We modified and optimized a first generation quadrupole time-of-flight (Q-TOF) 1 to perform tandem mass spectrometry on macromolecular protein complexes. The modified instrument allows isolation and subsequent dissociation of high-mass protein complexes through collisions with argon molecules. The modifications of the Q-TOF 1 include the introduction of (1) a flow-restricting sleeve around the first hexapole ion bridge, (2) a low-frequency ion-selecting quadrupole, (3) a high-pressure hexapole collision cell, (4) high-transmission grids in the multicomponent ion lenses, and (5) a low repetition rate pusher. Using these modifications, we demonstrate the experimental isolation of ions up to 12 800 mass-to-charge units and detection of product ions up to 38 150 Da, enabling the investigation of the gas-phase stability, protein complex topology, and quaternary structure of protein complexes. Some of the data reveal a so-far unprecedented new mechanism in gas-phase dissociation of protein oligomers whereby a tetramer complex dissociates into two dimers. These data add to the current debate whether gas-phase structures of protein complexes do retain some of the structural features of the corresponding species in solution. The presented low-cost modifications on a Q-TOF 1 instrument are of interest to everyone working in the fields of macromolecular mass spectrometry and more generic structural biology.

The pentose metabolism of Archaea is largely unknown. Here, we have employed an integrated genomics approach including DNA microarray and proteomics analyses to elucidate the catabolic pathway for D-arabinose in Sulfolobus solfataricus. During growth on this sugar, a small set of genes appeared to be differentially expressed compared with growth on D-glucose. These genes were heterologously overexpressed in Escherichia coli, and the recombinant proteins were purified and biochemically studied. This showed that D-arabinose is oxidized to 2-oxoglutarate by the consecutive action of a number of previously uncharacterized enzymes, including a D-arabinose dehydrogenase, a D-arabinonate dehydratase, a novel 2-keto-3-deoxy-D-arabinonate dehydratase, and a 2,5-dioxopentanoate dehydrogenase. Promoter analysis of these genes revealed a palindromic sequence upstream of the TATA box, which is likely to be involved in their concerted transcriptional control. Integration of the obtained biochemical data with genomic context analysis strongly suggests the occurrence of pentose oxidation pathways in both Archaea and Bacteria, and predicts the involvement of additional enzyme components. Moreover, it revealed striking genetic similarities between the catabolic pathways for pentoses, hexaric acids, and hydroxyproline degradation, which support the theory of metabolic pathway genesis by enzyme recruitment.Pentose sugars are a ubiquitous class of carbohydrates with diverse biological functions. Ribose and deoxyribose are major constituents of nucleic acids, whereas arabinose and xylose are building blocks of several plant cell wall polysaccharides. Many prokaryotes, as well as yeasts and fungi, are able to degrade these polysaccharides, and use the released five-carbon sugars as a sole carbon and energy source. At present, three main catabolic pathways have been described for pentoses. The first is present in Bacteria and uses isomerases, kinases, and epimerases to convert D-and L-arabinose (Ara) and D-xylose (Xyl) into D-xylulose 5-phosphate (Fig. 1A), which is further metabolized by the enzymes of the phosphoketolase or pentose phosphate pathway. The genes encoding the pentose-converting enzymes are often located in gene clusters in bacterial genomes, for example, the araBAD operon for L-Ara (1), the xylAB operon for D-Xyl (2), and the darK-fucPIK gene cluster for D-Ara (3). The second catabolic pathway for pentoses converts D-Xyl into D-xylulose 5-phosphate as well, but the conversions are catalyzed by reductases and dehydrogenases instead of isomerases and epimerases (Fig. 1B). This pathway is commonly found in yeasts, fungi, mammals, and plants, but also in some bacteria (4 -6). In a third pathway, pentoses such as L-Ara, D-Xyl, D-ribose, and D-Ara are metabolized non-phosphorylatively to either 2-oxoglutarate (2-OG) 4 or to pyruvate and glycolaldehyde (Fig. 1C). The conversion to 2-OG, which is a tricarboxylic acid cycle intermediate, proceeds via the subsequent action of a pentose dehydrogenase, a pentonolactonase, a pentoni...

The subunit architecture of the yeast vacuolar ATPase (V-ATPase) was analyzed by single particle transmission electron microscopy and electrospray ionization (ESI) tandem mass spectrometry. A three-dimensional model of the intact V-ATPase was calculated from two-dimensional projections of the complex at a resolution of 25 angstroms. Images of yeast V-ATPase decorated with monoclonal antibodies against subunits A, E, and G position subunit A within the pseudo-hexagonal arrangement in the V1, the N terminus of subunit G in the V1-V0 interface, and the C terminus of subunit E at the top of the V1 domain. ESI tandem mass spectrometry of yeast V1-ATPase showed that subunits E and G are most easily lost in collision-induced dissociation, consistent with a peripheral location of the subunits. An atomic model of the yeast V-ATPase was generated by fitting of the available x-ray crystal structures into the electron microscopy-derived electron density map. The resulting atomic model of the yeast vacuolar ATPase serves as a framework to help understand the role the peripheral stalk subunits are playing in the regulation of the ATP hydrolysis driven proton pumping activity of the vacuolar ATPase.

Type I cyclic guanosine 3',5'-monophosphate (cGMP)-dependent protein kinase (PKG) is involved in the nitric oxide/cGMP signaling pathway. PKG has been identified in many different species, ranging from unicelölular organisms to mammals. The enzyme serves as one of the major receptor proteins for intracellular cGMP and controls a variety of cellular responses, ranging from smooth-muscle relaxation to neuronal synaptic plasticity. In the absence of a crystal structure, the three-dimensional structure of the homodimeric 152-kDa kinase PKG is unknown; however, there is evidence that the kinase adopts a distinct cGMP-dependent active conformation when compared to the inactive conformation. We performed mass-spectrometry-based hydrogen/deuterium exchange experiments to obtain detailed information on the structural changes in PKG I alpha induced by cGMP activation. Site-specific exchange measurements confirmed that the autoinhibitory domain and the hinge region become more solvent exposed, whereas the cGMP-binding domains become more protected in holo-PKG (dimeric PKG saturated with four cGMP molecules bound). More surprisingly, our data revealed a specific disclosure of the substrate-binding region of holo-PKG, shedding new light into the kinase-activation process of PKG.

The stoichiometry of yeast V 1 -ATPase peripheral stalk subunits E and G was determined by two independent approaches using mass spectrometry (MS). First, the subunit ratio was inferred from measuring the molecular mass of the intact V 1 -ATPase complex and each of the individual protein components, using native electrospray ionization-MS. The major observed intact complex had a mass of 593,600 Da, with minor components displaying masses of 553,550 and 428,300 Da, respectively. Second, defined amounts of V 1 -ATPase purified from yeast grown on 14 N-containing medium were titrated with defined amounts of 15 N-labeled E and G subunits as internal standards. Following protease digestion of subunit bands, 14 Nand 15 N-containing peptide pairs were used for quantification of subunit stoichiometry using matrix-assisted laser desorption/ ionization-time of flight MS. Results from both approaches are in excellent agreement and reveal that the subunit composition of yeast V 1 -ATPase is A 3 B 3 DE 3 FG 3 H.Vacuolar ATPases (V-ATPases, 3 V 1 V 0 -ATPases) are ATP hydrolysis-driven proton pumps found in the endomembrane system of eukaryotic organisms, where they function to acidify the interior of subcellular organelles such as lysosomes, early and late endosomes, clathrin-coated vesicles, the Golgi, the plant tonoplast, and the yeast vacuole (1-4). In higher organisms, the V-ATPase complex can also be found in the plasma membrane of polarized cells involved in acid secretion such as the ruffled membrane of bone osteoclasts or the apical membrane of renal intercalated cells. The vacuolar ATPase is a large, multisubunit complex, which can be divided into a water-soluble ATPase domain and a membrane-bound proton pore. The two domains are termed V 1 and V 0 , respectively, in analogy to the F 1 and F 0 of the related F 1 F 0 -ATP synthase. In yeast, the V 1 -ATPase domain contains subunits AB(C)DEFGH, whereas the membrane-bound V 0 is made of subunits accЈcЉde. Much like the F-ATP synthase, the V-ATPase is a rotary molecular motor enzyme (5, 6); ATP hydrolysis taking place on the A subunits of the A 3 B 3 catalytic domain is coupled to proton translocation across the membrane domain via rotation of a central stalk made of subunits D, F, and d and a proteolipid ring (subunits c, cЈ, and cЉ). The remaining subunits C, E, G, and H are involved in forming a peripheral stator domain that provides a structural link between the catalytic domain (A 3 B 3 ) and the membrane-bound a subunit. In the related F-ATP synthase, it is now well established that there is a single peripheral stalk, which, in the case of the bacterial enzyme, is formed by two copies of the membrane-anchored b subunits and the ␦ subunit (7). The situation in the vacuolar ATPase, however, is more complicated in that there appear to be multiple peripheral stalks that connect the catalytic domain to the membranebound a subunit, possibly via the V-ATPase-specific H and C subunits. Using electron microscopy and single particle image analysis, we have previously shown ...

Combined analysis by SAXS, NMR and homology modeling reveals the structure of an apo module from a trans-acyltransferase polyketide synthase.

The flavoenzyme vanillyl alcohol oxidase (VAO, EC 1.1.3.38) from Penicillium simplicissimum is active on a range of phenolic compounds [1,2]. It contains a covalently linked FAD cofactor, and the holoprotein forms stable octamers. VAO was the first histidyl-FAD-containing flavoprotein for which the crystal structure was determined [3], and serves as a prototype for a specific flavoprotein family [4]. Mutagenesis studies have shown that the covalent flavin-protein bond is crucial for efficient catalysis, and that covalent flavinylation of the apoprotein proceeds via an autocatalytic event [5,6]. As well as oxidizing alcohols, the fungal enzyme is also able to perform amine oxidations, enantioselective hydroxylations, and oxidative ether-cleavage reactions [7,8]. Several substrates can serve as vanillin precursors (e.g. vanillyl alcohol, vanillyl amine and creosol) [9,10]. Recently, VAO has been used in metabolic engineering experiments with the aim of creating a bacterial whole cell biocatalyst that is able to form vanillin from eugenol [11,12]. However, VAO is poorly expressed in bacteria, resulting in a relatively low intracellular VAO activity [12] and low yields of A gene encoding a eugenol oxidase was identified in the genome from Rhodococcus sp. strain RHA1. The bacterial FAD-containing oxidase shares 45% amino acid sequence identity with vanillyl alcohol oxidase from the fungus Penicillium simplicissimum. Eugenol oxidase could be expressed at high levels in Escherichia coli, which allowed purification of 160 mg of eugenol oxidase from 1 L of culture. Gel permeation experiments and macromolecular MS revealed that the enzyme forms homodimers. Eugenol oxidase is partly expressed in the apo form, but can be fully flavinylated by the addition of FAD. Cofactor incorporation involves the formation of a covalent protein-FAD linkage, which is formed autocatalytically. Modeling using the vanillyl alcohol oxidase structure indicates that the FAD cofactor is tethered to His390 in eugenol oxidase. The model also provides a structural explanation for the observation that eugenol oxidase is dimeric whereas vanillyl alcohol oxidase is octameric. The bacterial oxidase efficiently oxidizes eugenol into coniferyl alcohol (K M ¼ 1.0 lm, k cat ¼ 3.1 s )1 ).Vanillyl alcohol and 5-indanol are also readily accepted as substrates, whereas other phenolic compounds (vanillylamine, 4-ethylguaiacol) are converted with relatively poor catalytic efficiencies. The catalytic efficiencies with the identified substrates are strikingly different when compared with vanillyl alcohol oxidase. The ability to efficiently convert eugenol may facilitate biotechnological valorization of this natural aromatic compound.Abbreviations EUGO, eugenol oxidase; PCMH, p-cresol methylhydroxylase (EC 1.17.99.1); VAO, vanillyl alcohol oxidase (EC 1.1.3.38).

Halorespiration is a bacterial respiratory process in which haloorganic compounds act as terminal electron acceptors. This process is controlled at transcriptional level by CprK, a member of the ubiquitous CRP-FNR family. Here we present the crystal structures of oxidized CprK in presence of the ligand orthochlorophenolacetic acid and of reduced CprK in absence of this ligand. These structures reveal that highly specific binding of chlorinated, rather than the corresponding non-chlorinated, phenolic compounds in the NH 2 -terminal ␤-barrels causes reorientation of these domains with respect to the central ␣-helix at the dimer interface. Unexpectedly, the COOH-terminal DNA-binding domains dimerize in the non-DNA binding state. We postulate the ligand-induced conformational change allows formation of interdomain contacts that disrupt the DNA domain dimer interface and leads to repositioning of the helixturn-helix motifs. These structures provide a structural framework for further studies on transcriptional control by CRP-FNR homologs in general and of halorespiration regulation by CprK in particular.Past and present industrial and agricultural activities have led to the ever increasing presence of haloorganic compounds such as chlorophenols and chlorinated ethenes in the environment (1). Due to both toxicity and recalcitrant nature, increasing amounts of these xenobiotics threaten the integrity of the environment and human health (2). In recent years, it has emerged that several haloorganic compounds are also naturally produced (3) and that several species of strictly anaerobic bacteria are able to conserve energy via the reductive dehalogenation of these compounds by respiratory metabolism (4, 5). In view of their favorable degrading capacities, e.g. high dehalogenation rate and low residual concentration of the contaminant, it has been anticipated that halorespiring microorganisms should be of utmost significance for efficient biological remediation of halogenated hydrocarbons in anoxic environments (6, 7). The versatile, strictly anaerobic Gram-positive bacterium Desulfitobacterium dehalogenans and the closely related Desulfitobacterium hafniense have the capacity of degrading ortho-chlorophenol. Both have been used as model organisms in halorespiration studies, representing one of the most significant groups of halorespiring isolates (8). In these organisms, proteins involved in halorespiration are encoded by the cpr (chlorophenol reductive dehalogenase) operon, of which multiple copies are present within the genome. This potentially allows for reductive dehalogenation of a wide range of haloorganic compounds by the use of a series of paralogous enzymes (9).The cpr operon is transcriptionally regulated by CprK, a member of the CRP-FNR family of regulators that is ubiquitous in bacteria (10). Recent in vivo and in vitro studies reveal that CprK binds 3-chloro-4-hydroxyphenylacetate (CHPA) 2 with micromolar affinity promoting a tight interaction with a specific DNA sequence in the promoter region of the cprencod...