymes that catalyze the condensation of malonyl extender units iteratively, followed by chain modifications, to produce the aromatic polyketides (3,4). † This work is supported by the Pew Foundation and National Institute of General Medicinal Sciences (NIGMS R01GM076330). ‡ The atomic coordinates have been deposited in the Protein Data Bank (accession code 2RH4, 2RHC, and 2RHR).* Author to whom correspondence should be addressed. Phone 949-824-4486, e-mail sctsai@uci.edu, fax 949-824-8552. ⊥ Department of Molecular Biology and Biochemistry. § Department of Chemistry.1 Abbreviations: KR, ketoreductase; FabG, β-ketoacyl [acyl carrier protein] reductase; Act, actinorhodin; PKS, polyketide synthase; NADP, nicotinamide adenine dinucleotide phosphate; NADPH, reduced nicotinamide adenine dinucleotide phosphate; SDR, short-chain dehydrogenase/reductase; T3HNR, 1,3,8-trihydro...

Polyketide natural products possess diverse architectures and biological functions and share a subset of biosynthetic steps with fatty acid synthesis. The final transformation catalyzed by both polyketide synthases (PKSs) and fatty acid synthases is most often carried out by a thioesterase (TE). The synthetic versatility of TE domains in fungal nonreducing, iterative PKSs (NR-PKSs) has been shown to extend to Claisen cyclase (CLC) chemistry by catalyzing C–C ring closure reactions as opposed to thioester hydrolysis or O–C/N–C macrocyclization observed in previously reported TE structures. Catalysis of C–C bond formation as a product release mechanism dramatically expands the synthetic potential of PKSs, but how this activity was acquired has remained a mystery. We report the biochemical and structural analyses of the TE/CLC domain in polyketide synthase A, the multidomain PKS central to the biosynthesis of aflatoxin B 1 , a potent environmental carcinogen. Mutagenesis experiments confirm the predicted identity of the catalytic triad and its role in catalyzing the final Claisen-type cyclization to the aflatoxin precursor, norsolorinic acid anthrone. The 1.7 Å crystal structure displays an α/β-hydrolase fold in the catalytic closed form with a distinct hydrophobic substrate-binding chamber. We propose that a key rotation of the substrate side chain coupled to a protein conformational change from the open to closed form spatially governs substrate positioning and C–C cyclization. The biochemical studies, the 1.7 Å crystal structure of the TE/CLC domain, and intermediate modeling afford the first mechanistic insights into this widely distributed C–C bond-forming class of TEs.