Metabolism of halogenated compounds in the white rot fungus Bjerkandera adusta studied by membrane inlet mass spectrometry and tandem mass spectrometry

Membrane inlet mass spectrometry has been used for the characterization of halogenated organic compounds produced by the fungus Bjerkandera adusta. Using this technique we obtained electron impact-, chemical ionization-, electron capture negative chemical ionization-mass spectra and tandem mass spectra directly from the growth medium. Through this direct analysis of the samples we identified novel bioconversion products and confirmed recently published data on the production of both chlorinated and brominated methoxybenzaldehyde metabolites. Growth profiles of the culture grown on a defined medium showed that the production of secondary metabolites starts after approximately 6 days and reaches maximal concentrations of 25-250 muM after 15-20 days. Although delayed, the production of secondary metabolites paralleled a depletion of glucose from the medium and stopped shortly after all glucose had been consumed. Experiments in which fluoro- and bromo-labeled 4-methoxybenzaldehydes were added to the medium at day 8 showed biotransformation of these compounds into chloro-3-fluoro-4-methoxy-benzaldehyde and chloro-3-bromo-4-methoxybenzaldehyde, respectively. No dichlorinated products were observed, suggesting that halogenation takes place only at the meta position on the 4-methoxybenzaldehydes. These experiments are the first to bring direct evidence of a halogenation mechanism, where the enzymatic attack takes place directly on the 4-methoxybenzaldehyde intermediates. (c) John Wiley & Sons, Inc.     

Modification of yeast metabolism by immobilization onto porous glass

Summary Porous glass beads are used to immobilizeSaccharomyces carlsbergensis cells. If silica pores are large enough, adsorption occurs. On the other hand, activation of the silica by glutaraldehyde allows the cells to bind onto the support. In the other case, the most porous glass has the greatest retention capacities. In all cases, 15 min is sufficient for support saturation by microorganisms.The study of glucose fermentation in immobilized cells shows that immobilization modifies the metabolism. Adsorption leads to an acceleration of metabolism, while a slowing down of the cell's activity follows covalent binding. Nevertheless, in both cases yield of glucose converted into ethanol increases while yield of glucose converted into carbon dioxide decreases.     

Methanogenesis from methanol in Methanosarcina barkeri studied using membrane inlet mass spectrometry

Abstract A mass spectrometer with membrane inlet was used to study methanol metabolism by Methanosarcina barkeri strain MS. The addition of methanol to methanol grown culture samples in the mass spectrometer vessel stimulated methanogenesis and hydrogen production. The apparent K _s for methanol was determined as 0.5 mM and the V _max as 8.14 mmol g (dry weight) h⁻¹. The V _max for methane production was fairly constant during growth of the culture on methanol implying that growth is tightly coupled to methanogenesis. The addition of methanol to culture samples in the mass spectrometer vessel stimulated methanogenesis with no lag which indicated that methanogenesis can be uncoupled from growth. Exposure of the culture sample in the mass spectrometer vessel to an atmosphere of 2 kPa oxygen for 80 min resulted in a decrease in the rate of methanogenesis from methanol but on returning the atmosphere to nitrogen the addition of further methanol stimulated methanogenesis. The effect of other inhibitors of methanogenesis (2-bromoethane sulphonate and monensin); K _j values 21.5 μM and 0.3 mM, respectively) were also studied.     

Tyrosine residues modification studied by MALDI-TOF mass spectrometry

Amino acid residue-specific reactivity in proteins is of great current interest in structural biology as it provides information about solvent accessibility and reactivity of the residue and, consequently, about protein structure and possible interactions. In the work presented tyrosine residues of three model proteins with known spatial structure are modified with two tyrosine-specific reagents: tetranitromethane and iodine. Modified proteins were specifically digested by proteases and the mass of resulting peptide fragments was determined using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry. Our results show that there are only small differences in the extent of tyrosine residues modification by tetranitromethane and iodine. However, data dealing with accessibility of reactive residues obtained by chemical modifications are not completely identical with those obtained by nuclear magnetic resonance and X-ray crystallography. These interesting discrepancies can be caused by local molecular dynamics and/or by specific chemical structure of the residues surrounding.     

Interaction of the K+ channel KcsA with membrane phospholipids as studied by ESI mass spectrometry

In this study we have used electrospray ionization mass spectrometry (ESI-MS) to investigate interactions between the bacterial K(+) channel KcsA and membrane phospholipids. KcsA was reconstituted into lipid vesicles of variable lipid composition. These vesicles were directly analyzed by ESI-MS or mixed with trifluoroethanol (TFE) before analysis. In the resulting mass spectra, non-covalent complexes of KcsA and phospholipids were observed with an interesting lipid specificity. The anionic phosphatidylglycerol (PG), and, to a lesser extent, the zwitterionic phosphatidylethanolamine (PE), which both are abundant bacterial lipids, were found to preferentially associate with KcsA as compared to the zwitterionic phosphatidylcholine (PC). These preferred interactions may reflect the differences in affinity of these phospholipids for KcsA in the membrane.     

Charting the protein complexome in yeast by mass spectrometry

It has become evident over the past few years that many complex cellular processes, including control of the cell cycle and ubiquitin-dependent proteolysis, are carried out by sophisticated multisubunit protein machines that are dynamic in abundance, post-translational modification state, and composition. To understand better the nature of the macromolecular assemblages that carry out the cell cycle and ubiquitin-dependent proteolysis, we have used mass spectrometry extensively over the past few years to characterize both the composition of various protein complexes and the modification states of their subunits. In this article we review some of our recent efforts, and describe a promising new approach for using mass spectrometry to dissect protein interaction networks.     

Measurement of plasma membrane potentials of yeast cells with glass microelectrodes

Glass microelectrodes were used to measure the electrical potential difference (Δψ) across plasma membrane of the yeast Pichia humboldtii. The cells were captured in the neck of a glass microfunnel and impaled with a glass microelectrode. The measurements were reproducible and stable for several minutes. The highest Δψ values were obtained in cells metabolizing glucose at pH 6. Δψ in cells deenergized by uncouplers or in dead cells was reduced to about one third of the maximal value. This residual Δψ probably represented Donnan potential. Δψ also was reduced by increasing concentrations of K⁺ in the medium. Other monovalent cations were distinctly less effective: Li⁺ ⪡ Na⁺ < K⁺, and Ca²⁺ was without effect. These experiments prove the applicability of the electrophysiological technique on yeast cells and thus open the way for direct determination of the electrical component of the plasma membrane electrochemical proton gradient.     

Tetramolecular G-quadruplex formation pathways studied by electrospray mass spectrometry

Electrospray mass spectrometry was used to investigate the mechanism of tetramolecular G-quadruplex formation by the DNA oligonucleotide dTG₅T, in ammonium acetate. The intermediates and products were separated according to their mass (number of strands and inner cations) and quantified. The study of the temporal evolution of each species allows us to propose the following formation mechanism. (i) Monomers, dimers and trimers are present at equilibrium already in the absence of ammonium acetate. (ii) The addition of cations promotes the formation of tetramers and pentamers that incorporate ammonium ions and therefore presumably have stacked guanine quartets in their structure. (iii) The pentamers eventually disappear and tetramers become predominant. However, these tetramers do not have their four strands perfectly aligned to give five G-quartets: the structures contain one ammonium ion too few, and ion mobility spectrometry shows that their conformation is more extended. (iv) At 4°C, the rearrangement of the kinetically trapped tetramers with presumably slipped strand(s) into the perfect G-quadruplex structure is extremely slow (not complete after 4 months). We also show that the addition of methanol to the monomer solution significantly accelerates the cation-induced G-quadruplex assembly.     

The methane mono-oxygenase reaction system studied in vivo by membrane-inlet mass spectrometry.

A membrane-inlet mass spectrometer connected to an open-system cuvette was used for direct measurement of dissolved methane and O2 in bacterial samples of strain OU-4-1, a type II methanotrophic bacterium. A technique was applied for keeping the concentration of dissolved methane or O2 in the sample constant while the concentration of the other dissolved gas was varied. This allowed the reaction mechanism of methane mono-oxygenase to be studied in vivo. The enzyme was found to follow a random bi-reactant mechanism with respect to binding of methane and O2. Binding of one substrate decreased the affinity for the other. The true binding constants were 1 microM for methane and 0.14 microM for O2. Studies of HCN inhibition confirmed the random bi-reactant mechanism. HCN was found to be a non-exclusive inhibitor with a binding constant of 0.4 microM.     

Protein-protein and protein-ligand interactions studied by electrospray-ionization mass spectrometry

Preservation of non-covalent interactions in biopolymer mass spectrometry offers new approaches to binding analysis. Recent work from our laboratory is reviewed here and discussed with reference to recent literature in the field. Three issues are considered in particular: hydrophobically stabilized complexes, pH-dependent transitions, and linked protein-ligand and protein-protein binding equilibria.     

Glycolysis and respiration in yeasts. The Pasteur effect studied by mass spectrometry.

Simultaneous and continuous measurements of changes in CO2 and O2 concentrations in glucose-metabolizing yeast suspensions by mass spectrometry enabled a study of the Pasteur effect (aerobic inhibition of glycolysis) in Saccharomyces uvarum and Schizosaccharomyces pombe. A different control mechanism operates in Candida utilis to give a damped oscillation after the anaerobic-aerobic transition. The apparent Km values for respiration of the three yeasts were in the range 1.3-1.8 microM-O2. The apparent Km values for O2 of the Pasteur effect were 5 and 13 microM for catabolite-repressed and derepressed S. uvarum respectively and 7 microM for Sch. pombe. These results are discussed with respect to currently accepted mechanisms for the control of glycolysis.     

The application of mass spectrometry to membrane proteomics

Membrane proteins perform some of the most important functions in the cell, including the regulation of cell signaling through surface receptors, cell-cell interactions, and the intracellular compartmentalization of organelles. Recent developments in proteomic strategies have focused on the inclusion of membrane proteins in high-throughput analyses. While slow and steady progress continues to be made in gel-based technologies, significant advances have been reported in non-gel shotgun methods using liquid chromatography coupled to mass spectrometry (LC/MS). These latter strategies facilitate the identification of large numbers of membrane proteins and modifications, and have the potential to provide insights into protein topology and orientation in membranes.     

Decolourisation of molasses wastewater by cells of Pseudomonas fluorescens immobilised on porous cellulose carrier

Pseudomonas fluorescens isolated from soil samples contaminated with molasses, decolourised molasses wastewater (MWW) samples up to 76% under non-sterile conditions in four days at 30 degrees C. Immobilised cells could be reused for decolourisation activity. However, in subsequent cycles, this was found to decrease from 76% to 50% and from 50% to 24%. Decolourisation activity was regenerated from 30% to 45% by recultivating the immobilised cells in a fresh growth medium. Cellulose carrier coated with collagen was found to be most effective carrier, which produced the highest decolourisation activity of 94% in a 4-day process. This carrier could be reused with 50% of the decolourisation activity retained until the seventh day.     

The conformational dynamics of a metastable serpin studied by hydrogen exchange and mass spectrometry

Serpins are a class of protease inhibitors that initially fold to a metastable structure and subsequently undergo a large conformational change to a stable structure when they inhibit their target proteases. How serpins are able to achieve this remarkable conformational rearrangement is still not understood. To address the question of how the dynamic properties of the metastable form may facilitate the conformational change, hydrogen/deuterium exchange and mass spectrometry were employed to probe the conformational dynamics of the serpin human alpha(1)-antitrypsin (alpha(1)AT). It was found that the F helix, which in the crystal structure appears to physically block the conformational change, is highly dynamic in the metastable form. In particular, the C-terminal half of the F helix appears to spend a substantial fraction of time in a partially unfolded state. In contrast, beta-strands 3A and 5A, which must separate to accommodate insertion of the reactive center loop (RCL), are not conformationally flexible in the metastable state but are rigid and stable. The conformational lability required for loop insertion must therefore be triggered during the inhibition reaction. Beta-strand 1C, which anchors the distal end of the RCL and thus prevents transition to the so-called latent form, is also stable, consistent with the observation that alpha(1)AT does not spontaneously adopt the latent form. A surprising degree of flexibility is seen in beta-strand 6A, and it is speculated that this flexibility may deter the formation of edge-edge polymers.     

Protein folding mechanisms studied by pulsed oxidative labeling and mass spectrometry

Deciphering the mechanisms of protein folding remains a considerable challenge. In this review we discuss the application of pulsed oxidative labeling for tracking protein structural changes in a time-resolved fashion. Exposure to a microsecond OH pulse at selected time points during folding induces the oxidation of solvent-accessible side chains, whereas buried residues are protected. Oxidative modifications can be detected by mass spectrometry. Folding is associated with dramatic accessibility changes, and therefore this method can provide detailed mechanistic insights. Solvent accessibility patterns are complementary to H/D exchange investigations, which report on the extent of hydrogen bonding. This review highlights the application of pulsed OH labeling to soluble proteins as well as membrane proteins.     

Growth kinetics of gel-immobilized yeast cells studied by on-line microscopy

A microscope reactor was used to study online the dynamics of gel immobilized cell systems. The applicability of the reactor is demonstrated by a study of the growth kinetics of Saccharomyces cerevisiae entrapped in 2% calcium alginate. The specific growth rates of single immobilized cells and free cells were measured. The growth of a microcolony in Ca-alginate was followed and the specific growth rate of the cells in the microcolony determined. A simple growth model was used to estimate the cell volume fraction of the yeast cells in the microcolony. As internal and external mass transfer limitations can be neglected and immobilized cell growth rates were found to be identical to those of free cells, one may conclude that immobilization does not influence cell growth under our experimental conditions.