Crystal Structures of the CERT START Domain with Inhibitors Provide Insights into the Mechanism of Ceramide Transfer

The cytosolic protein CERT transfers ceramide from the endoplasmic reticulum to the Golgi apparatus where ceramide is converted to SM. The C-terminal START (steroidogenic acute regulatory protein-related lipid transfer) domain of CERT binds one ceramide molecule in its central amphiphilic cavity. (1R,3R)-N-(3-Hydroxy-1-hydroxymethyl-3-phenylpropyl)alkanamide (HPA), a synthesized analogue of ceramide, inhibits ceramide transfer by CERT. Here we report crystal structures of the CERT START domain in complex with HPAs of varying acyl chain lengths. In these structures, one HPA molecule is buried in the amphiphilic cavity where the amide and hydroxyl groups of HPA form a hydrogen-bond network with specific amino acid residues. The Ω1 loop, which has been suggested to function as a gate of the cavity, adopts a different conformation when bound to HPA than when bound to ceramide. In the Ω1 loop region, Trp473 shows the largest difference between these two structures. This residue exists inside of the cavity in HPA-bound structures, while it is exposed to the outside of the protein in the apo-form and ceramide-bound complex structures. Surface plasmon resonance experiments confirmed that Trp473 is important for interaction with membranes. These results provide insights into not only the molecular mechanism of inhibition by HPAs but also possible mechanisms by which CERT interacts with ceramide.     

Crystal and EM Structures of Human Phosphoribosyl Pyrophosphate Synthase I (PRS1) Provide Novel Insights into the Disease-Associated Mutations

Human PRS1, which is indispensable for the biosynthesis of nucleotides, deoxynucleotides and their derivatives, is associated directly with multiple human diseases because of single base mutation. However, a molecular understanding of the effect of these mutations is hampered by the lack of understanding of its catalytic mechanism. Here, we reconstruct the 3D EM structure of the PRS1 apo state. Together with the native stain EM structures of AMPNPP, AMPNPP and R5P, ADP and the apo states with distinct conformations, we suggest the hexamer is the enzymatically active form. Based on crystal structures, sequence analysis, mutagenesis, enzyme kinetics assays, and MD simulations, we reveal the conserved substrates binding motifs and make further analysis of all pathogenic mutants.     

Crystal Structure and Computational Analyses Provide Insights into the Catalytic Mechanism of 2,4-Diacetylphloroglucinol Hydrolase PhlG from Pseudomonas fluorescens

2,4-Diacetylphloroglucinol hydrolase PhlG from Pseudomonas fluorescens catalyzes hydrolytic carbon-carbon (C–C) bond cleavage of the antibiotic 2,4-diacetylphloroglucinol to form monoacetylphloroglucinol, a rare class of reactions in chemistry and biochemistry. To investigate the catalytic mechanism of this enzyme, we determined the three-dimensional structure of PhlG at 2.0 Å resolution using x-ray crystallography and MAD methods. The overall structure includes a small N-terminal domain mainly involved in dimerization and a C-terminal domain of Bet v1-like fold, which distinguishes PhlG from the classical α/β-fold hydrolases. A dumbbell-shaped substrate access tunnel was identified to connect a narrow interior amphiphilic pocket to the exterior solvent. The tunnel is likely to undergo a significant conformational change upon substrate binding to the active site. Structural analysis coupled with computational docking studies, site-directed mutagenesis, and enzyme activity analysis revealed that cleavage of the 2,4-diacetylphloroglucinol C–C bond proceeds via nucleophilic attack by a water molecule, which is coordinated by a zinc ion. In addition, residues Tyr¹²¹, Tyr²²⁹, and Asn¹³², which are predicted to be hydrogen-bonded to the hydroxyl groups and unhydrolyzed acetyl group, can finely tune and position the bound substrate in a reactive orientation. Taken together, these results revealed the active sites and zinc-dependent hydrolytic mechanism of PhlG and explained its substrate specificity as well.     

Crystal Structures of Human HMG-CoA Synthase Isoforms Provide Insights into Inherited Ketogenesis Disorders and Inhibitor Design

3-Hydroxy-3-methylglutaryl coenzyme A (CoA) synthase (HMGCS) catalyzes the condensation of acetyl-CoA and acetoacetyl-CoA into 3-hydroxy-3-methylglutaryl CoA. It is ubiquitous across the phylogenetic tree and is broadly classified into three classes. The prokaryotic isoform is essential in Gram-positive bacteria for isoprenoid synthesis via the mevalonate pathway. The eukaryotic cytosolic isoform also participates in the mevalonate pathway but its end product is cholesterol. Mammals also contain a mitochondrial isoform; its deficiency results in an inherited disorder of ketone body formation. Here, we report high-resolution crystal structures of the human cytosolic (hHMGCS1) and mitochondrial (hHMGCS2) isoforms in binary product complexes. Our data represent the first structures solved for human HMGCS and the mitochondrial isoform, allowing for the first time structural comparison among the three isoforms. This serves as a starting point for the development of isoform-specific inhibitors that have potential cholesterol-reducing and antibiotic applications. In addition, missense mutations that cause mitochondrial HMGCS deficiency have been mapped onto the hHMGCS2 structure to rationalize the structural basis for the disease pathology.     

Two Crystal Structures of Pneumococcal Pilus Sortase C Provide Novel Insights into Catalysis and Substrate Specificity

The respiratory tract pathogen Streptococcus pneumoniae is a primary cause of morbidity and mortality worldwide. Pili enhance initial adhesion as well as the capacity of pneumococci to cause pneumonia and bacteremia. Pilus-associated sortases (SrtB, SrtC, and SrtD) are involved in the biogenesis of pneumococcal pili, composed of repeating units of RrgB that create the stalk to which the RrgA adhesin and the preferential pilus tip subunit RrgC are covalently associated. Using single sortase-expressing strains, we demonstrate that both pilin-polymerizing sortases SrtB and SrtC can covalently link pili to the peptidoglycan cell wall, a property shared with the non-pilus-polymerizing enzyme SrtD and the housekeeping sortase SrtA. Comparative analysis of the crystal structures of S. pneumoniae SrtC and SrtB revealed structural differences explaining the incapacity of SrtC, but not of SrtB, to incorporate RrgC into the pilus. Accordingly, site-directed mutagenesis of Thr¹⁶⁰ in SrtB to an arginine as in SrtC (Arg¹⁶⁰) partially converted its substrate specificity into that of SrtC. Solving two crystal structures for SrtC suggests that an opening of a flexible lid and a concomitant cysteine rotation are important for catalysis and the activation of the catalytic cysteine of pilus-associated sortases.     

Insights into the Catalytic Mechanism of 16S rRNA Methyltransferase RsmE (mU1498) from Crystal and Solution Structures

RsmE is the founding member of a new RNA methyltransferase (MTase) family responsible for methylation of U1498 in 16S ribosomal RNA in Escherichia coli. It is well conserved across bacteria and plants and may play an important role in ribosomal intersubunit communication. The crystal structure in monomer showed that it consists of two distinct but structurally related domains: the PUA (pseudouridine synthases and archaeosine‐specific transglycosylases)-like RNA recognition and binding domain and the conserved MTase domain with a deep trefoil knot. Analysis of small-angle X-ray scattering data revealed that RsmE forms a flexible dimeric conformation that may be essential for substrate binding. The S‐adenosyl‐l‐methionine (AdoMet)-binding characteristic determined by isothermal titration calorimetry suggested that there is only one AdoMet molecule bound in the subunit of the homodimer. In vitro methylation assay of the mutants based on the RsmE-AdoMet-uridylic acid complex model showed key residues involved in substrate binding and catalysis. Comprehensive comparisons of RsmE with closely related MTases, combined with the biochemical experiments, indicated that the MTase domain of one subunit in dimeric RsmE is responsible for binding of one AdoMet molecule and catalytic process while the PUA-like domain in the other subunit is mainly responsible for recognition of one substrate molecule (the ribosomal RNA fragment and ribosomal protein complex). The methylation process is required by collaboration of both subunits, and dimerization is functionally critical for catalysis. In general, our study provides new information on the structure-function relationship of RsmE and thereby suggests a novel catalytic mechanism.     

Canine CNGA3 Gene Mutations Provide Novel Insights into Human Achromatopsia-Associated Channelopathies and Treatment

Cyclic nucleotide-gated (CNG) ion channels are key mediators underlying signal transduction in retinal and olfactory receptors. Genetic defects in CNGA3 and CNGB3, encoding two structurally related subunits of cone CNG channels, lead to achromatopsia (ACHM). ACHM is a congenital, autosomal recessive retinal disorder that manifests by cone photoreceptor dysfunction, severely reduced visual acuity, impaired or complete color blindness and photophobia. Here, we report the first canine models for CNGA3-associated channelopathy caused by R424W or V644del mutations in the canine CNGA3 ortholog that accurately mimic the clinical and molecular features of human CNGA3-associated ACHM. These two spontaneous mutations exposed CNGA3 residues essential for the preservation of channel function and biogenesis. The CNGA3-R424W results in complete loss of cone function in vivo and channel activity confirmed by in vitro electrophysiology. Structural modeling and molecular dynamics (MD) simulations revealed R424-E306 salt bridge formation and its disruption with the R424W mutant. Reversal of charges in a CNGA3-R424E-E306R double mutant channel rescued cGMP-activated currents uncovering new insights into channel gating. The CNGA3-V644del affects the C-terminal leucine zipper (CLZ) domain destabilizing intersubunit interactions of the coiled-coil complex in the MD simulations; the in vitro experiments showed incompetent trimeric CNGA3 subunit assembly consistent with abnormal biogenesis of in vivo channels. These newly characterized large animal models not only provide a valuable system for studying cone-specific CNG channel function in health and disease, but also represent prime candidates for proof-of-concept studies of CNGA3 gene replacement therapy for ACHM patients.     

Structural Insights into the Unique Activation Mechanisms of a Non-classical Calpain and Its Disease-Causing Variants

1. Download : Download high-res image (214KB)
2. Download : Download full-size image

     

Integrative Analyses of De Novo Mutations Provide Deeper Biological Insights into Autism Spectrum Disorder

1. Download high-res image (188KB)
2. Download full-size image

     

Ligand-bound Structures Provide Atomic Snapshots for the Catalytic Mechanism of d-Amino Acid Deacylase

d-tyrosyl-tRNA^Tyr deacylase (DTD) is an editing enzyme that removes d-amino acids from mischarged tRNAs. We describe an in-depth analysis of the malaria parasite Plasmodium falciparum DTD here. Our data provide structural insights into DTD complexes with adenosine and d-amino acids. Bound adenosine is proximal to the DTD catalysis site, and it represents the authentic terminal adenosine of charged tRNA. DTD-bound d-amino acids cluster at three different subsites within the overall active site pocket. These subsites, called transition, active, and exit subsites allow docking, re-orientation, chiral selection, catalysis, and exit of the free d-amino acid from DTD. Our studies reveal variable modes of d-amino acid recognition by DTDs, suggesting an inherent plasticity that can accommodate all d- amino acids. An in-depth analysis of native, ADP-bound, and d- amino acid-complexed DTD structures provide the first atomic snapshots of ligand recognition and subsequent catalysis by this enzyme family. We have mapped sites for the deacylation reaction and mark possible routes for entry and egress of all substrates and products. We have also performed structure-based inhibitor discovery and tested lead compounds against the malaria parasite P. falciparum using growth inhibition assays. Our studies provide a comprehensive structural basis for the catalytic mechanism of DTD enzymes and have implications for inhibition of this enzyme in P. falciparum as a route to inhibiting the parasite.     

Structural Insights into the Catalytic Mechanism of Bacterial Guanosine-diphospho-d-mannose Pyrophosphorylase and Its Regulation by Divalent Ions

GMP catalyzes the formation of GDP-Man, a fundamental precursor for protein glycosylation and bacterial cell wall and capsular polysaccharide biosynthesis. Crystal structures of GMP from the thermophilic bacterium Thermotoga maritima in the apo form, in complex with the substrates mannose-1-phosphate or GTP and bound with the end product GDP-Man in the presence of the essential divalent cation Mg²⁺, were solved in the 2.1–2.8 Å resolution range. The T. maritima GMP molecule is organized in two separate domains: a N-terminal Rossman fold-like domain and a C-terminal left-handed β-helix domain. Two molecules associate into a dimer through a tail-to-tail arrangement of the C-terminal domains. Comparative analysis of the structures along with characterization of enzymatic parameters reveals the bases of substrate specificity of this class of sugar nucleotidyltransferases. In particular, substrate and product binding are associated with significant changes in the conformation of loop regions lining the active center and in the relative orientation of the two domains. Involvement of both the N- and C-terminal domains, coupled to the catalytic role of a bivalent metal ion, highlights the catalytic features of bacterial GMPs compared with other members of the pyrophosphorylase superfamily.     

Theoretical Insights into Catalytic Mechanism of Protein Arginine Methyltransferase 1

Protein arginine methyltransferase 1 (PRMT1), the major arginine asymmetric dimethylation enzyme in mammals, is emerging as a potential drug target for cancer and cardiovascular disease. Understanding the catalytic mechanism of PRMT1 will facilitate inhibitor design. However, detailed mechanisms of the methyl transfer process and substrate deprotonation of PRMT1 remain unclear. In this study, we present a theoretical study on PRMT1 catalyzed arginine dimethylation by employing molecular dynamics (MD) simulation and quantum mechanics/molecular mechanics (QM/MM) calculation. Ternary complex models, composed of PRMT1, peptide substrate, and S-adenosyl-methionine (AdoMet) as cofactor, were constructed and verified by 30-ns MD simulation. The snapshots selected from the MD trajectory were applied for the QM/MM calculation. The typical S_N2-favored transition states of the first and second methyl transfers were identified from the potential energy profile. Deprotonation of substrate arginine occurs immediately after methyl transfer, and the carboxylate group of E144 acts as proton acceptor. Furthermore, natural bond orbital analysis and electrostatic potential calculation showed that E144 facilitates the charge redistribution during the reaction and reduces the energy barrier. In this study, we propose the detailed mechanism of PRMT1-catalyzed asymmetric dimethylation, which increases insight on the small-molecule effectors design, and enables further investigations into the physiological function of this family.     

Crystal Structures of Physcomitrella patens AOC1 and AOC2: Insights into the Enzyme Mechanism and Differences in Substrate Specificity

In plants, oxylipins regulate developmental processes and defense responses. The first specific step in the biosynthesis of the cyclopentanone class of oxylipins is catalyzed by allene oxide cyclase (AOC) that forms cis(+)-12-oxo-phytodienoic acid. The moss Physcomitrella patens has two AOCs (PpAOC1 and PpAOC2) with different substrate specificities for C₁₈- and C₂₀-derived substrates, respectively. To better understand AOC’s catalytic mechanism and to elucidate the structural properties that explain the differences in substrate specificity, we solved and analyzed the crystal structures of 36 monomers of both apo and ligand complexes of PpAOC1 and PpAOC2. From these data, we propose the following intermediates in AOC catalysis: (1) a resting state of the apo enzyme with a closed conformation, (2) a first shallow binding mode, followed by (3) a tight binding of the substrate accompanied by conformational changes in the binding pocket, and (4) initiation of the catalytic cycle by opening of the epoxide ring. As expected, the substrate dihydro analog cis-12,13S-epoxy-9Z,15Z-octadecadienoic acid did not cyclize in the presence of PpAOC1; however, when bound to the enzyme, it underwent isomerization into the corresponding trans-epoxide. By comparing complex structures of the C₁₈ substrate analog with in silico modeling of the C₂₀ substrate analog bound to the enzyme allowed us to identify three major molecular determinants responsible for the different substrate specificities (i.e. larger active site diameter, an elongated cavity of PpAOC2, and two nonidentical residues at the entrance of the active site).Oxylipins comprise a large family of oxidized fatty acids and metabolites thereof (Acosta and Farmer, 2010). They are abundant in mammals (Funk, 2001) and flowering plants (Creelman and Mulpuri, 2002). In addition, they have been found in fungi (Brodhun and Feussner, 2011) as well as in nonflowering plants like mosses and algae (Andreou et al., 2009). In plants, these lipids serve as signaling molecules regulating developmental processes and mediating defense reactions (Howe and Jander, 2008; Browse, 2009; Acosta and Farmer, 2010). The first committed step in oxylipin biosynthesis is the peroxidation of a polyunsaturated fatty acid containing a 1Z,4Z-pentadiene system by lipoxygenase (LOX) or the peroxidation at the C2 position of a fatty acid by α-dioxygenase. These reactions start the so-called LOX or oxylipin pathway (Feussner and Wasternack, 2002) and are followed by further enzymatic reactions in which the hydroperoxy fatty acid is converted to a set of different secondary products. In the case of LOX-derived hydroperoxy fatty acids, such conversions are mainly catalyzed by members of the cytochrome P450 subfamily Cyp74 (i.e. fatty acid hydroperoxide lyase, divinyl ether synthase, epoxy alcohol synthase, and allene oxide synthase [AOS]; Stumpe and Feussner, 2006; Lee et al., 2008). Additional conversions of the fatty acid hydroperoxide are catalyzed by other proteins, such as LOX or peroxygenase (Mosblech et al., 2009).Jasmonic acid (JA) biosynthesis is one specific branch of the oxylipin pathway. It may start with the release of α-linolenic acid [18:3(n-3)] from membrane lipids by a lipase (Schaller and Stintzi, 2009). This free fatty acid is subsequently oxidized by a 13-LOX to yield 13-hydroperoxy octadecatrienoic acid (13-HPOTE) and converted by the action of AOS into the unstable allene oxide 12,13S-epoxy-9Z,11E,15Z-octadecatrienoic acid (12,13-EOT; Fig. 1). 12,13-EOT is then cyclized by allene oxide cyclase (AOC) to the cyclopentenone derivative cis(+)-12-oxo-phytodienoic acid [cis(+)-OPDA]. In the absence of AOC, the epoxide is hydrolyzed into ketols and racemic 12-oxo-phytodienoic acid (OPDA). cis(+)-OPDA is the first cyclic and biologically active compound in that pathway (Dave and Graham, 2012). While the reactions leading from 18:3(n-3) to cis(+)-OPDA occur in the plastid, all further enzymatic steps resulting in the formation of JA are localized in the peroxisomes (Wasternack, 2007). Here, cis(+)-OPDA is reduced in a NADPH-dependent reaction by cis(+)-OPDA reductase isoform 3 to 3-oxo-2(2′Z-pentenyl)-cyclopentane-1-octanoic acid. This step is followed by activation of the carboxyl group and three steps of β-oxidation and finally leads to the formation of (+)-7-iso-JA (Dave and Graham, 2012).Open in a separate windowFigure 1.Overview of the enzymatic steps in JA biosynthesis with molecular focus (box) on the reaction catalyzed by AOC. JA biosynthesis may start with the release of 18:3(n-3) or roughanic acid from a lipid. Next, the fatty acid is oxidized by a 13-LOX, yielding the 13-hydroperoxy derivative. This serves as a substrate for a subsequent conversion catalyzed by AOS and AOC, yielding the cyclopentenone derivatives cis(+)-OPDA and cis(+)-dinorOPDA, respectively, via an unstable allene oxide. Cyclization of the allene oxide seems to be initiated by one particular Glu residue in the active site of AOC that leads to an opening of the epoxy ring, conformational changes, and a concerted pericyclic ring closure (details are explained in the text). After reduction of the cyclopentenone by cis(+)-OPDA reductase isoform 3 (OPR3), the octanoic or hexanoic side chain is shortened by β-oxidation cycles.The conversion of 13-HPOTE into cis(+)-OPDA was first observed using a flaxseed (Linum usitatissimum) acetone powder preparation and was suggested to take place via a hypothetical epoxide intermediate (Vick et al., 1980). Later studies unequivocally demonstrated that 12,13-EOT (Hamberg, 1987; Brash et al., 1988), an allene oxide formed from 13-HPOTE by AOS (Song and Brash, 1991; Song et al., 1993), serves as substrate for the cyclization reaction catalyzed by AOC (Hamberg and Fahlstadius, 1990). The enzyme was purified (Ziegler et al., 1997), characterized with regard to the substrate specificity (Ziegler et al., 1999), and cloned and recombinantly expressed (Ziegler et al., 2000; Stenzel et al., 2003). In 2006, the crystal structure of an AOC from Arabidopsis (Arabidopsis thaliana; AtAOC2) was solved (Hofmann et al., 2006), and the reaction mechanism as well as the subcellular localization were studied (Schaller et al., 2008). The enzyme crystallized as a homotrimer, with each subunit forming an eight-stranded antiparallel β-barrel harboring a hydrophobic cavity in which the active site of the enzyme is located. While the exterior loops showed a high degree of flexibility, the central part of the enzyme was very rigid, and no induced-fit mechanism could be observed upon binding of a substrate analog (Hofmann et al., 2006). Based on the structure of AtAOC2 in complex with vernolic acid [cis(+/−)-12,13-epoxy-9Z-octadecenoic acid (12,13-EOM)] as an inert substrate analog, the following reaction mechanism has been proposed (Fig. 1, box): the allene oxide substrate binds with its fatty acid backbone deep in the barrel, where it interacts with hydrophobic amino acid residues, while the polar carboxy head group is located on the exterior of the cavity. One particular Glu residue (Glu-23 in AtAOC2) pointing to the Δ^15Z-double bond of the substrate may induce a partial charge separation that leads to a delocalization of the π-electron system, thereby facilitating opening of the epoxide ring. The oxyanion thus formed is stabilized via polar interactions with a catalytic, structurally conserved water molecule that is positioned in the polar cavity of the enzyme formed by two Asn residues (Asn-25 and Asn-53 in AtAOC2, respectively), one Ser (Ser-31 in AtAOC2), and one Pro (Pro-32 in AtAOC2). The ring closure that leads to the formation of the cyclopentenone derivative is achieved by a conformational reorganization of the C10-C11 substrate bond from the trans- to the cis-geometry. Due to steric limitations in the active site, this rotation may be accompanied by a cis/trans-isomerization of the C8-C9 substrate bond. Since the enzyme dictates the stereochemistry of the final ring closure, the released product is exclusively the (+)-enantiomer, cis(+)-OPDA (Schaller et al., 2008). Notably, this reaction competes with the spontaneous decomposition of the allene oxide substrate that leads to the formation of racemic OPDA as well as α-ketols and γ-ketols. This hints toward a low-energy barrier of the cyclization reaction and suggests that AOC does not need much of a catalytic functionality in terms of lowering this barrier (Schaller and Stintzi, 2009). It has been proposed that the enzymatic cyclization reaction is achieved according to the rules of Hoffmann and Woodward (1970) via a concerted pericyclic ring closure while spontaneous cyclization proceeds through a dipolar ring closure (Grechkin et al., 2002). The facts that the allene oxide formed by AOS has a very short half-life in aqueous solution and that natural OPDA is found in its enantiopure cis(+)-configuration suggest that AOS and AOC are coupled. However, no physical interaction of both enzymes may be necessary to form cis(+)-OPDA in vitro (Zerbe et al., 2007).Recently, it was shown that the moss Physcomitrella patens harbors and metabolizes not only C₁₈ but also C₂₀ polyunsaturated fatty acids to form oxylipins (Fig. 2; Stumpe et al., 2010). In particular, it was shown that (12S)-hydroperoxy eicosatetraenoic acid (12-HPETE) is endogenously formed by a bifunctional LOX as the major hydroperoxy fatty acid of arachidonic acid [20:4(n-6)] (Wichard et al., 2004). 12-HPETE serves as a substrate for further conversions either leading to the formation of C₈- and C₉-volatiles (e.g. octenals, octenols, and nonenals) or the cyclopentenone derivative 11-oxo prostatrienoic acid (11-OPTA; Stumpe et al., 2010). Whereas the volatiles are formed by at least two bifunctional LOXs with an additional hydroperoxide lyase activity (Wichard et al., 2004; Senger et al., 2005; Anterola et al., 2009) or by a Cyp74-derived hydroperoxide lyase (Stumpe et al., 2006), 11-OPTA is formed in analogy to the octadecanoids by one particular AOC, PpAOC2, via the allene oxide intermediate formed by PpAOS (Bandara et al., 2009). In contrast, PpAOC1 does not accept the 12-HPETE-derived C₂₀-allene oxide and thus converts only the 13-HPOTE-derived allene oxide.Open in a separate windowFigure 2.AOS/AOC pathways in P. patens. 13-HPOTE is converted by PpAOS to 12,13-EOT, which may either hydrolyze in the absence of PpAOC1 or PpAOC1 to ketols and racemic OPDA or, in the presence of PpAOC1 and PpAOC2, cyclize to cis(+)-OPDA. 12-HPETE is converted by PpAOS to 11,12-EET, which again may either hydrolyze in the absence of PpAOC2 to ketols and racemic OPDA or, in the presence of PpAOC2, cyclize to 11-OPTA.In this study, the crystal structures of PpAOC1 and PpAOC2 were solved. Data were also obtained for mutated forms of PpAOC1 and for PpAOC1 and PpAOC2 in complex with the allene oxide stable analog 12,13-EOD. In this way, detailed information about the allene oxide-to-cyclopentenone conversions promoted by the two AOCs was obtained.     

hnRNPDL Phase Separation Is Regulated by Alternative Splicing and Disease-Causing Mutations Accelerate Its Aggregation

1. Download : Download high-res image (189KB)
2. Download : Download full-size image

     

A Population-Based Study of Autosomal-Recessive Disease-Causing Mutations in a Founder Population

The decreasing cost of whole-genome and whole-exome sequencing has resulted in a renaissance for identifying Mendelian disease mutations, and for the first time it is possible to survey the distribution and characteristics of these mutations in large population samples. We conducted carrier screening for all autosomal-recessive (AR) mutations known to be present in members of a founder population and revealed surprisingly high carrier frequencies for many of these mutations. By utilizing the rich demographic, genetic, and phenotypic data available on these subjects and simulations in the exact pedigree that these individuals belong to, we show that the majority of mutations were most likely introduced into the population by a single founder and then drifted to the high carrier frequencies observed. We further show that although there is an increased incidence of AR diseases overall, the mean carrier burden is likely to be lower in the Hutterites than in the general population. Finally, on the basis of simulations, we predict the presence of 30 or more undiscovered recessive mutations among these subjects, and this would at least double the number of AR diseases that have been reported in this isolated population.     

Crystal Structure of the N-Acetyltransferase Domain of Human N-Acetyl-L-Glutamate Synthase in Complex with N-Acetyl-L-Glutamate Provides Insights into Its Catalytic and Regulatory Mechanisms

N-acetylglutamate synthase (NAGS) catalyzes the conversion of AcCoA and L-glutamate to CoA and N-acetyl-L-glutamate (NAG), an obligate cofactor for carbamyl phosphate synthetase I (CPSI) in the urea cycle. NAGS deficiency results in elevated levels of plasma ammonia which is neurotoxic. We report herein the first crystal structure of human NAGS, that of the catalytic N-acetyltransferase (hNAT) domain with N-acetyl-L-glutamate bound at 2.1 Å resolution. Functional studies indicate that the hNAT domain retains catalytic activity in the absence of the amino acid kinase (AAK) domain. Instead, the major functions of the AAK domain appear to be providing a binding site for the allosteric activator, L-arginine, and an N-terminal proline-rich motif that is likely to function in signal transduction to CPS1. Crystalline hNAT forms a dimer similar to the NAT-NAT dimers that form in crystals of bifunctional N-acetylglutamate synthase/kinase (NAGS/K) from Maricaulis maris and also exists as a dimer in solution. The structure of the NAG binding site, in combination with mutagenesis studies, provide insights into the catalytic mechanism. We also show that native NAGS from human and mouse exists in tetrameric form, similar to those of bifunctional NAGS/K.     

Chromosomal Passports Provide New Insights into Diffusion of Emmer Wheat

Emmer wheat, Triticum dicoccon schrank (syn. T. dicoccum (schrank) schÜbl.), is one of the earliest domesticated crops, harboring a wide range of genetic diversity and agronomically valuable traits. The crop, however, is currently largely neglected. We provide a wealth of karyotypic information from a comprehensive collection of emmer wheat and related taxa. In addition to C-banding polymorphisms, we identified 43 variants of chromosomal rearrangements in T. dicoccon; among them 26 (60.4%) were novel. The T7A:5B translocation was most abundant in Western Europe and the Mediterranean. The plant genetic resources investigated here might become important in the future for wheat improvement. Based on cluster analysis four major karyotypic groups were discriminated within the T. dicoccon genepool, each harboring characteristic C-banding patterns and translocation spectra: the balkan, asian, european and ethiopian groups. We postulate four major diffusion routes of the crop and discuss their migration out of the Fertile Crescent considering latest archaeobotanical findings.     

禾本科植物分蘖为茎的分枝研究提供新视角

分蘖(或分枝)是作物产量的一个主要决定因素,受植物激素、自身生长发育和环境等因素的调控。近年报道的单子叶植物新的分蘖(或分枝)基因和调控机制深化了对植物分蘖的认知。对以禾本科植物为代表的单子叶植物的分蘖(或分枝)相关基因和调控机制进行了综述,从激素、基因、转录等几方面比较了单子叶植物分蘖和双子叶植物分枝调控机制的异同,为植物产量形成、适应环境及提高生存竞争能力的研究提供理论依据。