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Triacylglycerols store metabolic energy in organisms and have industrial uses as foods and fuels. Excessive accumulation of triacylglycerols in humans causes obesity and is associated with metabolic diseases.sup.1. Triacylglycerol synthesis is catalysed by acyl-CoA diacylglycerol acyltransferase (DGAT) enzymes.sup.2-4, the structures and catalytic mechanisms of which remain unknown. Here we determined the structure of dimeric human DGAT1, a member of the membrane-bound O-acyltransferase (MBOAT) family, by cryo-electron microscopy at approximately 3.0 Å resolution. DGAT1 forms a homodimer through N-terminal segments and a hydrophobic interface, with putative active sites within the membrane region. A structure obtained with oleoyl-CoA substrate resolved at approximately 3.2 Å shows that the CoA moiety binds DGAT1 on the cytosolic side and the acyl group lies deep within a hydrophobic channel, positioning the acyl-CoA thioester bond near an invariant catalytic histidine residue. The reaction centre is located inside a large cavity, which opens laterally to the membrane bilayer, providing lipid access to the active site. A lipid-like density--possibly representing an acyl-acceptor molecule--is located within the reaction centre, orthogonal to acyl-CoA. Insights provided by the DGAT1 structures, together with mutagenesis and functional studies, provide the basis for a model of the catalysis of triacylglycerol synthesis by DGAT. Cryo-electron microscopy structures and functional and mutagenesis studies provide insights into the catalysis of triacylglycerol synthesis by human acyl-CoA diacylglycerol acyltransferase at its intramembrane active site., Author(s): Xuewu Sui [sup.1] [sup.2] , Kun Wang [sup.1] [sup.2] , Nina L. Gluchowski [sup.1] [sup.2] [sup.3] , Shane D. Elliott [sup.1] [sup.2] , Maofu Liao [sup.2] , Tobias C. [...]

Small RNAs such as small interfering RNAs (siRNAs) and microRNAs (miRNAs) silence the expression of their complementary target messenger RNAs.sup.1,2 via the formation of effector RNA-induced silencing complexes (RISCs), which contain Argonaute (Ago) family proteins at their core. Although loading of siRNA duplexes into Drosophila Ago2 requires the Dicer-2-R2D2 heterodimer.sup.3,4,5 and the Hsc70/Hsp90 (Hsp90 also known as Hsp83) chaperone machinery.sup.6,7,8, the details of RISC assembly remain unclear. Here we reconstitute RISC assembly using only Ago2, Dicer-2, R2D2, Hsc70, Hsp90, Hop, Droj2 (an Hsp40 homologue) and p23. By following the assembly of single RISC molecules, we find that, in the absence of the chaperone machinery, an siRNA bound to Dicer-2-R2D2 associates with Ago2 only transiently. The chaperone machinery extends the dwell time of the Dicer-2-R2D2-siRNA complex on Ago2, in a manner dependent on recognition of the 5'-phosphate on the siRNA guide strand. We propose that the chaperone machinery supports a productive state of Ago2, allowing it to load siRNA duplexes from Dicer-2-R2D2 and thereby assemble RISC. The assembly of single Drosophila RNA-induced silencing complexes (RISCs) is reconstituted using seven purified proteins, revealing that chaperones help stabilize the interaction of the protein heterodimer Dicer-2-R2D2 bound to the short interfering RNA with Ago2. A functioning RISC complex reconstituted The central effector involved in RNA silencing is RISC, the RNA-induced silencing complex, composed of an Argonaute (Ago) protein bound to a small interfering RNA (siRNA). Yukihide Tomari and colleagues have now reconstituted the assembly of single Drosophila RISC molecules using eight purified proteins. They find that the chaperones help to stabilize the interaction of the siRNA, which is initially bound to a complex of Dicer-2 and R2D2, with Ago2. This stabilization allows Ago2 to recognize the 5'-phosphate of the siRNA guide strand, after which the guide strand is transferred onto Ago2., Author(s): Shintaro Iwasaki [sup.1] [sup.4] , Hiroshi M. Sasaki [sup.1] , Yuriko Sakaguchi [sup.2] , Tsutomu Suzuki [sup.2] , Hisashi Tadakuma [sup.3] , Yukihide Tomari [sup.1] [sup.3] Author Affiliations: (1) [...]

This study shows the structural and biochemical characterization of a new type of polyketide synthase module that catalyses the vinylogous addition of a malonyl unit to an unsaturated thioester, generating a branch in the growing polyketide chain; this characterization provides a mechanism by which the structural diversity of polyketide natural products can be increased. A new route to polyketides Polyketide synthases (PKSs) are modular enzyme complexes that produce polyketides, a large class of secondary metabolites -- otherwise known as natural products. In this manuscript, the authors identify and characterize a novel PKS module from the endofungal bacterium Burkholderia rhizoxinica that catalyses a vinylogous addition of a malonyl unit to an unsaturated thioester, generating a branch in the growing polyketide chain. This enzymological activity is very unusual; all previously studied PKS modules catalyse the head-to-tail fusion of acyl and malonyl units, yielding a linear polyketide chain. This newly discovered reaction provides a simple mechanism by which structural diversity of polyketide natural products can be increased. Bacteria use modular polyketide synthases (PKSs) to assemble complex polyketides, many of which are leads for the development of clinical drugs, in particular anti-infectives and anti-tumoral agents.sup.1. Because these multifarious compounds are notoriously difficult to synthesize, they are usually produced by microbial fermentation. During the past two decades, an impressive body of knowledge on modular PKSs.sup.2,3 has been gathered that not only provides detailed insight into the biosynthetic pathways but also allows the rational engineering of enzymatic processing lines to yield structural analogues.sup.4,5. Notably, a hallmark of all PKS modules studied so far is the head-to-tail fusion of acyl and malonyl building blocks, which leads to linear backbones. Yet, structural diversity is limited by this uniform assembly mode. Here we demonstrate a new type of PKS module from the endofungal bacterium Burkholderia rhizoxinica that catalyses a Michael-type acetyl addition to generate a branch in the carbon chain. In vitro reconstitution of the entire PKS module, X-ray structures of a ketosynthase-branching didomain and mutagenesis experiments revealed a crucial role of the ketosynthase domain in branching the carbon chain. We present a trapped intermediary state in which acyl carrier protein and ketosynthase are covalently linked by the branched polyketide and suggest a new mechanism for chain alkylation, which is functionally distinct from terpenoid-like [beta]-branching. For the rice seedling blight toxin rhizoxin, one of the strongest known anti-mitotic agents, the non-canonical polyketide modification is indispensable for phytotoxic and anti-tumoral activities. We propose that the formation of related pharmacophoric groups follows the same general scheme and infer a unifying vinylogous branching reaction for PKS modules with a ketosynthase-branching-acyl-carrier-protein architecture. This study unveils the structure and function of a new PKS module that broadens the biosynthetic scope of polyketide biosynthesis and sets the stage for rationally creating structural diversity., Author(s): Tom Bretschneider [sup.1] , Joel B. Heim [sup.2] , Daniel Heine [sup.1] , Robert Winkler [sup.1] , Benjamin Busch [sup.1] , Björn Kusebauch [sup.1] , Thilo Stehle [sup.2] [sup.3] [...]

Oxygen is critically important to metazoan life, and the EGL-9/PHD enzymes are key regulators of hypoxia (low oxygen) response. When oxygen levels are high, the EGL-9/PHD proteins hydroxylate hypoxia-inducible factor (HIF) transcription factors. Once hydroxylated, HIF? subunits bind to von Hippel-Lindau (VHL) E3 ligases and are degraded. Prior genetic analyses in Caenorhabditis elegans had shown that EGL-9 also acted through a vhl-1-independent pathway to inhibit HIF-1 transcriptional activity. Here, we characterize this novel EGL-9 function. We employ an array of complementary methods to inhibit EGL-9 hydroxylase activity in vivo. These include hypoxia, hydroxylase inhibitors, mutation of the proline in HIF-1 that is normally modified by EGL-9, and mutation of the EGL-9 catalytic core. Remarkably, we find that each of these treatments or mutations eliminates oxygen-dependent degradation of HIF-1 protein, but none of them abolishes EGL-9-mediated repression of HIF-1 transcriptional activity. Further, analyses of new egl-9 alleles reveal that the evolutionarily conserved EGL-9 MYND zinc finger domain does not have a major role in HIF-1 regulation. We conclude that C. elegans EGL-9 is a bifunctional protein. In addition to its well-established role as the oxygen sensor that regulates HIF-1 protein levels, EGL-9 inhibits HIF-1 transcriptional activity via a pathway that has little or no requirement for hydroxylase activity or for the EGL-9 MYND domain.

The yellow seed characteristic in Brassica napus L. is desirable because of its association with higher oil content and better quality of oil-extracted meal. YN01-429 is a yellow-seeded canola-quality germplasm developed in Canada arising from several years of research. Seed-coat pigmentation is due to oxidized proanthocyanidins (PA; condensed tannins) derived from phenylpropanoids and malonyl CoA. We found PA accumulation to be most robust in young seed coats (20 d post anthesis; dpa) of a related black-seeded line N89-53 and only very little PA in YN01-429, which also contained much less extractable phenolics. The flavonol content, however, did not show as great a difference between these two lines. Furthermore, sinapine, a product of the general phenylpropanoid metabolism, was present at comparable levels in the embryos of both lines. Dihydroflavonol reductase (DFR) activity that commits phenolics to PA synthesis was lower in YN01-429 seed coats. The results of Southern blot and in silico analyses were indicative of two copies of the DFR gene in B. napus. Both copies were functional in YN01-429, ruling out homeoallelic repression or silencing, but together they showed very low expression levels (17-fold fewer transcripts) relative to DFR activity in N89-53 seed coats. These results collectively suggest that YN01-429 differs in regulatory circuits that impact the PA synthesis branch much more than the flavonol synthesis branch in the seed coats and such circuits do not impinge upon general phenylpropanoid metabolism in the embryos. Key words: condensed tannin, dihydroflavonol reductase, flavonoids, oilseed rape, rapeseed, transparent testa. Le jaune caracteristique des grains du Brassica napus L. est desirable, compte tenu de son association avec une haute teneur en huile et une meilleure qualite de la farine extraite de l'huile. Le YN01-429 constitue un germplasme a graine jaune de qualite canola resultant de plusieurs annees de recherches. La pigmentation des teguments est attribuee a des proanthocyanidines oxydees (PA; tannins condenses) derivees de phenylpropanoides et du CoA du malonyl. Les auteurs ont trouve une plus forte accumulation de PA chez les jeunes teguments (20 jours apres l'anthese; dpa) d'une lignee apparentee a grains noirs N89-53 et tres peu de PA chez la lignee YN01-429 qui contient a la fois beaucoup moins de phenols extractibles. Cependant, la teneur en flavonols ne montre pas autant de differences entre ces deux lignees. De plus, la sinapine, un produit du metabolisme general des phenylpropanoides, se retrouve a des teneurs comparables chez les embryons des deux lignees. L'activite de la reductase du dihydroflavonol (DFR) qui pousse les phenols vers la synthese de PA, est plus faible dans les teguments du YN01-429. Les resultats du transfert Southern et des analyses in silico indiquent la presence de deux copies du gene DFR chez le B. napus. Les deux copies sont fonctionnelles chez le YNO1429, ce qui elimine la repression ou le silencing homeoallelique, mais ensemble elles ne montrent que de tres faibles degres d'expression (transcriptions 17 fois inferieures) par rapport a l'activite du DFR chez les teguments du N89-53. Ces resultats pris dans leur ensemble suggerent que le YN01-429 differe a l'echelle des circuits qui agissent sur la ligne de synthese des PA beaucoup plus que sur la ligne de synthese des flavonols dans les teguments, et de tels circuits n'affectent pas le metabolisme general des phenylpropanoides chez les embryons. Mots-cles : tannin condense, reductase du dihydroflavonol, flavonoides, colza, oleagineux, colza, teguments transparents. [Traduit par la Redaction], Introduction Canola refers to lines of Brassica napus L. and Brassica rapa L. that have much reduced erucic acid ( The importance of the seed coat was recognized very early [...]

We have investigated the hitherto unexplored possibility that differences in the catalytic efficiencies of thymidylate synthases ThyX and ThyA, enzymes that produce the essential DNA precursor dTMP, have influenced prokaryotic genome evolution. We demonstrate that DNA replication speed in bacteria and archaea that contain the low-activity ThyX enzyme is up to 10-fold decreased compared with species that contain the catalytically more efficient ThyA. Our statistical studies of >400 genomes indicated that ThyA proteins are preferred for the replication of large genomes, providing further evidence that the thymidylate metabolism is limiting expansion of prokaryotic genomes. Because both ThyX and ThyA participate in frequent reciprocal gene replacement events, our observations indicate that the bacterial metabolism continues to modulate the size and composition of prokaryotic genomes. We also propose that the increased kinetic efficiency of thymidylate synthesis has contributed to extending the prokaryotic evolutionary potential. genomeevolution | genereplacement | nucleotidemetabolisml PBCV-1 | thymidylate synthesis

Choline cytidylyltransferase (CCT) is the rate-limiting enzyme in the phosphatidylcholine biosynthetic pathway. Here, we demonstrate that CCT[alpha]-mediated phosphatidylcholine synthesis is required to maintain normal Golgi structure and function as well as cytokine secretion from the Golgi complex. CCT[alpha] is localized to the trans-Golgi region and its expression is increased in lipopolysaccharide (LPS)-stimulated wild-type macrophages. Although LPS triggers transient reorganization of Golgi morphology in wild-type macrophages, similar structural alterations persist in CCT[alpha]-deficient cells. Pro--tumor necrosis factor [alpha] and interleukin-6 remain lodged in the secretory compartment of CCT[alpha]-deficient macrophages after LPS stimulation. However, the lysosomal-mediated secretion pathways for interleukin-1[beta] secretion and constitutive apolipoprotein E secretion are unaltered. Exogenous lysophosphatidylcholine restores LPS-stimulated secretion from CCT[alpha]-deficient cells, and elevated diacylglycerol levels alone do not impede secretion of pro-tumor necrosis factor [alpha] or interleukin-6. These results identify CCT[alpha] as a key component in membrane biogenesis during LPS-stimulated cytokine secretion from the Golgi complex.

The design of new enzymes for reactions not catalysed by naturally occurring biocatalysts is a challenge for protein engineering and is a critical test of our understanding of enzyme catalysis. [...]

The significance and the role of the ancestral kinase, aarF-domain-containing kinase 3 gene (ADCK3) in the ubiquinone synthesis are discussed. ADCK3 mutations are found to regulate the production of ATP, hence leading to the deficiency of coenzyme [Q.sub.10].

The significance of the presence of CABC1 genes for the functioning of the ubiquinone biosynthesis pathway is discussed. The CABC1 gene mutations are found to lead to severe changes in the protein function, hence leading to the ubiquinone deficiency with cerebellar ataxia and seizures.

Capsazepine (CPZ), a synthetic capsaicin analogue, inhibits ATP hydrolysis by Na,K-ATPase in the presence but not in the absence of [K.sup.+]. Studies with purified membranes revealed that CPZ reduced [Na.sup.+]-dependent phosphorylation by interference with [Na.sup.+] binding from the intracellular side of the membrane. Kinetic analyses showed that CPZ stabilized an enzyme species that constitutively occluded [K.sup.+]. Low-affinity ATP interaction with the enzyme was strongly reduced after CPZ treatment; in contrast, indirectly measured interaction with ADP was much increased, which suggests that composite regulatory communication with nucleotides takes place during turnover. Studies with lipid vesicles revealed that CPZ reduced ATP-dependent digitoxigenin-sensitive [sup.22][Na.sup.+] influx into [K.sup.+]-loaded vesicles only at saturating ATP concentrations. The drug apparently abolishes the regulatory effect of ATP on the pump. Drawing on previous homology modeling studies of Na,K-ATPase to atomic models of sarcoplasmic reticulum Ca-ATPase and on kinetic data, we propose that CPZ uncouples an [Na.sup.+] cycle from an [Na.sup.+]/[K.sup.+] cycle in the pump. The [Na.sup.+] cycle possibly involves transport through the recently characterized [Na.sup.+]-specific site. A shift to such an uncoupled mode is believed to produce pumps mediating uncoupled [Na.sup.+] efflux by modifying the transport stoichiometry of single pump units. sodium pump | uncoupling | potassium occlusion

The sex pheromone of the female processionary moth, Thaumetopoea pityocampa, is a unique C16 enyne acetate that is biosynthesized from paimitic acid. Three consecutive desaturation reactions transform this saturated precursor into the triunsaturated fatty acyl intermediate: formation of (Z)-11-hexadecenoic acid, acetylenation to 11-hexadecynoic acid, and final [[DELTA].sup.13] desaturation to (Z)-13-hexadecen-11-ynoic acid. By using degenerate primers common to all reported insect desaturases, a single cDNA sequence was isolated from total RNA of T. pityocampa female pheromone glands. The full-length transcript of this putative desaturase was expressed in elo1[DELTA]/ole1[DELTA] yeast mutants (both elongase 1 and [[DELTA].sup.9] desaturase-deficient) for functional assays. The construct fully rescued the [DELTA]ole1 yeast phenotype, confirming its desaturase activity. Analysis of the unsaturated products from transformed yeast extracts demonstrated that the cloned enzyme showed 411 desaturase, [[DELTA].sup.11] acetylenase, and [[DELTA].sup.13] desaturase activities. Therefore, this single desaturase may account for the three desaturation steps involved in the sex pheromone biosynthetic pathway of the processionary moth. acetylene | cloning | enyne | Thaumetopoea pityocampa | yeast

The biosynthesis of rebeccamycin, an antitumor compound, involves the remarkable eight-electron oxidation of chlorinated chromopyrrolic acid. Although one rebeccamycin biosynthetic enzyme is capable of generating low levels of the eight-electron oxidation product on its own, a second protein, RebC, is required to accelerate product formation and eliminate side reactions. However, the mode of action of RebC was largely unknown. Using crystallography, we have determined a likely function for RebC as a flavin hydroxylase, captured two snapshots of its dynamic catalytic cycle, and trapped a reactive molecule, a putative substrate, in its binding pocket. These studies strongly suggest that the role of RebC is to sequester a reactive intermediate produced by its partner protein and to react with it enzymatically, preventing its conversion to a suite of degradation products that includes, at low levels, the desired product. flavin enzymes | x-ray crystallography | indolocarbazoles | antitumor

A metabolic pathway for biosynthesis of the nonreducing disaccharide mannosylfructose ([beta]-fructofuranosyl-[alpha]-mannopyranoside), an important osmolyte in Agrobacterium tumefaciens, was discovered. We have identified and functionally characterized two ORFs that correspond to genes (named mfpsA and mfppA) encoding the rare enzymes mannosylfructose-phosphate synthase and mannosylfructose-phosphate phosphatase, an associated phosphohydrolase. The mfpsA and mfppA genes are arranged in an operon structure, whose transcription is up-regulated by NaCI, resulting in the accumulation of mannosylfructose in the cells. Not only is the biosynthesis of mannosylfructose mechanistically similar to that of sucrose, but the corresponding genes for the biosynthesis of both disaccharides are also phylogenetic close relatives. Importantly, a protein phylogeny analysis indicated that mannosylfructose-phosphate synthase defines a unique group of mannosyltransferases. glycosyltransferase evolution | functional diversification | salt stress response | Agrobacterium-plant interaction | sucrose

The article presents a detailed analysis of the history of the arginine biosynthetic enzyme. The results reveal that the added, as well as the internally produced arginine highly represses the enzyme formation, as the potential required for this enzyme synthesis is found to be in excess of what is required.