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Objectives: We sought to determine whether patients with congestive heart failure (CHF) avoid the left lateral decubitus (LLD) position during sleep and, if so, whether this avoidance would be more pronounced in those with greater degrees of cardiomegaly.Background: Anecdotal reports suggest that, in patients with CHF, the LLD position is associated with discomfort due to the enlarged apical heart beat and greater degree of dyspnea (trepopnea) than other positions. It has also been suggested that the LLD position is associated with increased sympathetic nervous activity.Methods: A total of 75 patients with CHF and 75 control subjects underwent nocturnal polysomnography with monitoring of body position. Echocardiography was performed in all patients with CHF to determine left ventricular end-diastolic diameter (LVEDD). A total of 40 patients underwent cardiac catheterization from which pulmonary capillary wedge pressure (PCWP) and cardiac output (CO) were obtained.Results: Patients with CHF spent significantly less time in the LLD position than in the right lateral decubitus position. No such difference was observed among control subjects. Among patients with CHF, those with larger LVEDD, higher PCWP, and lower CO spent less time in the LLD position.Conclusions: Patients with CHF avoid the LLD position spontaneously during sleep. This may be a protective strategy to avoid discomfort from the enlarged apical heart beat or further hemodynamic or autonomic compromise. [ABSTRACT FROM AUTHOR]

Abstract Background Among next generation sequence technologies, platforms such as Illumina and SOLiD produce short reads but with higher coverage and lower cost per sequenced nucleotide than 454 or Sanger. A challenge now is to develop efficient strategies to use short-read length platforms for de novo assembly and marker development. The scope of this study was to develop a de novo assembly of carrot ESTs from multiple genotypes using the Illumina platform, and to identify polymorphisms. Results A de novo assembly of transcriptome sequence from four genetic backgrounds produced 58,751 contigs and singletons. Over 50% of these assembled sequences were annotated allowing detection of transposable elements and new carrot anthocyanin genes. Presence of multiple genetic backgrounds in our assembly allowed the identification of 114 computationally polymorphic SSRs, and 20,058 SNPs at a depth of coverage of 20× or more. Polymorphisms were predominantly between inbred lines except for the cultivated x wild RIL pool which had high intra-sample polymorphism. About 90% and 88% of tested SSR and SNP primers amplified a product, of which 70% and 46%, respectively, were of the expected size. Out of verified SSR and SNP markers 84% and 82% were polymorphic. About 25% of SNPs genotyped were polymorphic in two diverse mapping populations. Conclusions This study confirmed the potential of short read platforms for de novo EST assembly and identification of genetic polymorphisms in carrot. In addition we produced the first large-scale transcriptome of carrot, a species lacking genomic resources.

Site-specific covalent conjugation offers a powerful tool to identify and understand protein-protein interactions. In this study, we discover that sulfur fluoride exchange (SuFEx) warheads effectively crosslink the Escherichia coli acyl carrier protein (AcpP) with its partner BioF, a key pyridoxal 5'-phosphate (PLP)-dependent enzyme in the early steps of biotin biosynthesis by targeting a tyrosine residue proximal to the active site. We identify the site of crosslink by MS/MS analysis of the peptide originating from both partners. We further evaluate the BioF-AcpP interface through protein crystallography and mutational studies. Among the AcpP-interacting BioF surface residues, three critical arginine residues appear to be involved in AcpP recognition so that pimeloyl-AcpP can serve as the acyl donor for PLP-mediated catalysis. These findings validate an evolutionary gain-of-function for BioF, allowing the organism to build biotin directly from fatty acid biosynthesis through surface modifications selective for salt bridge formation with acidic AcpP residues.

Structural biology efforts using cryogenic electron microscopy are frequently stifled by specimens adopting "preferred orientations" on grids, leading to anisotropic map resolution and impeding structure determination. Tilting the specimen stage during data collection is a generalizable solution but has historically led to substantial resolution attenuation. Here, we develop updated data collection and image processing workflows and demonstrate, using multiple specimens, that resolution attenuation is negligible or significantly reduced across tilt angles. Reconstructions with and without the stage tilted as high as 60° are virtually indistinguishable. These strategies allowed the reconstruction to 3 Å resolution of a bacterial RNA polymerase with preferred orientation, containing an unnatural nucleotide for studying novel base pair recognition. Furthermore, we present a quantitative framework that allows cryo-EM practitioners to define an optimal tilt angle during data acquisition. These results reinforce the utility of employing stage tilt for data collection and provide quantitative metrics to obtain isotropic maps., (© 2024. The Author(s).)

Over the past decade, advances in genomics have identified thousands of additional protein-coding small open reading frames (smORFs) missed by traditional gene finding approaches. These smORFs encode peptides and small proteins, commonly termed micropeptides or microproteins. Several of these newly discovered microproteins have biological functions and operate through interactions with proteins and protein complexes within the cell. CYREN1 is a characterized microprotein that regulates double-strand break repair in mammalian cells through interaction with Ku70/80 heterodimer. Ku70/80 binds to and stabilizes double-strand breaks and recruits the machinery needed for nonhomologous end join repair. In this study, we examined the biochemical properties of CYREN1 to better understand and explain its cellular protein interactions. Our findings support that CYREN1 is an intrinsically disordered microprotein and this disordered structure allows it to enriches several proteins, including a newly discovered interaction with SF3B1 via a distinct short linear motif (SLiMs) on CYREN1. Since many microproteins are predicted to be disordered, CYREN1 is an exemplar of how microproteins interact with other proteins and reveals an unknown scaffolding function of this microprotein that may link NHEJ and splicing.

Single-particle analysis (SPA) by cryo-electron microscopy (cryo-EM) is now a mainstream technique for high-resolution structural biology. Structure determination by SPA relies upon obtaining multiple distinct views of a macromolecular object vitrified within a thin layer of ice. Ideally, a collection of uniformly distributed random projection orientations would amount to all possible views of the object, giving rise to reconstructions characterized by isotropic directional resolution. However, in reality, many samples suffer from preferentially oriented particles adhering to the air-water interface. This leads to non-uniform angular orientation distributions in the dataset and inhomogeneous Fourier-space sampling in the reconstruction, translating into maps characterized by anisotropic resolution. Tilting the specimen stage provides a generalizable solution to overcoming resolution anisotropy by virtue of improving the uniformity of orientation distributions, and thus the isotropy of Fourier space sampling. The present protocol describes a tilted-stage automated data collection strategy using Leginon, a software for automated image acquisition. The procedure is simple to implement, does not require any additional equipment or software, and is compatible with most standard transmission electron microscopes (TEMs) used for imaging biological macromolecules.

Objective: To determine if maternal plasma CRH and preterm birth history were associated with recurrent preterm birth risk in a high-risk cohort., Study Design: Secondary analysis of pregnant women with a prior preterm birth ≤35 weeks receiving 17-alpha hydroxyprogesterone caproate for the prevention of recurrent spontaneous preterm birth. All women with a 24-week blood sample were included. Maternal plasma CRH level at 24- and 32-weeks' gestation was measured using both enzyme-linked immunosorbent assay (ELISA) and extracted radioimmunoassay (RIA) technologies. The primary outcome was spontaneous preterm birth <37 weeks. The association of CRH, prior preterm birth history, and the two combined was assessed in relation to recurrent preterm birth risk., Results: Recurrent preterm birth in this cohort of 169 women was 24.9%. Comparing women who subsequently delivered <37 versus ≥37 weeks, mean levels of CRH measured by RIA were significantly different at 24 weeks (111.1±87.5 vs. 66.1±45.4 pg/mL, P = .002) and 32 weeks (440.9±275.6 vs. 280.2±214.5 pg/mL, P = .003). The area under the receiver operating curve (AUC) at 24 and 32 weeks for (1) CRH level was 0.68 (95% CI 0.59-0.78) and 0.70 (95% CI 0.59-0.81), (2) prior preterm birth history was 0.75 (95% CI 0.67-0.83) and 0.78 (95% CI 0.69-0.87), and (3) combined was 0.81 (95% CI 0.73-0.88, P = .001) and 0.81 (95% CI 0.72-0.90, P = .01) respectively for delivery <37 weeks. CRH measured by ELISA failed to correlate with gestational age or other clinical parameters., Conclusion: In women with a prior preterm birth, CRH levels were higher and had an earlier rise in women who experienced recurrent preterm birth. Second trimester CRH may be useful in identifying a sub-group of women with preterm birth due to early activation of the placenta-fetal adrenal axis. Assay methodology is a variable that contributes to difficulties in reproducibility of CRH levels in the obstetric literature., Competing Interests: The authors have declared that no competing interests exist.

In plants, phenylalanine biosynthesis occurs via two compartmentally separated pathways. Overexpression of petunia chorismate mutase 2 (PhCM2), which catalyzes the committed step of the cytosolic pathway, increased flux in cytosolic phenylalanine biosynthesis, but paradoxically decreased the overall levels of phenylalanine and phenylalanine-derived volatiles. Concomitantly, the levels of auxins, including indole-3-acetic acid and its precursor indole-3-pyruvic acid, were elevated. Biochemical and genetic analyses revealed the existence of metabolic crosstalk between the cytosolic phenylalanine biosynthesis and tryptophan-dependent auxin biosynthesis mediated by an aminotransferase that uses a cytosolic phenylalanine biosynthetic pathway intermediate, phenylpyruvate, as an amino acceptor for auxin formation.

Introduction: The aim of this study was to examine the association between plasma hormone concentrations, cervical length, and preterm delivery in twin pregnancies, including the effect of progesterone treatment., Material and Methods: This study included 191 women pregnant with twins from a randomized placebo-controlled trial. A baseline blood sample was collected at 18-24 weeks before treatment with vaginal progesterone (n = 95) or placebo pessaries (n = 96), and 167 (87.4%) women had a second sample collected after 4-8 weeks of treatment. At baseline, 155 (81.2%) women had their cervical length measured. Progesterone, estradiol, and unconjugated estriol concentration was measured, and the association between hormone concentrations, cervical length, and gestational age at delivery was examined. Hormone concentrations were compared in the placebo and progesterone group. Statistical analysis included Spearman's rho, Mann-Whitney U test, Cuzick's test for trends, and linear regression analyses., Results: A short cervical length was associated with preterm delivery. Cervical length and hormone concentrations were not associated (Spearman's rho; progesterone -.05, estradiol .04, estriol .08). Decreasing gestational age at delivery was associated with higher progesterone and estradiol concentrations at baseline (P trend; progesterone 0.04, estradiol 0.02) but not in the second sample or in the weekly change between samples. Progesterone treatment did not increase the progesterone concentration., Conclusions: Plasma concentrations of progesterone, estradiol, and unconjugated estriol at 18-24 weeks are not associated with cervical length or preterm delivery in twin pregnancies. Vaginal progesterone treatment does not increase the circulating progesterone concentration in twin pregnancies. Cervical length, but not hormone concentration, is predictive of preterm delivery in twin gestations., (© 2018 Nordic Federation of Societies of Obstetrics and Gynecology.)

Polyketides are attractive compounds for uses ranging from biorenewable chemical precursors to high-value therapeutics. In many cases, synthesis in a heterologous host is required to produce these compounds in industrially relevant quantities. The type III polyketide synthase 2-pyrone synthase (2-PS) from Gerbera hybrida was used for the production of triacetic acid lactone (TAL) in Saccharomyces cerevisiae. Initial in vitro characterization of 2-PS led to the identification of active site variants with improved kinetic properties relative to wildtype. Further in vivo evaluation in S. cerevisiae suggested certain 2-PS mutations altered enzyme stability during fermentation. In vivo experiments also revealed beneficial cysteine to serine mutations that were not initially explored due to their distance from the active site of 2-PS, leading to the design of additional 2-PS enzymes. While these variants showed varying catalytic efficiencies in vitro, they exhibited up to 2.5-fold increases in TAL production when expressed in S. cerevisiae. Coupling of the 2-PS variant [C35S,C372S] to an engineered S. cerevisiae strain led to over 10 g/L TAL at 38% of theoretical yield following fed-batch fermentation, the highest reported to date. Our studies demonstrate the success of a coupled in vitro/in vivo approach to engineering enzymes and provide insight on cysteine-rich enzymes and design principles toward their use in non-native microbial hosts., (© 2018 Wiley Periodicals, Inc.)

The peroxisome proliferator-activated receptor (PPAR) family comprises three subtypes: PPARα, PPARγ, and PPARδ. PPARδ transcriptionally modulates lipid metabolism and the control of energy homeostasis; therefore, PPARδ agonists are promising agents for treating a variety of metabolic disorders. In the present study, we develop a panel of rationally designed PPARδ agonists. The modular motif affords efficient syntheses using building blocks optimized for interactions with subtype-specific residues in the PPARδ ligand-binding domain (LBD). A combination of atomic-resolution protein X-ray crystallographic structures, ligand-dependent LBD stabilization assays, and cell-based transactivation measurements delineate structure-activity relationships (SARs) for PPARδ-selective targeting and structural modulation. We identify key ligand-induced conformational transitions of a conserved tryptophan side chain in the LBD that trigger reorganization of the H2'-H3 surface segment of PPARδ. The subtype-specific conservation of H2'-H3 sequences suggests that this architectural remodeling constitutes a previously unrecognized conformational switch accompanying ligand-dependent PPARδ transcriptional regulation.

Objective: The objective of the study was to analyse placental hormone profiles in twin pregnancies to determine if they could be used to predict preterm birth., Study Design: Progesterone, estradiol, estriol and corticotropin-releasing hormone were measured using competitive immunoassay and radioimmunoassay in serum and saliva samples of 98 women with twin pregnancies,at 3 or more gestational timepoints. Hormone profiles throughout gestation were compared between very preterm (<34 weeks; n = 8), preterm (<37 weeks; n = 40) and term (37+ weeks; n = 50) deliveries., Results: No significant differences were found between preterm and term deliveries in either absolute hormone concentrations or ratios. Estimated hormone concentrations and ratios at 26 weeks did not appear to predict preterm delivery. Salivary and serum hormone concentrations were generally poorly correlated., Conclusion: Our results suggest that serial progesterone, estradiol, estriol and corticotropin-releasing hormone measurements in saliva and serum are not robust biomarkers for preterm birth in twin pregnancies.

Poor litter quality is a potential challenge to footpad health as well as the primary cause of ammonia volatilization. High ambient ammonia concentration is one of the most significant factors negatively affecting poultry production today. Some minerals have been reported to reduce ammonia release from poultry litter. Silicon dioxide, a highly pure and natural mineral, shows promise in decreasing ammonia volatilization and improving litter quality. The objective of the current study was to investigate the effects of feed-borne silicon dioxide on litter quality and how this impacts bird performance, general health and footpad health throughout a 12-wk posthatching turkey study. Supplementing the diet with silicon dioxide was found to significantly improve turkey BW gain and the efficiency of feed conversion. The severity of footpad dermatitis was monitored throughout the experimental period but no significant effect of diet was seen. The feeding of silicon dioxide reduced litter pH which decreased the conversion of NH4⁺ to NH3 thereby reducing nitrogen losses from litter. It was concluded that, under our study conditions, the feeding of 0.02% silicon dioxide offers potential economic benefits to turkey producers., (© 2015 Poultry Science Association Inc.)

The enzymes cinnamoyl-CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the two key reduction reactions in the conversion of cinnamic acid derivatives into monolignol building blocks for lignin polymers in plant cell walls. Here, we describe detailed functional and structural analyses of CCRs from Medicago truncatula and Petunia hybrida and of an atypical CAD (CAD2) from M. truncatula. These enzymes are closely related members of the short-chain dehydrogenase/reductase (SDR) superfamily. Our structural studies support a reaction mechanism involving a canonical SDR catalytic triad in both CCR and CAD2 and an important role for an auxiliary cysteine unique to CCR. Site-directed mutants of CAD2 (Phe226Ala and Tyr136Phe) that enlarge the phenolic binding site result in a 4- to 10-fold increase in activity with sinapaldehyde, which in comparison to the smaller coumaraldehyde and coniferaldehyde substrates is disfavored by wild-type CAD2. This finding demonstrates the potential exploitation of rationally engineered forms of CCR and CAD2 for the targeted modification of monolignol composition in transgenic plants. Thermal denaturation measurements and structural comparisons of various liganded and unliganded forms of CCR and CAD2 highlight substantial conformational flexibility of these SDR enzymes, which plays an important role in the establishment of catalytically productive complexes of the enzymes with their NADPH and phenolic substrates., (© 2014 American Society of Plant Biologists. All rights reserved.)

We examined the rates and risk factors for surgical site infections (SSIs) following delayed sternal closure after pediatric cardiac surgery by way of retrospective review of prospectively collected infection control data. Of 130 patients, 13.7% developed an SSI, and 6.9% developed mediastinitis following delayed sternal closure. There was a trend toward increased SSIs in patients undergoing delayed sternal closure in beds in the open bay of a pediatric intensive care unit., (Copyright © 2013 Association for Professionals in Infection Control and Epidemiology, Inc. Published by Mosby, Inc. All rights reserved.)

The C-terminal domain (CTD) of eukaryotic RNA polymerase II is an essential regulator for RNA polymerase II-mediated transcription. It is composed of multiple repeats of a consensus sequence Tyr(1)Ser(2)Pro(3)Thr(4)Ser(5)Pro(6)Ser(7). CTD regulation of transcription is mediated by both phosphorylation of the serines and prolyl isomerization of the two prolines. Interestingly, the phosphorylation sites are typically close to prolines, and thus the conformation of the adjacent proline could impact the specificity of the corresponding kinases and phosphatases. Experimental evidence of cross-talk between these two regulatory mechanisms has been elusive. Pin1 is a highly conserved phosphorylation-specific peptidyl-prolyl isomerase (PPIase) that recognizes the phospho-Ser/Thr (pSer/Thr)-Pro motif with CTD as one of its primary substrates in vivo. In the present study, we provide structural snapshots and kinetic evidence that support the concept of cross-talk between prolyl isomerization and phosphorylation. We determined the structures of Pin1 bound with two substrate isosteres that mimic peptides containing pSer/Thr-Pro motifs in cis or trans conformations. The results unequivocally demonstrate the utility of both cis- and trans-locked alkene isosteres as close geometric mimics of peptides bound to a protein target. Building on this result, we identified a specific case in which Pin1 differentially affects the rate of dephosphorylation catalyzed by two phosphatases (Scp1 and Ssu72) that target the same serine residue in the CTD heptad repeat but have different preferences for the isomerization state of the adjacent proline residue. These data exemplify for the first time how modulation of proline isomerization can kinetically impact signal transduction in transcription regulation.

Specialized metabolic enzymes biosynthesize chemicals of ecological importance, often sharing a pedigree with primary metabolic enzymes. However, the lineage of the enzyme chalcone isomerase (CHI) remained unknown. In vascular plants, CHI-catalysed conversion of chalcones to chiral (S)-flavanones is a committed step in the production of plant flavonoids, compounds that contribute to attraction, defence and development. CHI operates near the diffusion limit with stereospecific control. Although associated primarily with plants, the CHI fold occurs in several other eukaryotic lineages and in some bacteria. Here we report crystal structures, ligand-binding properties and in vivo functional characterization of a non-catalytic CHI-fold family from plants. Arabidopsis thaliana contains five actively transcribed genes encoding CHI-fold proteins, three of which additionally encode amino-terminal chloroplast-transit sequences. These three CHI-fold proteins localize to plastids, the site of de novo fatty-acid biosynthesis in plant cells. Furthermore, their expression profiles correlate with those of core fatty-acid biosynthetic enzymes, with maximal expression occurring in seeds and coinciding with increased fatty-acid storage in the developing embryo. In vitro, these proteins are fatty-acid-binding proteins (FAPs). FAP knockout A. thaliana plants show elevated α-linolenic acid levels and marked reproductive defects, including aberrant seed formation. Notably, the FAP discovery defines the adaptive evolution of a stereospecific and catalytically 'perfected' enzyme from a non-enzymatic ancestor over a defined period of plant evolution.

Lignin forms from the polymerization of phenylpropanoid-derived building blocks (the monolignols), whose modification through hydroxylation and O-methylation modulates the chemical and physical properties of the lignin polymer. The enzyme caffeic acid O-methyltransferase (COMT) is central to lignin biosynthesis. It is often targeted in attempts to engineer the lignin composition of transgenic plants for improved forage digestibility, pulping efficiency, or utility in biofuel production. Despite intensive investigation, the structural determinants of the regiospecificity and substrate selectivity of COMT remain poorly defined. Reported here are x-ray crystallographic structures of perennial ryegrass (Lolium perenne) COMT (Lp OMT1) in open conformational state, apo- and holoenzyme forms and, most significantly, in a closed conformational state complexed with the products S-adenosyl-L-homocysteine and sinapaldehyde. The product-bound complex reveals the post-methyl-transfer organization of COMT's catalytic groups with reactant molecules and the fully formed phenolic-ligand binding site. The core scaffold of the phenolic ligand forges a hydrogen-bonding network involving the 4-hydroxy group that anchors the aromatic ring and thereby permits only metahydroxyl groups to be positioned for transmethylation. While distal from the site of transmethylation, the propanoid tail substituent governs the kinetic preference of ryegrass COMT for aldehydes over alcohols and acids due to a single hydrogen bond donor for the C9 oxygenated moiety dictating the preference for an aldehyde.

Cinnamoyl CoA-reductase (CCR) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the biosynthesis of monolignols, which serve as building blocks in the formation of plant lignin. We identified candidate genes encoding these two enzymes in perennial ryegrass (Lolium perenne) and show that the spatio-temporal expression patterns of these genes in planta correlate well with the developmental profile of lignin deposition. Downregulation of CCR1 and caffeic acid O-methyltransferase 1 (OMT1) using an RNA interference-mediated silencing strategy caused dramatic changes in lignin level and composition in transgenic perennial ryegrass plants grown under both glasshouse and field conditions. In CCR1-deficient perennial ryegrass plants, metabolic profiling indicates the redirection of intermediates both within and beyond the core phenylpropanoid pathway. The combined results strongly support a key role for the OMT1 gene product in the biosynthesis of both syringyl- and guaiacyl-lignin subunits in perennial ryegrass. Both field-grown OMT1-deficient and CCR1-deficient perennial ryegrass plants showed enhanced digestibility without obvious detrimental effects on either plant fitness or biomass production. This highlights the potential of metabolic engineering not only to enhance the forage quality of grasses but also to produce optimal feedstock plants for biofuel production.

Context: Clinical prediction of preterm delivery is largely ineffective, and the mechanism mediating progesterone (P) withdrawal and estrogen activation at the onset of human labor is unclear., Objectives: Our objectives were to determine associations of rates of change of circulating maternal CRH in midpregnancy with preterm delivery, CRH with estriol (E3) concentrations in late pregnancy, and predelivery changes in the ratios of E3, estradiol (E2), and P., Design and Setting: A cohort of 500 pregnant women was followed from first antenatal visits to delivery during the period 2000-2004 at John Hunter Hospital, New South Wales, Australia, a tertiary care obstetric hospital., Patients: Unselected subjects were recruited (including women with multiple gestations) and serial blood samples obtained., Main Outcome Measures: CRH daily percentage change in term and preterm singletons at 26 wk, ratios E3/E2, P/E3, and P/E2 and the association between E3 and CRH concentrations in the last month of pregnancy (with spontaneous labor onset) were assessed., Results: CRH percentage daily change was significantly higher in preterm than term singletons at 26 wk (medians 3.09 and 2.73; P = 0.003). In late pregnancy, CRH and E3 concentrations were significantly positively associated (P = 0.003). E3/E2 increased, P/E3 decreased, and P/E2 was unchanged in the month before delivery (medians: E3/E2, 7.04 and 10.59, P < 0.001; P/E3, 1.55 and 0.98, P < 0.001; P/E2, 11.78 and 10.79, P = 0.07)., Conclusions: The very rapid rise of CRH in late pregnancy is associated with an E3 surge and critically altered P/E3 and E3/E2 ratios that create an estrogenic environment at the onset of labor. Our evidence provides a rationale for the use of CRH in predicting preterm birth and informs approaches to delaying labor using P supplementation.

Many plants synthesize the volatile phenylpropene compounds eugenol and isoeugenol to serve in defense against herbivores and pathogens and to attract pollinators. Clarkia breweri flowers emit a mixture of eugenol and isoeugenol, while Petunia hybrida flowers emit mostly isoeugenol with small amounts of eugenol. We recently reported the identification of a petunia enzyme, isoeugenol synthase 1 (PhIGS1) that catalyzes the formation of isoeugenol, and an Ocimum basilicum (basil) enzyme, eugenol synthase 1 (ObEGS1), that produces eugenol. ObEGS1 and PhIGS1 both utilize coniferyl acetate, are 52% sequence identical, and belong to a family of NADPH-dependent reductases involved in secondary metabolism. Here we show that C. breweri flowers have two closely related proteins (96% identity), CbIGS1 and CbEGS1, that are similar to ObEGS1 (58% and 59% identity, respectively) and catalyze the formation of isoeugenol and eugenol, respectively. In vitro mutagenesis experiments demonstrate that substitution of only a single residue can substantially affect the product specificity of these enzymes. A third C. breweri enzyme identified, CbEGS2, also catalyzes the formation of eugenol from coniferyl acetate and is only 46% identical to CbIGS1 and CbEGS1 but more similar (>70%) to other types of reductases. We also found that petunia flowers contain an enzyme, PhEGS1, that is highly similar to CbEGS2 (82% identity) and that converts coniferyl acetate to eugenol. Our results indicate that plant enzymes with EGS and IGS activities have arisen multiple times and in different protein lineages.

Plants grown at high densities perceive a decrease in the red to far-red (R:FR) ratio of incoming light, resulting from absorption of red light by canopy leaves and reflection of far-red light from neighboring plants. These changes in light quality trigger a series of responses known collectively as the shade avoidance syndrome. During shade avoidance, stems elongate at the expense of leaf and storage organ expansion, branching is inhibited, and flowering is accelerated. We identified several loci in Arabidopsis, mutations in which lead to plants defective in multiple shade avoidance responses. Here we describe TAA1, an aminotransferase, and show that TAA1 catalyzes the formation of indole-3-pyruvic acid (IPA) from L-tryptophan (L-Trp), the first step in a previously proposed, but uncharacterized, auxin biosynthetic pathway. This pathway is rapidly deployed to synthesize auxin at the high levels required to initiate the multiple changes in body plan associated with shade avoidance.

Phenylpropenes, a large group of plant volatile compounds that serve in multiple roles in defense and pollinator attraction, contain a propenyl side chain. Eugenol synthase (EGS) catalyzes the reductive displacement of acetate from the propenyl side chain of the substrate coniferyl acetate to produce the allyl-phenylpropene eugenol. We report here the structure determination of EGS from basil (Ocimum basilicum) by protein x-ray crystallography. EGS is structurally related to the short-chain dehydrogenase/reductases (SDRs), and in particular, enzymes in the isoflavone-reductase-like subfamily. The structure of a ternary complex of EGS bound to the cofactor NADP(H) and a mixed competitive inhibitor EMDF ((7S,8S)-ethyl (7,8-methylene)-dihydroferulate) provides a detailed view of the binding interactions within the EGS active site and a starting point for mutagenic examination of the unusual reductive mechanism of EGS. The key interactions between EMDF and the EGS-holoenzyme include stacking of the phenyl ring of EMDF against the cofactor's nicotinamide ring and a water-mediated hydrogen-bonding interaction between the EMDF 4-hydroxy group and the side-chain amino moiety of a conserved lysine residue, Lys132. The C4 carbon of nicotinamide resides immediately adjacent to the site of hydride addition, the C7 carbon of cinnamyl acetate substrates. The inhibitor-bound EGS structure suggests a two-step reaction mechanism involving the formation of a quinone-methide prior to reduction. The formation of this intermediate is promoted by a hydrogen-bonding network that favors deprotonation of the substrate's 4-hydroxyl group and disfavors binding of the acetate moiety, akin to a push-pull catalytic mechanism. Notably, the catalytic involvement in EGS of the conserved Lys132 in preparing the phenolic substrate for quinone methide formation through the proton-relay network appears to be an adaptation of the analogous role in hydrogen bonding played by the equivalent lysine residue in other enzymes of the SDR family.

Human Pin1 is a key regulator of cell-cycle progression and plays growth-promoting roles in human cancers. High-affinity inhibitors of Pin1 may provide a unique opportunity for disrupting oncogenic pathways. Here we report two high-resolution X-ray crystal structures of human Pin1 bound to non-natural peptide inhibitors. The structures of the bound high-affinity peptides identify a type-I beta-turn conformation for Pin1 prolyl peptide isomerase domain-peptide binding and an extensive molecular interface for high-affinity recognition. Moreover, these structures suggest chemical elements that may further improve the affinity and pharmacological properties of future peptide-based Pin inhibitors. Finally, an intramolecular hydrogen bond observed in both peptide complexes mimics the cyclic conformation of FK506 and rapamycin. Both FK506 and rapamycin are clinically important inhibitors of other peptidyl-prolyl cis-trans isomerases. This comparative discovery suggests that a cyclic peptide polyketide bridge, like that found in FK506 and rapamycin or a similar linkage, may significantly improve the binding affinity of structure-based Pin1 inhibitors.

Phenylalanine ammonia lyase (PAL) catalyzes the deamination of phenylalanine to cinnamate and ammonia. While PALs are common in terrestrial plants where they catalyze the first committed step in the formation of phenylpropanoids, only a few prokaryotic PALs have been identified to date. Here we describe for the first time PALs from cyanobacteria, in particular, Anabaena variabilis ATCC 29413 and Nostoc punctiforme ATCC 29133, identified by screening the genome sequences of these organisms for members of the aromatic amino acid ammonia lyase family. Both PAL genes associate with secondary metabolite biosynthetic gene clusters as observed for other eubacterial PAL genes. In comparison to eukaryotic homologues, the cyanobacterial PALs are 20% smaller in size but share similar substrate selectivity and kinetic activity toward L-phenylalanine over L-tyrosine. Structure elucidation by protein X-ray crystallography confirmed that the two cyanobacterial PALs are similar in tertiary and quatenary structure to plant and yeast PALs as well as the mechanistically related histidine ammonia lyases.

Aromatic amino acid ammonia-lyases catalyze the deamination of L-His, L-Phe, and L-Tyr, yielding ammonia plus aryl acids bearing an alpha,beta-unsaturated propenoic acid. We report crystallographic analyses of unliganded Rhodobacter sphaeroides tyrosine ammonia-lyase (RsTAL) and RsTAL bound to p-coumarate and caffeate. His 89 of RsTAL forms a hydrogen bond with the p-hydroxyl moieties of coumarate and caffeate. His 89 is conserved in TALs but replaced in phenylalanine ammonia-lyases (PALs) and histidine ammonia-lyases (HALs). Substitution of His 89 by Phe, a characteristic residue of PALs, yields a mutant with a switch in kinetic preference from L-Tyr to L-Phe. Structures of the H89F mutant in complex with the PAL product, cinnamate, or the PAL-specific inhibitor, 2-aminoindan-2-phosphonate (AIP), support the role of position 89 as a specificity determinant in the family of aromatic amino acid ammonia-lyases and aminomutases responsible for beta-amino acid biosynthesis.

Differentiation-inducing factors (DIFs) are well known to modulate formation of distinct communal cell types from identical Dictyostelium discoideum amoebas, but DIF biosynthesis remains obscure. We report complimentary in vivo and in vitro experiments identifying one of two approximately 3,000-residue D. discoideum proteins, termed 'steely', as responsible for biosynthesis of the DIF acylphloroglucinol scaffold. Steely proteins possess six catalytic domains homologous to metazoan type I fatty acid synthases (FASs) but feature an iterative type III polyketide synthase (PKS) in place of the expected FAS C-terminal thioesterase used to off load fatty acid products. This new domain arrangement likely facilitates covalent transfer of steely N-terminal acyl products directly to the C-terminal type III PKS active sites, which catalyze both iterative polyketide extension and cyclization. The crystal structure of a steely C-terminal domain confirms conservation of the homodimeric type III PKS fold. These findings suggest new bioengineering strategies for expanding the scope of fatty acid and polyketide biosynthesis.

Protein folding barriers result from a combination of factors including unavoidable energetic frustration from nonnative interactions, natural variation and selection of the amino acid sequence for function, and/or selection pressure against aggregation. The rate-limiting step for human Pin1 WW domain folding is the formation of the loop 1 substructure. The native conformation of this six-residue loop positions side chains that are important for mediating protein-protein interactions through the binding of Pro-rich sequences. Replacement of the wild-type loop 1 primary structure by shorter sequences with a high propensity to fold into a type-I' beta-turn conformation or the statistically preferred type-I G1 bulge conformation accelerates WW domain folding by almost an order of magnitude and increases thermodynamic stability. However, loop engineering to optimize folding energetics has a significant downside: it effectively eliminates WW domain function according to ligand-binding studies. The energetic contribution of loop 1 to ligand binding appears to have evolved at the expense of fast folding and additional protein stability. Thus, the two-state barrier exhibited by the wild-type human Pin1 WW domain principally results from functional requirements, rather than from physical constraints inherent to even the most efficient loop formation process.

Corticotropin-releasing hormone (CRH), a potent neuropeptide, is produced by the placenta of anthropoid primates. No other mammals, including prosimian primates, are known to produce placental CRH. In humans, placental CRH appears to play an important role in the progression of pregnancy to parturition. Maternal circulating CRH begins to rise early in pregnancy and increases until parturition. Gorillas and chimpanzees share this pattern of increasing maternal CRH during pregnancy with humans. In humans, chimpanzees, and gorillas, maternal CRH and estradiol concentrations are correlated, consistent with the hypothesis that CRH is involved in the biosynthetic pathway for placental estrogen production. In contrast, in baboons, maternal circulating CRH rises precipitously early in pregnancy and then declines, though CRH is detectable until birth. This research was designed to investigate the pattern of maternal circulating CRH in the common marmoset during pregnancy. Blood samples were taken across gestation from nine subjects over 11 pregnancies, and the plasma was assayed for CRH. The pattern of maternal circulating CRH in the common marmoset was similar to that of the baboon, with a rapid rise starting at about 50 days postconception and a peak at approximately 70 days postconception. By 110 days postconception, CRH concentration had plateaued at a significantly lower value. The peak and mean values for CRH were associated with fetal number (e.g., females gestating triplets had higher values than females gestating twins). Urinary estradiol showed no association with plasma CRH concentration. Marmosets appear to differ from the great apes in this regard, and to share a pattern of maternal CRH during pregnancy with the baboon, indicating that the baboon and marmoset pattern may be ancestral. The function of the early rapid rise of CRH in baboons and marmosets, and the significance of this difference between monkeys and apes, are not known., (Copyright 2006 Wiley-Liss, Inc.)

In subjects with sinus rhythm, respiration has a profound effect on heart rate variability (HRV) at high frequencies (HF). Because this HF respiratory arrhythmia is lost in atrial fibrillation (AF), it has been assumed that respiration does not influence the ventricular response. However, previous investigations have not considered the possibility that respiration might influence HRV at lower frequencies. We hypothesized that Cheyne-Stokes respiration with central sleep apnea (CSR-CSA) would entrain HRV at very low frequency (VLF) in AF by modulating atrioventricular (AV) nodal refractory period and concealed conduction. Power spectral analysis of R-wave-to-R-wave (R-R) intervals and respiration during sleep were performed in 13 subjects with AF and CSR-CSA. As anticipated, no modulation of HRV was detected at HF during regular breathing. In contrast, VLF HRV was entrained by CSR-CSA [coherence between respiration and HRV of 0.69 (SD 0.22) at VLF during CSR-CSA vs. 0.20 (SD 0.19) at HF during regular breathing, P < 0.001]. Comparison of R-R intervals during CSR-CSA demonstrated a shorter AV node refractory period during hyperpnea than apnea [minimum R-R of 684 (SD 126) vs. 735 ms (SD 147), P < 0.001] and a lesser degree of concealed conduction [scatter of 178 (SD 56) vs. 246 ms (SD 72), P = 0.001]. We conclude that CSR-CSA entrains the ventricular response to AF, even in the absence of HF respiratory arrhythmia, by inducing rhythmic oscillations in AV node refractoriness and the degree of concealed conduction that may be a function of autonomic modulation of the AV node.

4,2',4',6'-Tetrahydroxychalcone (chalcone) and 4,2',4'-trihydroxychalcone (deoxychalcone) serve as precursors of ecologically important flavonoids and isoflavonoids. Deoxychalcone formation depends on chalcone synthase and chalcone reductase; however, the identity of the chalcone reductase substrate out of the possible substrates formed during the multistep reaction catalyzed by chalcone synthase remains experimentally elusive. We report here the three-dimensional structure of alfalfa chalcone reductase bound to the NADP+ cofactor and propose the identity and binding mode of its substrate, namely the non-aromatized coumaryl-trione intermediate of the chalcone synthase-catalyzed cyclization of the fully extended coumaryl-tetraketide thioester intermediate. In the absence of a ternary complex, the quality of the refined NADP+-bound chalcone reductase structure serves as a template for computer-assisted docking to evaluate the likelihood of possible substrates. Interestingly, chalcone reductase adopts the three-dimensional structure of the aldo/keto reductase superfamily. The aldo/keto reductase fold is structurally distinct from all known ketoreductases of fatty acid biosynthesis, which instead belong to the short-chain dehydrogenase/reductase superfamily. The results presented here provide structural support for convergent functional evolution of these two ketoreductases that share similar roles in the biosynthesis of fatty acids/polyketides. In addition, the chalcone reductase structure represents the first protein structure of a member of the aldo/ketoreductase 4 family. Therefore, the chalcone reductase structure serves as a template for the homology modeling of other aldo/keto-reductase 4 family members, including the reductase involved in morphine biosynthesis, namely codeinone reductase.

In bacteria, a structurally simple type III polyketide synthase (PKS) known as 1,3,6,8-tetrahydroxynaphthlene synthase (THNS) catalyzes the iterative condensation of five CoA-linked malonyl units to form a pentaketide intermediate. THNS subsequently catalyzes dual intramolecular Claisen and aldol condensations of this linear intermediate to produce the fused ring tetrahydroxynaphthalene (THN) skeleton. The type III PKS-catalyzed polyketide extension mechanism, utilizing a conserved Cys-His-Asn catalytic triad in an internal active site cavity, is fairly well understood. However, the mechanistic basis for the unusual production of THN and dual cyclization of its malonyl-primed pentaketide is obscure. Here we present the first bacterial type III PKS crystal structure, that of Streptomyces coelicolor THNS, and identify by mutagenesis, structural modeling, and chemical analysis the unexpected catalytic participation of an additional THNS-conserved cysteine residue in facilitating malonyl-primed polyketide extension beyond the triketide stage. The resulting new mechanistic model, involving the use of additional cysteines to alter and steer polyketide reactivity, may generally apply to other PKS reaction mechanisms, including those catalyzed by iterative type I and II PKS enzymes. Our crystal structure also reveals an unanticipated novel cavity extending into the "floor" of the traditional active site cavity, providing the first plausible structural and mechanistic explanation for yet another unusual THNS catalytic activity: its previously inexplicable extra polyketide extension step when primed with a long acyl starter. This tunnel allows for selective expansion of available active site cavity volume by sequestration of aliphatic starter-derived polyketide tails, and further suggests another distinct protection mechanism involving maintenance of a linear polyketide conformation.

Escherichia coli 2-C-methyl-D-erythritol-4-phosphate cytidyltransferase (YgbP or IspD) catalyzes the conversion of 2-C-methyl-D-erythritol 4-phosphate (MEP) and cytidine triphosphate (CTP) to 4-diphosphocytidyl-2-C-methylerythritol (CDPME). Pulse chase experiments established that the reaction involves an ordered sequential mechanism with mandatory initial binding of CTP. On the basis of analysis of the previously reported crystal structures of apo-YgbP as well as YgbP complexed with both CTP.Mg(2+) and CDPME.Mg(2+) [Richard, S. B., Bowman, M. E., Kwiatkowski, W., Kang, I., Chow, C., Lillo, A. M., Cane, D. E., and Noel, J. P. (2001) Nat. Struct. Biol. 8, 641-648], a group of active site residues were selected for site-directed mutagenesis and steady-state kinetic analysis. Both Lys27 and Lys213 were shown to be essential to catalytic activity, consistent with their proposed role in stabilization of a pentacoordinate phosphate transition state resulting from in-line attack of the MEP phosphate on the alpha-phosphate of CTP. In addition, Thr140, Arg109, Asp106, and Thr165 were all shown to play critical roles in the binding and proper orientation of the MEP substrate.

Stilbene synthase (STS) and chalcone synthase (CHS) each catalyze the formation of a tetraketide intermediate from a CoA-tethered phenylpropanoid starter and three molecules of malonyl-CoA, but use different cyclization mechanisms to produce distinct chemical scaffolds for a variety of plant natural products. Here we present the first STS crystal structure and identify, by mutagenic conversion of alfalfa CHS into a functional stilbene synthase, the structural basis for the evolution of STS cyclization specificity in type III polyketide synthase (PKS) enzymes. Additional mutagenesis and enzymatic characterization confirms that electronic effects rather than steric factors balance competing cyclization specificities in CHS and STS. Finally, we discuss the problematic in vitro reconstitution of plant stilbenecarboxylate pathways, using insights from existing biomimetic polyketide cyclization studies to generate a novel mechanistic hypothesis to explain stilbenecarboxylate biosynthesis.

The farnesoid X receptor (FXR) functions as a bile acid (BA) sensor coordinating cholesterol metabolism, lipid homeostasis, and absorption of dietary fats and vitamins. However, BAs are poor reagents for characterizing FXR functions due to multiple receptor independent properties. Accordingly, using combinatorial chemistry we evolved a small molecule agonist termed fexaramine with 100-fold increased affinity relative to natural compounds. Gene-profiling experiments conducted in hepatocytes with FXR-specific fexaramine versus the primary BA chenodeoxycholic acid (CDCA) produced remarkably distinct genomic targets. Highly diffracting cocrystals (1.78 A) of fexaramine bound to the ligand binding domain of FXR revealed the agonist sequestered in a 726 A(3) hydrophobic cavity and suggest a mechanistic basis for the initial step in the BA signaling pathway. The discovery of fexaramine will allow us to unravel the FXR genetic network from the BA network and selectively manipulate components of the cholesterol pathway that may be useful in treating cholesterol-related human diseases.

Ubiquitin ligases (E3) select proteins for ubiquitylation, a modification that directs altered subcellular trafficking and/or degradation of the target protein. HECT domain E3 ligases not only recognize, but also directly catalyze, ligation of ubiquitin to their protein substrates. The crystal structure of the HECT domain of the human ubiquitin ligase WWP1/AIP5 maintains a two-lobed structure like the HECT domain of the human ubiquitin ligase E6AP. While the individual N and C lobes of WWP1 possess very similar folds to those of E6AP, the organization of the two lobes relative to one another is different from E6AP due to a rotation about a polypeptide hinge linking the N and C lobes. Mutational analyses suggest that a range of conformations achieved by rotation about this hinge region is essential for catalytic activity.

In flavonoid, isoflavonoid, and anthocyanin biosynthesis, chalcone isomerase (CHI) catalyzes the intramolecular cyclization of chalcones into (S)-flavanones with a second-order rate constant that approaches the diffusion-controlled limit. The three-dimensional structures of alfalfa CHI complexed with different flavanones indicate that two sets of hydrogen bonds may possess critical roles in catalysis. The first set of interactions includes two conserved amino acids (Thr48 and Tyr106) that mediate a hydrogen bond network with two active site water molecules. The second set of hydrogen bonds occurs between the flavanone 7-hydroxyl group and two active site residues (Asn113 and Thr190). Comparison of the steady-state kinetic parameters of wild-type and mutant CHIs demonstrates that efficient cyclization of various chalcones into their respective flavanones requires both sets of contacts. For example, the T48A, T48S, Y106F, N113A, and T190A mutants exhibit 1550-, 3-, 30-, 7-, and 6-fold reductions in k(cat) and 2-3-fold changes in K(m) with 4,2',4'-trihydroxychalcone as a substrate. Kinetic comparisons of the pH-dependence of the reactions catalyzed by wild-type and mutant enzymes indicate that the active site hydrogen bonds contributed by these four residues do not significantly alter the pK(a) of the intramolecular cyclization reaction. Determinations of solvent kinetic isotope and solvent viscosity effects for wild-type and mutant enzymes reveal a change from a diffusion-controlled reaction to one limited by chemistry in the T48A and Y106F mutants. The X-ray crystal structures of the T48A and Y106F mutants support the assertion that the observed kinetic effects result from the loss of key hydrogen bonds at the CHI active site. Our results are consistent with a reaction mechanism for CHI in which Thr48 polarizes the ketone of the substrate and Tyr106 stabilizes a key catalytic water molecule. Hydrogen bonds contributed by Asn113 and Thr190 provide additional stabilization in the transition state. Conservation of these residues in CHIs from other plant species implies a common reaction mechanism for enzyme-catalyzed flavanone formation in all plants.

Type III polyketide synthases (PKS) generate an array of natural products by condensing multiple acetyl units derived from malonyl-CoA to thioester-linked starter molecules covalently bound in the PKS active site. One strategy adopted by Nature for increasing the functional diversity of these biosynthetic enzymes involves modifying polyketide assembly by altering the preference for starter molecules. Chalcone synthase (CHS) is a ubiquitous plant PKS and the first type III PKS described functionally and structurally. Guided by the three-dimensional structure of CHS, Phe-215 and Phe-265, which are situated at the active site entrance, were targeted for site-directed mutagenesis to diversify CHS activity. The resulting mutants were screened against a panel of aliphatic and aromatic CoA-linked starter molecules to evaluate the degree of starter molecule specificity in CHS. Although wild-type CHS accepts a number of natural CoA thioesters, it does not use N-methylanthraniloyl-CoA as a substrate. Substitution of Phe-215 by serine yields a CHS mutant that preferentially accepts this CoA-thioester substrate to generate a novel alkaloid, namely N-methylanthraniloyltriacetic acid lactone. These results demonstrate that a point mutation in CHS dramatically shifts the molecular selectivity of this enzyme. This structure-based approach to metabolic redesign represents an initial step toward tailoring the biosynthetic activity of plant type III PKS.

The enzyme 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (MECDP) synthase catalyzes the conversion of 4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (CDP-ME2P) to MECDP, a highly unusual cyclodiphosphate-containing intermediate on the mevalonate-independent pathway to isopentenyl diphosphate and dimethylallyl diphosphate. We now report two x-ray crystal structures of MECDP synthase refined to 2.8-A resolution. The first structure contains a bound Mn(2+) cation, and the second structure contains CMP, MECDP, and Mn(2+). The protein adopts a homotrimeric quaternary structure built around a central hydrophobic cavity and three externally facing active sites. Each of these active sites is located between two adjacent monomers. A tetrahedrally arranged transition metal binding site, potentially occupied by Mn(2+), sits at the base of the active site cleft. A phosphate oxygen of MECDP and the side chains of Asp(8), His(10), and His(42) occupy the metal ion coordination sphere. These structures reveal for the first time the structural determinants underlying substrate, product, and Mn(2+) recognition and the likely catalytic mechanism accompanying the biosynthesis of the cyclodiphosphate-containing isoprenoid precursor, MECDP.

Chalcone synthase (CHS) belongs to the family of type III polyketide synthases (PKS) that catalyze formation of structurally diverse polyketides. CHS synthesizes a tetraketide by sequential condensation of three acetyl anions derived from malonyl-CoA decarboxylation to a p-coumaroyl moiety attached to an active site cysteine. Gly256 resides on the surface of the CHS active site that is in direct contact with the polyketide chain derived from malonyl-CoA. Thus, position 256 serves as an ideal target to probe the link between cavity volume and polyketide chain-length determination in type III PKS. Functional examination of CHS G256A, G256V, G256L, and G256F mutants reveals altered product profiles from that of wild-type CHS. With p-coumaroyl-CoA as a starter molecule, the G256A and G256V mutants produce notably more tetraketide lactone. Further restrictions in cavity volume such as that seen in the G256L and G256F mutants yield increasing levels of the styrylpyrone bis-noryangonin from a triketide intermediate. X-ray crystallographic structures of the CHS G256A, G256V, G256L, and G256F mutants establish that these substitutions reduce the size of the active site cavity without significant alterations in the conformations of the polypeptide backbones. The side chain volume of position 256 influences both the number of condensation reactions during polyketide chain extension and the conformation of the triketide and tetraketide intermediates during the cyclization reaction. These results viewed in conjunction with the natural sequence variation of residue 256 suggest that rapid diversification of product specificity without concomitant loss of substantial catalytic activity in related CHS-like enzymes can occur by site-specific evolution of side chain volume at position 256.

Polyketide synthases (PKS) produce an array of natural products with different biological activities and pharmacological properties by varying the starter and extender molecules that form the final polyketide. Recent studies of the simplest PKS, the chalcone synthase (CHS)-like enzymes involved in the biosynthesis of flavonoids, anthocyanin pigments, and antimicrobial phytoalexins, have yielded insight on the molecular basis of this biosynthetic versatility. Understanding the structure-function relationship in these PKS provides a foundation for manipulating polyketide formation and suggests strategies for further increasing the scope of polyketide biosynthetic diversity.

The YgbP protein of Escherichia coli encodes the enzyme 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME) synthetase, a member of the cytidyltransferase family of enzymes. CDP-ME is an intermediate in the mevalonate-independent pathway for isoprenoid biosynthesis in a number of prokaryotic organisms, algae, the plant plastids and the malaria parasite. Because vertebrates synthesize isoprenoid precursors using a mevalonate pathway, CDP-ME synthetase and other enzymes of the mevalonate-independent pathway for isoprenoid production represent attractive targets for the structure-based design of selective antibacterial, herbicidal and antimalarial drugs. The high-resolution structures of E. coli CDP-ME synthetase in the apo form and complexed with both CTP-Mg2+ and CDP-ME-Mg2+ reveal the stereochemical principles underlying both substrate and product recognition as well as catalysis in CDP-ME synthetase. Moreover, these complexes represent the first experimental structures for any cytidyltransferase with both substrates and products bound.

In humans, placental corticotropin-releasing hormone (CRH) production has been linked to the determination of gestational length, and a late gestational fall in CRH-binding protein (CRH-BP) has been linked to the onset of parturition. Expression of placental CRH mRNA is limited to primates, and only in man has a circulating CRH-BP been described. As the fall in CRH-BP in late gestation has been associated with parturition in humans, we sought to determine whether a CRH-BP circulated in the plasma of other primates. It is unclear whether maternal plasma CRH concentrations are elevated in New World monkeys and prosimians. We have therefore performed CRH plasma measurements in the blood of pregnant marmosets, in several species of lemur, and in pregnant and fetal rhesus monkeys as a positive control. Using gel chromatography, CRH-BP was detected in the human, gorilla, chimpanzee, orangutan, gibbon, macaque, squirrel monkey, and marmoset, but was absent in the mandrill, spider monkey, and lemur. CRH was detected in the plasma of pregnant marmosets and rhesus monkeys. CRH was also detected in the fetal rhesus monkey, but at lower concentrations than in maternal plasma. CRH immunoreactivity was not detectable in the plasma of pregnant lemurs or in extracts of lemur placenta. In conclusion, a circulating binding protein for CRH exists in all species of apes but occurs variably among New World and Old World monkeys and is absent in lemurs. The variable occurrence of the CRH-BP does not support a role for this protein in the mechanism of parturition in primates. Maternal CRH is elevated in the pregnant marmoset and rhesus, and may play a role in the pregnancy of New and Old World monkeys.

Background: Polyketide synthases (PKSs) generate molecular diversity by utilizing different starter molecules and by controlling the final length of the polyketide. Although exploitation of this mechanistic variability has produced novel polyketides, the structural foundation of this versatility is unclear. Plant-specific PKSs are essential for the biosynthesis of anti-microbial phytoalexins, anthocyanin floral pigments, and inducers of Rhizobium nodulation genes. 2-Pyrone synthase (2-PS) and chalcone synthase (CHS) are plant-specific PKSs that share 74% amino acid sequence identity. 2-PS forms the triketide methylpyrone from an acetyl-CoA starter molecule and two malonyl-CoAs. CHS uses a p-coumaroyl-CoA starter molecule and three malonyl-CoAs to produce the tetraketide chalcone. Our goal was to elucidate the molecular basis of starter molecule selectivity and control of polyketide length in this class of PKS., Results: The 2.05 A resolution crystal structure of 2-PS complexed with the reaction intermediate acetoacetyl-CoA was determined by molecular replacement. 2-PS and CHS share a common three-dimensional fold, a set of conserved catalytic residues, and similar CoA binding sites. However, the active site cavity of 2-PS is smaller than the cavity in CHS. Of the 28 residues lining the 2-PS initiation/elongation cavity, four positions vary in CHS. Point mutations at three of these positions in CHS (T197L, G256L, and S338I) altered product formation. Combining these mutations in a CHS triple mutant (T197L/G256L/S338I) yielded an enzyme that was functionally identical to 2-PS., Conclusions: Structural and functional characterization of 2-PS together with generation of a CHS mutant with an initiation/elongation cavity analogous to 2-PS demonstrates that cavity volume influences the choice of starter molecule and controls the final length of the polyketide. These results provide a structural basis for control of polyketide length in other PKSs, and suggest strategies for further increasing the scope of polyketide biosynthetic diversity.

Chalcone isomerase (CHI) catalyzes the intramolecular cyclization of chalcone synthesized by chalcone synthase (CHS) into (2S)-naringenin, an essential compound in the biosynthesis of anthocyanin pigments, inducers of Rhizobium nodulation genes, and antimicrobial phytoalexins. The 1.85 A resolution crystal structure of alfalfa CHI in complex with (2S)-naringenin reveals a novel open-faced beta-sandwich fold. Currently, proteins with homologous primary sequences are found only in higher plants. The topology of the active site cleft defines the stereochemistry of the cyclization reaction. The structure and mutational analysis suggest a mechanism in which shape complementarity of the binding cleft locks the substrate into a constrained conformation that allows the reaction to proceed with a second-order rate constant approaching the diffusion controlled limit. This structure raises questions about the evolutionary history of this structurally unique plant enzyme.