Structure of ATP citrate lyase and the origin of citrate synthase in the Krebs cycle.

Across different kingdoms of life, ATP citrate lyase (ACLY, also known as ACL) catalyses the ATP-dependent and coenzyme A (CoA)-dependent conversion of citrate, a metabolic product of the Krebs cycle, to oxaloacetate and the high-energy biosynthetic precursor acetyl-CoA1. The latter fuels pivotal biochemical reactions such as the synthesis of fatty acids, cholesterol and acetylcholine2, and the acetylation of histones and proteins3,4. In autotrophic prokaryotes, ACLY is a hallmark enzyme of the reverse Krebs cycle (also known as the reductive tricarboxylic acid cycle), which fixates two molecules of carbon dioxide in acetyl-CoA5,6. In humans, ACLY links carbohydrate and lipid metabolism and is strongly expressed in liver and adipose tissue1 and in cholinergic neurons2,7. The structural basis of the function of ACLY remains unknown. Here we report high-resolution crystal structures of bacterial, archaeal and human ACLY, and use distinct substrate-bound states to link the conformational plasticity of ACLY to its multistep catalytic itinerary. Such detailed insights will provide the framework for targeting human ACLY in cancer8-11 and hyperlipidaemia12,13. Our structural studies also unmask a fundamental evolutionary relationship that links citrate synthase, the first enzyme of the oxidative Krebs cycle, to an ancestral tetrameric citryl-CoA lyase module that operates in the reverse Krebs cycle. This molecular transition marked a key step in the evolution of metabolism on Earth.

Differential proteomic analysis of the response of Stenotrophomonas maltophilia to imipenem.

This study represents two different large-scale proteomic experiments analyzing the antibiotic response and the mechanisms of production of ß-lactamases in the nosocomial pathogen Stenotrophomonas maltophilia. Two-dimensional gel electrophoresis on the cytoplasmic protein fraction, together with iTRAQ® differential labeling and 2-D liquid chromatographic separation (2D-LC) MS/MS on the enriched membrane protein fraction, revealed 73 proteins with a change in abundance upon imipenem challenge. These proteins belong to several different functional pathways. We observe an increase in ß-lactamase production as well as in proteins important for their function in the periplasm. The up-regulation of the L1 and L2 ß-lactamases, along with their activator LysR transcriptional factor AmpR, is linked to an increase in proteins responsible for peptidoglycan remodeling and stress response. The interesting identification of an increase in abundance after treatment of the two-component GGDEF signaling protein and an integral membrane sensor signal transduction histidine kinase, indicates that induction of the ß-lactamases is not restricted to the ampR-ampD-ampG pathway. This is the first proteomic study in S. maltophilia upon imipenem stimulation to further unravel the cellular adaptation resulting in ß-lactamase production.

Asymmetric bioreduction of activated carbon-carbon double bonds using Shewanella yellow enzyme (SYE-4) as novel enoate reductase.

Shewanella yellow enzyme (SYE-4), a novel recombinant enoate reductase, was screened against a variety of different substrates bearing an activated double bond, such as unsaturated cyclic ketones, diesters, and substituted imides. Dimethyl- and ethyl esters of 2-methylmaleic acid were selectively reduced to (R)-configured succinic acid derivatives and various N-substituted maleimides furnished the desired (R)-products in up to >99% enantiomeric excess. Naturally occurring (+)-carvone was selectively reduced to (-)-cis-dihydrocarvone and (-)-carvone was converted to the diastereomeric product, respectively. Overall SYE-4 proved to be a useful biocatalyst for the selective reduction of activated C = C double bonds and complements the pool of synthetic valuable enoate reductases.

Glycan structures of the structural subunit (HtH1) of Haliotis tuberculata hemocyanin.

The oligosaccharide structures of the structural subunit HtH1 of Haliotis tuberculata hemocyanin (HtH) were studied by mass spectral sequence analysis of the glycans. The proposed structures are based on MALDI-TOF-MS data before and after treatment with the specific exoglycosidases ß1-3,4,6-galactosidase and α1-6(>2,3,4) fucosidase followed by sequence analysis via electrospray ionization MS/MS-spectra. In total, 15 glycans were identified as a highly heterogeneous group of structures. As in most molluscan hemocyanins, the glycans of HtH1 contain a terminal MeHex, but more interestingly, a novel structural motif was observed: MeHex[Fuc(α1-3)-]GlcNAc, including thus MeHex and (α1-3)-Fuc residues being linked to an internal GlcNAc residue. While the functional unit (FU) c (HtH1-c) is completely lacking any potential glycosylation site, FU-h possesses a second exposed sugar attachment site between beta-strands 8 and 9 within the beta sandwich domain compared to the other FUs. The glycosylation pattern/sites show a high degree of conservation. In FU-h two prominent potential glycosylation sites can be detected. The finding that HtH1 is not able to form multidecameric structures in vivo could be explained by the presence of the exposed glycan on the surface of FU-h.

A proteome map of the pituitary melanotrope cell activated by black-background adaptation of Xenopus laevis.

Upon transfer of Xenopus laevis from a white to a black background, the melanotrope cells in the pituitary pars intermedia secrete alpha-melanocyte-stimulating hormone, which stimulates dispersion of melanin pigment in skin melanophores. This adaptive behavior is under the control of neurotransmitters and neuropeptides of hypothalamic origin. The alpha-melanocyte-stimulating hormone-producing cells and their hypothalamic control system provide an interesting model to study proteins required for biosynthetic and secretory processes involved in peptide hormone production and for brain-pituitary signaling. We present a 2-D PAGE-based proteome map of melanotrope cells from black-adapted animals, identifying 204 different proteins by MS analysis.

Evidence from the structure and function of cytochromes c(2) that nonsulfur purple bacterial photosynthesis followed the evolution of oxygen respiration.

Cytochromes c(2) are the nearest bacterial homologs of mitochondrial cytochrome c. The sequences of the known cytochromes c(2) can be placed in two subfamilies based upon insertions and deletions, one subfamily is most like mitochondrial cytochrome c (the small C2s, without significant insertions and deletions), and the other, designated large C2, shares 3- and 8-residue insertions as well as a single-residue deletion. C2s generally function between cytochrome bc(1) and cytochrome oxidase in respiration (ca 80 examples known to date) and between cytochrome bc(1) and the reaction center in nonsulfur purple bacterial photosynthesis (ca 21 examples). However, members of the large C2 subfamily are almost always involved in photosynthesis (12 of 14 examples). In addition, the gene for the large C2 (cycA) is associated with those for the photosynthetic reaction center (pufBALM). We hypothesize that the insertions in the large C2s, which were already functioning in photosynthesis, allowed them to replace the membrane-bound tetraheme cytochrome, PufC, that otherwise mediates between the small C2 or other redox proteins and photosynthetic reaction centers. Based upon our analysis, we propose that the involvement of C2 in nonsulfur purple bacterial photosynthesis was a metabolic feature subsequent to the evolution of oxygen respiration.

Crystallization and X-ray diffraction studies of inverting trehalose phosphorylase from Thermoanaerobacter sp.

Disaccharide phosphorylases are attractive enzymatic platforms for tailor-made sugar synthesis owing to their ability to catalyze both the synthesis and the breakdown of disaccharides. Trehalose phosphorylase from Thermoanaerobacter sp. (TP) is a glycoside hydrolase family 65 enzyme which catalyzes the reversible breakdown of trehalose [D-glucopyranosyl-alpha(1,1)alpha-D-glucopyranose] to beta-D-glucose 1-phosphate and D-glucose. Recombinant purified protein was produced in Escherichia coli and crystallized in space group P2(1)2(1)2(1). Crystals of recombinant TP were obtained in their native form and were soaked with glucose, with n-octyl-beta-D-glucoside and with trehalose. The crystals presented a number of challenges including an unusually large unit cell, with a c axis measuring 420 A, and variable diffraction quality. Crystal-dehydration protocols led to improvements in diffraction quality that were often dramatic, typically from 7-8 to 3-4 A resolution. The structure of recombinant TP was determined by molecular replacement to 2.8 A resolution, thus establishing a starting point for investigating the structural and mechanistic determinants of the disaccharide phosphorylase activity. To the best of our knowledge, this is the first crystal structure determination of an inverting trehalose phosphorylase.

Towards structural studies of the old yellow enzyme homologue SYE4 from Shewanella oneidensis and its complexes at atomic resolution.

Shewanella oneidensis is an environmentally versatile Gram-negative gamma-proteobacterium that is endowed with an unusually large proteome of redox proteins. Of the four old yellow enzyme (OYE) homologues found in S. oneidensis, SYE4 is the homologue most implicated in resistance to oxidative stress. SYE4 was recombinantly expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to the orthorhombic space group P2(1)2(1)2(1) and were moderately pseudo-merohedrally twinned, emulating a P422 metric symmetry. The native crystals of SYE4 were of exceptional diffraction quality and provided complete data to 1.10 A resolution using synchrotron radiation, while crystals of the reduced enzyme and of the enzyme in complex with a wide range of ligands typically led to high-quality complete data sets to 1.30-1.60 A resolution, thus providing a rare opportunity to dissect the structure-function relationships of a good-sized enzyme (40 kDa) at true atomic resolution. Here, the attainment of a number of experimental milestones in the crystallographic studies of SYE4 and its complexes are reported, including isolation of the elusive hydride-Meisenheimer complex.

Crystallization and X-ray diffraction studies of cellobiose phosphorylase from Cellulomonas uda.

Disaccharide phosphorylases are able to catalyze both the synthesis and the breakdown of disaccharides and have thus emerged as attractive platforms for tailor-made sugar synthesis. Cellobiose phosphorylase from Cellulomonas uda (CPCuda) is an enzyme that belongs to glycoside hydrolase family 94 and catalyzes the reversible breakdown of cellobiose [beta-D-glucopyranosyl-(1,4)-D-glucopyranose] to alpha-D-glucose-1-phosphate and D-glucose. Crystals of ligand-free recombinant CPCuda and of its complexes with substrates and reaction products yielded complete X-ray diffraction data sets to high resolution using synchrotron radiation but suffered from significant variability in diffraction quality. In at least one case an intriguing space-group transition from a primitive monoclinic to a primitive orthorhombic lattice was observed during data collection. The structure of CPCuda was determined by maximum-likelihood molecular replacement, thus establishing a starting point for an investigation of the structural and mechanistic determinants of disaccharide phosphorylase activity.

Simultaneous Genome Sequencing of Prosthecochloris ethylica and Desulfuromonas acetoxidans within a Syntrophic Mixture Reveals Unique Pili and Protein Interactions.

Strains of Chloropseudomonas ethylica, 2-K, N2, and N3 are known to be composed of a syntrophic mixture of a green sulfur bacterium and a sulfur-reducing colorless component. Upon sequence analysis, the green sulfur photosynthetic bacterial component of strain N3 was dominant and was readily sequenced, but the less abundant sulfur-reducing bacterial component was apparent only when analyzed by metagenomic binning. Whole-genome comparison showed that the green bacterium belonged to the genus Prosthecochloris and apparently was a species for which there was no genome sequence on file. For comparison, we also sequenced the genome of Prosthecochloris sp. DSM 1685, which had previously been isolated from the 2-K mixture in pure culture and have shown that all three Prosthecochloris genomes belong to a new species, which we propose to be named Prosthecochloris ethylica comb. nov. Whole genomes were also sequenced for the isolated Desulfuromonas strains DSM 1675 (from strain 2-K) and DSM 1676 (from strain N2) and shown to be nearly identical to the genome found in the N3 mixture. The genome of the green sulfur bacterium contains large genes for agglutination proteins, similar to the ones proposed to be involved in larger photosynthetic consortia of Chlorochromatium aggregatum. In addition, we also identified several unique "tight adhesion (tad)" pili genes that are presumably involved in the formation of cell-cell interactions. The colorless component, on the other hand, contained a unique large multiheme cytochrome C and unique genes for e-pili (geopilin) formation, genetically clustered with a conserved ferredoxin gene, which are all expected to play an electron transfer role in the closed sulfur cycle in the syntrophic mixture. The findings from the simultaneous genome sequencing of the components of Cp. ethylica have implications for the phenomenon of direct interspecies interactions and coupled electron transfer in photosynthetic symbionts. The mechanisms for such interactions appear to be more common in the environment than originally anticipated.

The agglutination protein AggA from Shewanella oneidensis MR-1 is a TolC-like protein and forms active channels in vitro.

Type I secretion systems (TISS) are associated with the virulence of Gram-negative bacteria and the secretion of pathogenic molecular determinants. The Shewanella oneidensis MR-1 outer-membrane protein AggA is part of a TISS. Recombinant AggA expressed in Escherichia coli as inclusion bodies can be efficiently refolded in vitro, and can form active channel-tunnels as shown by proteoliposome swelling assays and electrophysiological measurements in lipid bilayers. Structure-based sequence alignments identify AggA as a TolC-like protein, and point to a conserved structural framework among such proteins despite their marginal sequence similarity. Phylogenetic analysis reveals that clustering of TolC-like proteins can be correlated with their involvement in TISS, Resistance/Nodulation/Division (RND) or Major Facilitator Superfamily (MFS) complexes. Taken together, our results establish a first set of structure-function relationships for a bacterial outer-membrane protein likely to be exclusively involved in TISS, and may contribute towards a more accurate classification of Outer-Membrane Factor (OMF) family proteins.

Multiplicity of aspartic proteinases from Cynara cardunculus L.

Aspartic proteinases (AP) play major roles in physiologic and pathologic scenarios in a wide range of organisms from vertebrates to plants or viruses. The present work deals with the purification and characterisation of four new APs from the cardoon Cynara cardunculus L., bringing the number of APs that have been isolated, purified and biochemically characterised from this organism to nine. This is, to our knowledge, one of the highest number of APs purified from a single organism, consistent with a specific and important biological function of these protein within C. cardunculus. These enzymes, cardosins E, F, G and H, are dimeric, glycosylated, pepstatin-sensitive APs, active at acidic pH, with a maximum activity around pH 4.3. Their primary structures were partially determined by N- and C-terminal sequence analysis, peptide mass fingerprint analysis on a MALDI-TOF/TOF instrument and by LC-MS/MS analysis on a Q-TRAP instrument. All four enzymes are present on C. cardunculus L. pistils, along with cyprosins and cardosins A and B. Their micro-heterogeneity was detected by 2D-electrophoresis and mass spectrometry. The enzymes resemble cardosin A more than they resemble cardosin B or cyprosin, with cardosin E and cardosin G being more active than cardosin A, towards the synthetic peptide KPAEFF(NO(2))AL. The specificity of these enzymes was investigated and it is shown that cardosin E, although closely related to cardosin A, exhibits different specificity.

Identification of glycosylated sites in Rapana hemocyanin by mass spectrometry and gene sequence, and their antiviral effect.

Molluscan hemocyanins (Hcs) have recently received particular interest due to their significant immunostimulatory properties. This is mainly related to their high carbohydrate content and specific monosaccharide composition. We have now analyzed the oligosaccharides and the carbohydrate linkage sites of the Rapana venosa hemocyanin (RvH) using different approaches. We analyzed a number of glycopeptides by LC/ESI-MS/MS and identified the sugar chains and peptide sequences of 12 glycopeptides. Additionally, the potential carbohydrate linkage sites of 2 functional units, RvH-b and RvH-c, were determined by gene sequence analysis. Only RvH-c shows a potential N-glycosylation site. During this study, we discovered a highly conserved linker-intron, separating the coding exons of RVH-b and RvH-c. Following reports on antiviral properties from arthropod hemocyanin, we conducted a preliminary study of the antiviral activity of RvH and the functional units RvH-b and RvH-c. We show that the glycosylated FU RvH-c has antiviral properties against the respiratory syncytial virus (RSV), whereas native RvH and the nonglycosylated FU RvH-b have not. This is the first report of the fact that also molluscan hemocyanin functional units possess antiviral activity.

C-terminal sequence analysis of 2DE-separated proteins.

The overall study of post-translational modifications (PTMs) of proteins is gaining strong interest. Beside phosphorylation and glycosylation, truncations of the nascent polypeptide chain at the N- or C-terminus are by far the most common types of PTMs. Nevertheless, little attention has been paid to the development of approaches that allow a systematic analysis of these proteolytic processing events. Here we present a protocol that allows the identification of the C-terminal sequence of proteins separated by two-dimensional polyacrylamide gel electrophoresis (2DE). For each purified protein, a peptide mixture is generated by cleavage of the protein with cyanogen bromide. During incubation with carboxypeptidases only the original C-terminal fragment forms a ladder. Ladder readout is performed using MALDI mass spectrometry. 2DE-separated proteins from Shewanella oneidensis were chosen as a model system to investigate the effectiveness of the approach.

Mutational analysis of the zinc- and substrate-binding sites in the CphA metallo-beta-lactamase from Aeromonas hydrophila.

The subclass B2 CphA (Carbapenemase hydrolysing Aeromonas) beta-lactamase from Aeromonas hydrophila is a Zn(2+)-containing enzyme that specifically hydrolyses carbapenems. In an effort to evaluate residues potentially involved in metal binding and/or catalysis (His(118), Asp(120), His(196) and His(263)) and in substrate specificity (Val(67), Thr(157), Lys(224) and Lys(226)), site-directed mutants of CphA were generated and characterized. Our results confirm that the first zinc ion is in interaction with Asp(120) and His(263), and thus is located in the 'cysteine' zinc-binding site. His(118) and His(196) residues seem to be interacting with the second zinc ion, as their replacement by alanine residues has a negative effect on the affinity for this second metal ion. Val(67) plays a significant role in the binding of biapenem and benzylpenicillin. The properties of a mutant with a five residue (LFKHV) insertion just after Val(67) also reveals the importance of this region for substrate binding. This latter mutant has a higher affinity for the second zinc ion than wild-type CphA. The T157A mutant exhibits a significantly modified activity spectrum. Analysis of the K224Q and N116H/N220G/K224Q mutants suggests a significant role for Lys(224) in the binding of substrate. Lys(226) is not essential for the binding and hydrolysis of substrates. Thus the present paper helps to elucidate the position of the second zinc ion, which was controversial, and to identify residues important for substrate binding.

Variability of polymorphic families of three types of xylanase inhibitors in the wheat grain proteome.

Cereals contain proteinaceous inhibitors of endo-beta-1,4-xylanases (E.C.3.2.1.8, xylanases). Since these xylanase inhibitors (XIs) are only active against xylanases of microbial origin and do not interact with plant endogenous xylanases, they are believed to act as a defensive barrier against phytopathogenic attack. So far, three types of XIs have been identified, i.e. Triticum aestivum XI (TAXI), xylanase inhibiting protein (XIP), and thaumatin-like XI (TLXI) proteins. In this study the variation in XI forms present in wheat grain was elucidated using high-resolution 2-DE in combination with LC-ESI-MS/MS and biochemical techniques. Reproducible 2-DE fingerprints of TAXI-, XIP-, and TLXI-type XIs, selectively purified from whole meal of three European wheat cultivars using cation exchange chromatography followed by affinity chromatography, were obtained using a pH-gradient of 6 to 11 and a molecular mass range of 10 to 60 kDa. Large polymorphic XI families, not known to date, which exhibit different pI- and/or molecular mass values, were visualised by colloidal CBB staining. Identification of distinct genetic variants by MS/MS-analysis provides a partial explanation for the observed XI heterogeneity. Besides genetic diversity, PTMs, such as glycosylation, account for the additional complexity of the 2-DE patterns.

Crystal structure of a cold-adapted class C beta-lactamase.

In this study, the crystal structure of a class C beta-lactamase from a psychrophilic organism, Pseudomonas fluorescens, has been refined to 2.2 A resolution. It is one of the few solved crystal structures of psychrophilic proteins. The structure was compared with those of homologous mesophilic enzymes and of another, modeled, psychrophilic protein. The elucidation of the 3D structure of this enzyme provides additional insights into the features involved in cold adaptation. Structure comparison of the psychrophilic and mesophilic beta-lactamases shows that electrostatics seems to play a major role in low-temperature adaptation, with a lower total number of ionic interactions for cold enzymes. The psychrophilic enzymes are also characterized by a decreased number of hydrogen bonds, a lower content of prolines, and a lower percentage of arginines in comparison with lysines. All these features make the structure more flexible so that the enzyme can behave as an efficient catalyst at low temperatures.

Crystal structure of Sulfolobus acidocaldarius aspartate carbamoyltransferase in complex with its allosteric activator CTP.

Aspartate carbamoyltransferase (ATCase) is a paradigm for allosteric regulation of enzyme activity. B-class ATCases display very similar homotropic allosteric behaviour, but differ extensively in their heterotropic patterns. The ATCase from the thermoacidophilic archaeon Sulfolobus acidocaldarius, for example, is strongly activated by its metabolic pathway's end product CTP, in contrast with Escherichia coli ATCase which is inhibited by CTP. To investigate the structural basis of this property, we have solved the crystal structure of the S. acidocaldarius enzyme in complex with CTP. Structure comparison reveals that effector binding does not induce similar large-scale conformational changes as observed for the E. coli ATCase. However, shifts in sedimentation coefficients upon binding of the bi-substrate analogue PALA show the existence of structurally distinct allosteric states. This suggests that the so-called "Nucleotide-Perturbation model" for explaining heterotropic allosteric behaviour, which is based on global conformational strain, is not a general mechanism of B-class ATCases.

Structural investigation of cold activity and regulation of aspartate carbamoyltransferase from the extreme psychrophilic bacterium Moritella profunda.

Aspartate carbamoyltransferase (EC 2.1.3.2) is extensively studied as a model for cooperativity and allosteric regulation. The structure of the Escherichia coli enzyme has been thoroughly analyzed by X-ray crystallography, and recently the crystal structures of two hyperthermophilic ATCases of the same structural class have been characterized. We here report the detailed functional and structural investigation of the ATCase from the psychrophilic deep sea bacterium Moritella profunda. Our analysis indicates that the enzyme conforms to the E. coli model in that two allosteric states exist that are influenced by similar homotropic interactions. The heterotropic properties differ in that CTP and UTP inhibit the holoenzyme, but ATP seems to exhibit a dual regulatory pattern, activating the enzyme at low concentrations and inhibiting it in the mM range. The crystal structure of the unliganded M. profunda ATCase shows resemblance to a more extreme T state reported previously for an E. coli ATCase mutant. A detailed molecular analysis reveals potential features of adaptation to cold activity and cold regulation. Moreover, M. profunda ATCase presents similarities with certain mutants of E. coli ATCase altered in their kinetic properties or temperature relationships. Finally, structural and functional comparison of ATCases across the full physiological temperature range agrees with an important, but fundamentally different role for electrostatics in protein adaptation at both extremes, i.e. an increased stability through the formation of ion pairs and ion pair networks at high physiological temperatures, and an increased flexibility through enhanced protein solvation at low temperatures.

Proteomic analysis of the honey bee worker venom gland focusing on the mechanisms of protection against tissue damage.

Honey bee workers use venom for the defence of the colony and themselves when they are exposed to dangers and predators. It is produced by a long thin, convoluted, and bifurcated gland, and consists of several toxic proteins and peptides. The present study was undertaken in order to identify the mechanisms that protect the venom gland secretory cells against these harmful components. Samples of whole venom glands, including the interconnected reservoirs, were separated by two-dimensional gel electrophoresis and the most abundant protein spots were subjected to mass spectrometric identification using MALDI TOF/TOF-MS and LC MS/MS. This proteomic study revealed four antioxidant enzymes: CuZn superoxide dismutase (SOD1), glutathione-S-transferase sigma 1 isoform A (GSTS1), peroxiredoxin 2540 (PXR2540) and thioredoxin peroxidase 1 isoform A (TPX1). Although glutathione-S-transferase (GST) has also been associated with xenobiotic detoxification, the protein we found belongs to the GST Sigma class which is known to protect against oxidative stress only. Moreover, we could demonstrate that the GST and SOD activity of the venom gland was low and moderate, respectively, when compared to other tissues from the adult honey bee. Several proteins involved in other forms of stress were likewise found but it remains uncertain what their function is in the venom gland. In addition to major royal jelly protein 9 (MRJP9), already found in a previous proteomic study, we identified MRJP8 as second member of the MRJP protein family to be associated with the venom gland. Transcripts of both MRJPs were amplified and sequenced. Two endocuticular structural proteins were abundantly present in the 2D-gel and most probably represent a structural component of the epicuticular lining that protects the secretory cells from the toxins they produce.