The growing family of photoactive yellow proteins and their presumed functional roles.

For several years following the discovery and characterization of the first PYP, from Halorhodospira halophila, it was thought that this photoactive protein was quite unique, notwithstanding the isolation of two additional examples in rapid succession. Mainly because of genomic and metagenomic analyses, we have now learned that more than 140 PYP genes occur in a wide variety of bacteria and metabolic niches although the protein has not been isolated in most cases. The amino acid sequences and physical properties permit their organization into at least seven groups that are also likely to be functionally distinct. Based upon action spectra and the wavelength of maximum absorbance, it was speculated nearly 20 years ago but never proven that Hr. halophila PYP was involved in phototaxis. Nevertheless, in only one instance has the functional role and interaction partner for a PYP been experimentally proven, in Rs. centenum Ppr. Genetic context is one of several types of evidence indicating that PYP is potentially involved in a number of diverse functional roles. The interaction with other sensors to modulate their activity stands out as the single most prominent role for PYP. In this review, we have attempted to summarize the evidence for the functional roles and interaction partners for some 26 of the 35 named species of PYP, which should be considered the basis for further focused molecular and biochemical research.

The estimated impact of fungi on nutrient dynamics during decomposition of Phragmites australis leaf sheaths and stems.

Decomposition of culms (sheaths and stems) of the emergent macrophyte Phragmites australis (common reed) was followed for 16 months in the litter layer of a brackish tidal marsh along the river Scheldt (the Netherlands). Stems and leaf sheaths were separately analyzed for mass loss, litter-associated fungal biomass (ergosterol), nutrient (N and P), and cell wall polymer concentrations (cellulose and lignin). The role of fungal biomass in litter nutrient dynamics was evaluated by estimating nutrient incorporation within the living fungal mass. After 1 year of standing stem decay, substantial fungal colonization was found. This corresponded to an overall fungal biomass of 49 +/- 8.7 mg g(-1) dry mass. A vertical pattern of fungal colonization on stems in the canopy is suggested. The litter bag experiment showed that mass loss of stems was negligible during the first 6 months, whereas leaf sheaths lost almost 50% of their initial mass during that time. Exponential breakdown rates were -0.0039 +/- 0.0004 and -0.0026 +/- 0.0003 day(-1) for leaf sheaths and stems, respectively (excluding the initial lag period). In contrast to the stem tissue--which had no fungal colonization--leaf sheaths were heavily colonized by fungi (93 +/- 10 mg fungal biomass g(-1) dry mass) prior to placement in the litter layer. Once being on the sediment surface, 30% of leaf sheath's associated fungal biomass was lost, but ergosterol concentrations recovered the following months. In the stems, fungal biomass increased steadily after an initial lag period to reach a maximal biomass of about 120 mg fungal biomass g(-1) dry mass for both plant parts at the end of the experiment. Fungal colonizers are considered to contain an important fraction of nutrients within the decaying plant matter. Fungal N incorporation was estimated to be 64 +/- 13 and 102 +/- 15% of total available N pool during decomposition for leaf sheaths and stems, respectively. Fungal P incorporation was estimated to be 37 +/- 9 and 52 +/- 15% of total available P during decomposition for leaf sheaths and stems, respectively. Furthermore, within the stem tissue, fungi are suggested to be active immobilizers of nutrients from the external environment because fungi were often estimated to contain more than 100% of the original nutrient stock.

On-column sample enrichment for the high-sensitivity sheath-flow CE-MS analysis of peptides.

A sample enrichment technique to increase sensitivity in capillary electrophoresis-mass spectrometry (CE-MS) is described. Peptides or glycopeptides are retained and concentrated on a short (3-5-mm) reversed-phase (C18) packed-bed situated in the fused-silica separation capillary and are subsequently released for electrophoretic separation by injection of an organic elutant. The concentration limits of detection are in the high picomolar range with a sheath-flow CE-MS interface.

Oligosaccharide binding in family 8 glycosidases: crystal structures of active-site mutants of the beta-1,4-xylanase pXyl from Pseudoaltermonas haloplanktis TAH3a in complex with substrate and product.

The structures of inactive mutants D144A and E78Q of the glycoside hydrolase family 8 (GH-8) endo-beta-1,4-d-xylanase (pXyl) from the Antarctic bacterium Pseudoalteromonas haloplanktis TAH3a in complex with its substrate xylopentaose (at 1.95 A resolution) and product xylotriose (at 1.9 A resolution) have been determined by X-ray crystallography. A detailed comparative analysis of these with the apo-enzyme and with other GH-8 structures indicates an induced fit mechanism upon ligand binding whereby a number of conformational changes and, in particular, a repositioning of the proton donor into a more catalytically competent position occurs. This has also allowed for the description of protein-ligand interactions in this enzyme and for the demarcation of subsites -3 to +3. An in-depth analysis of each of these subsites gives an insight into the structure-function relationship of this enzyme and the basis of xylose/glucose discrimination in family 8 glycoside hydrolases. Furthermore, the structure of the -1/+1 subsite spanning complex reveals that the substrate is distorted from its ground state conformation. Indeed, structural analysis and in silico docking studies indicate that substrate hydrolysis in GH-8 members is preceded by a conformational change, away from the substrate ground-state chair conformation, to a pretransition state local minimum (2)S(O) conformation.

Selectivity of tryptophan residues in mediating photolysis of disulfide bridges in goat alpha-lactalbumin.

Goat alpha-lactalbumin (GLA) contains four tryptophan (Trp) residues and four disulfide bonds. Illumination with near-UV light results in the cleavage of disulfide bridges and in the formation of free thiols. To obtain information about the reaction products, the illuminated protein was carbamidomethylated and digested with trypsin and the peptides were analyzed by mass spectrometry. Peptides containing Cys120Cam, Cys61Cam, or Cys91Cam were detected, as well as two peptides containing a new Cys-Lys cross-link. In one, Cys6 was cross-linked to Lys122, while the cross-link in the second was either a Cys91-Lys79 or Cys73-Lys93 cross-link; however, the exact linkage could not be defined. The results demonstrate photolytic cleavage of the Cys6-Cys120, Cys61-Cys77, and Cys73-Cys91 disulfide bonds. While photolysis of Cys6-Cys120 and Cys73-Cys91 disulfide bonds in GLA has been reported, cleavage of the Cys61-Cys77 disulfide bonds has not been previously detected. To examine the contribution of the individual Trp residues, we constructed the GLA mutants, W26F, W60F, W104F, and W118F, by replacing single Trp residues with phenylalanine (Phe). The substitution of each Trp residue led to less thiol production compared to that for wild-type GLA, showing that each Trp residue in GLA contributed to the photolytic cleavage of disulfide bridges. The specificity was expressed by the nature of the reaction products. No cleavage of the Cys6-Cys120 disulfide bridge was detected when the W26F mutant was illuminated, and no cleavage of the Cys73-Cys91 disulfide bridge was seen following illumination of W26F or W104F. In contrast, Cys61Cam, resulting from the cleavage of the Cys61-Cys77 disulfide bridge, was found following illumination of any of the mutants.

Study of the active site residues of a glycoside hydrolase family 8 xylanase.

Site-directed mutagenesis and a comparative characterisation of the kinetic parameters, pH dependency of activity and thermal stability of mutant and wild-type enzymes have been used in association with crystallographic analysis to delineate the functions of several active site residues in a novel glycoside hydrolase family 8 xylanase. Each of the residues investigated plays an essential role in this enzyme: E78 as the general acid, D281 as the general base and in orientating the nucleophilic water molecule, Y203 in maintaining the position of the nucleophilic water molecule and in structural integrity and D144 in sugar ring distortion and transition state stabilization. Interestingly, although crystal structure analyses and the pH-activity profiles clearly identify the functions of E78 and D281, substitution of these residues with their amide derivatives results in only a 250-fold and 700-fold reduction in their apparent k(cat) values, respectively. This, in addition to the observation that the proposed general base is not conserved in all glycoside hydrolase family 8 enzymes, indicates that the mechanistic architecture in this family of inverting enzymes is more complex than is conventionally believed and points to a diversity in the identity of the mechanistically important residues as well as in the arrangement of the intricate microenvironment of the active site among members of this family.

Proteomic analysis of small heat shock protein isoforms in barley shoots.

The analysis of stress-responsiveness in plants is an important route to the discovery of genes conferring stress tolerance and their use in breeding programs. High temperature is one of the environmental stress factors that can affect the growth and quality characteristics of barley (Hordeum vulgare). In this study a proteomic analysis (2D-PAGE, MS) was used to detect the effects of heat shock on the protein pattern of an abiotic stress-tolerant (Mandolina) and an abiotic stress-susceptible (Jubilant) barley cultivar. Evaluation of two-dimensional gels revealed several proteins to be differentially expressed as a result of heat stress in both cultivars. The protein spots of interest were, after an in-gel tryptic digestion, further investigated by mass spectrometry. For the analysis of the peptide mixture, we both used a matrix-assisted laser desorption/ionization (MALDI) tandem time of flight mass spectrometer (TOF/TOF) and an automated nano-HPLC system coupled to an electrospray ionization-quadrupole linear ion trap (Q-TRAP) instrument. The hyphenation of the latter techniques proved to be a powerful technique as shown by the identification of six isoforms of a 16.9 kDa sHSP in one single spot. We observed that S-adenosylmethionine synthetase (SAM-S) was differentially expressed between the two cultivars. Recent results refer to the role of SAM-S as being involved in abiotic stress tolerance. Furthermore, comparison of the heat shock treated samples also revealed several small heat shock proteins (sHSP), of which distinct isoforms could be characterised.

Protein dynamics in the region of the sixth ligand methionine revealed by studies of imidazole binding to Rhodobacter capsulatus cytochrome c2 hinge mutants.

All class I c-type cytochromes studied to date undergo a dynamic process in the oxidized state, which results in the transient breaking of the iron-methionine-sulfur bond and sufficient movement to allow the binding of exogenous ligands (imidazole in this work). In the case of Rhodobacter capsulatus cytochrome c(2), the sixth heme ligand Met96 and up to 14 flanking residues (positions 88-100, termed the hinge region), located between two relatively rigid helical regions, may be involved in structural changes leading to a transient high-spin species able to bind ligands. We have examined 14 mutations at 9 positions in the hinge region of Rhodobacter capsulatus cytochrome c(2) and have determined the structure of the G95E mutant. Mutations near the N- and C-terminus of the hinge region do not affect the kinetics of movement but allow us to further define that portion of the hinge that moves away from the heme to the 93-100 region in the amino acid sequence. Mutations at positions 93 and 95 can alter the rate constant for hinge movement (up to 20-fold), presumably as a result of altering the structure of the native cytochrome to favor a more open conformation. The structure of one of these mutants, G95E, suggests that interactions within the hinge region are stabilized while interaction between the hinge and the heme are destabilized. In contrast, mutations at positions 98 and 99 alter imidazole binding kinetics but not the hinge movement. Thus, it appears that these mutations affect the structure of the cytochrome after the hinge region has moved away from the heme, resulting in increased solvent access to the bound imidazole or alter interactions between the protein and the bound imidazole.

Cross-linking of ubiquitin, HSP27, parkin, and alpha-synuclein by gamma-glutamyl-epsilon-lysine bonds in Alzheimer's neurofibrillary tangles.

The accumulation of misfolded proteins in intracellular inclusions is a generic feature of neurodegenerative disorders. Although heavily ubiquitylated, the aggregated proteins are not degraded by the proteasomes. A possible reason for this phenomenon may be a modification of deposited proteins by transglutaminases forming gamma-glutamyl-epsilon-lysine (GGEL) cross-links between distinct proteins. Here, we show that the frequency of GGEL cross-links is an order of magnitude higher in Alzheimer's brain cortex than in age-matched or younger controls. This difference is due to the accumulation of GGEL cross-links in ubiquitin-immunopositive protein particles present in both Alzheimer's brains and those from aged individuals. The highly cross-linked protein aggregates show immunoreactivity to antibodies against tau and neurofilament proteins, and partially also to alpha-synuclein, indicating that these structures are inherent in Alzheimer's neurofibrillary tangles and Lewy bodies. Using mass sequence analysis, we identified the same six pairs of peptide sequences cross-linked in both senile and Alzheimer's specimens: Gln31 and Gln190 of HSP27 protein are cross-linked with Lys29 and Lys48 of ubiquitin and HSP27 therefore may cross-link two (poly)ubiquitin chains. One lysine residue of parkin and one of alpha-synuclein were also found to be cross-linked. The data suggest that cross-linking of (poly)ubiquitin moieties via HSP27 may have a role in the stabilization of the intraneuronal protein aggregates by interference with the proteasomal elimination of unfolded proteins.

The Q-Trap mass spectrometer, a novel tool in the study of protein glycosylation.

The use of the recently introduced Q-Trap mass spectrometer in the study of protein glycosylation is described. The combined ion trap and triple quadrupole scan functions make it a powerful system in both oligosaccharide and glycopeptide analysis. Several oligosaccharides, both linear and branched, were analyzed to obtain information on sequence, linkage, and branching. Quadrupole like MS/MS spectra with ion trap sensitivity but without the typical ion trap low mass cut-off were obtained. To determine the origin of fragments and to reveal the existence of new ions, the MS(3) capabilities of the system proved to be useful. Glycopeptides were selectively detected in peptide mixtures using the triple quadrupole precursor ion scan function, either in off-line experiments or during LC/MS using information dependent acquisition (IDA).

Rhodobacter capsulatus photoactive yellow protein: genetic context, spectral and kinetics characterization, and mutagenesis.

A gene for photoactive yellow protein (PYP) was previously cloned from Rhodobacter capsulatus (Rc), and we have now found it to be associated with genes for gas vesicle formation in the recently completed genome sequence. However, the PYP had not been characterized as a protein. We have now produced the recombinant RcPYP in Escherichia coli as a glutathione-S-transferase (GST) fusion protein, along with the biosynthetic enzymes, resulting in the formation of holo-RcPYP following cleavage of the GST tag. The absorption spectrum (with characteristic peaks at 435 and 375 nm) and the photocycle kinetics, initiated by a laser flash at 445 nm, are generally similar to those of Rhodobacter sphaeroides (RsPYP) but are significantly different from those of the prototypic PYP from Halorhodospira halophila (HhPYP), which has a single peak at 446 nm and has slower recovery. RcPYP also is photoactive when excited with near-ultraviolet laser light, but the end point is then above the preflash baseline. This suggests that some of the PYP chromophore is present in the cis-protonated conformation in the resting state. The excess 435 nm form in RcPYP, built up from repetitive 365 nm laser flashes, returns to the preflash baseline with an estimated half-life of 2 h, which is markedly slower than that for the same reaction in RsPYP. Met100 has been reported to facilitate cis-trans isomerization in HhPYP, yet both Rc and RsPYPs have Lys and Gly substitutions at positions 99 and 100 (using HhPYP numbering throughout) and have 100-fold faster recovery kinetics than does HhPYP. However, the G100M and K99Q mutations of RcPYP have virtually no effect on kinetics. Apparently, the RcPYP M100 is in a different conformation, as was recently found for the PYP domain of Rhodocista centenaria Ppr. The cumulative results show that the two Rhodobacter PYPs are clearly distinct from the other species of PYP that have been characterized. These properties also suggest a different functional role, that we postulate to be in regulation of gas vesicle genes, which are known to be light-regulated in other species.

Role of Cys221 and Asn116 in the zinc-binding sites of the Aeromonas hydrophila metallo-beta-lactamase.

The CphA metallo-beta-lactamase produced by Aeromonas hydrophila exhibits two zinc-binding sites. Maximum activity is obtained upon binding of one zinc ion, whereas binding of the second zinc ion results in a drastic decrease in the hydrolytic activity. In this study, we analyzed the role of Asn116 and Cys221, two residues of the active site. These residues were replaced by site-directed mutagenesis and the different mutants were characterized. The C221S and C221A mutants were seriously impaired in their ability to bind the first, catalytic zinc ion and were nearly completely inactive, indicating a major role for Cys221 in the binding of the catalytic metal ion. By contrast, the binding of the second zinc ion was only slightly affected, at least for the C221S mutant. Mutation of Asn116 did not lead to a drastic decrease in the hydrolytic activity, indicating that this residue does not play a key role in the catalytic mechanism. However, the substitution of Asn116 by a Cys or His residue resulted in an approximately fivefold increase in the affinity for the second, inhibitory zinc ion. Together, these data suggested that the first zinc ion is located in the binding site involving the Cys221 and that the second zinc ion binds in the binding site involving Asn116 and, presumably, His118 and His196.

Amino acid sequences and distribution of high-potential iron-sulfur proteins that donate electrons to the photosynthetic reaction center in phototropic proteobacteria.

High-potential iron-sulfur protein (HiPIP) has recently been shown to function as a soluble mediator in photosynthetic electron transfer between the cytochrome bc1 complex and the reaction-center bacteriochlorophyll in some species of phototrophic proteobacteria, a role traditionally assigned to cytochrome c2. For those species that produce more than one high-potential electron carrier, it is unclear which protein functions in cyclic electron transfer and what characteristics determine reactivity. To establish how widespread the phenomenon of multiple electron donors might be, we have studied the electron transfer protein composition of a number of phototrophic proteobacterial species. Based upon the distribution of electron transfer proteins alone, we found that HiPIP is likely to be the electron carrier of choice in the purple sulfur bacteria in the families Chromatiaceae and Ectothiorhodospiraceae, but the majority of purple nonsulfur bacteria are likely to utilize cytochrome c2. We have identified several new species of phototrophic proteobacteria that may use HiPIP as electron donor and a few that may use cytochromes c other than c2. We have determined the amino acid sequences of 14 new HiPIPs and have compared their structures. There is a minimum of three sequence categories of HiPIP based upon major insertions and deletions which approximate the three families of phototrophic proteobacteria and each of them can be further subdivided prior to construction of a phylogenetic tree. The comparison of relationships based upon HiPIP and RNA revealed several discrepancies.

BlaB, a protein involved in the regulation of Streptomyces cacaoi beta-lactamases, is a penicillin-binding protein.

Streptomyces cacaoi beta-lactamase genes are controlled by two regulators named blaA and blaB. Whereas BlaA has been identified as a LysR-type activator, the function of BlaB is still unknown. Its primary structure is similar to that of the serine penicillin-recognizing enzymes (PREs). Indeed, the SXXK and KTG motifs are perfectly conserved in BlaB, whereas the common SXN element found in PREs is replaced by a SDG motif. Site-directed mutations were introduced in these motifs and they all disturb beta-lactamase regulation. A water-soluble form of BlaB was also overexpressed in the Streptomyces lividans TK24 cytoplasm and purified. To elucidate the activity of BlaB, several compounds recognized by PREs were tested. BlaB could be acylated by some of them, and it can therefore be considered as a penicillin-binding protein. BlaB is devoid of beta-lactamase, D-aminopeptidase, DD-carboxypeptidase or thiolesterase activity.

Microbial reduction and precipitation of vanadium by Shewanella oneidensis.

Shewanella oneidensis couples anaerobic oxidation of lactate, formate, and pyruvate to the reduction of vanadium pentoxide (V(V)). The bacterium reduces V(V) (vanadate ion) to V(IV) (vanadyl ion) in an anaerobic atmosphere. The resulting vanadyl ion precipitates as a V(IV)-containing solid.

Homozygous Gly555Glu mutation in the nuclear-encoded 70 kDa flavoprotein gene causes instability of the respiratory chain complex II.

A homozygous mutation in the flavoprotein (Fp) gene associated with complex II deficiency was demonstrated in a patient with consanguineous parents. She succumbed at 5(1/2) months of age following a respiratory infection. The c1664G-->A transition detected, predicted the substitution of the small uncharged glycine at position 555 by glutamic acid. Her clinical course was at variance with the Leigh syndrome in three previously reported patients due to Fp gene mutations. In this proband, CRM for flavoprotein as well as iron-containing protein (Ip) was decreased, CRM for the entire complex II (130 kDa) being reduced even more. This observation prompts speculation of a labile interaction between Ip and Fp polypeptides and of a key role of the amino acid at position 555 in the interacting domain.

Isolation and biochemical characterisation of enterocins produced by enterococci from different sources.

AIMS: Comparison of enterocins produced by six Enterococcus faecium strains and one Ent. faecalis strain isolated from different origin with regard to their microbiological and biochemical characteristics in view of their technological potential and practical use. METHODS AND RESULTS: The seven enterococci were sensitive to the glycopeptide antibiotics vancomycin and teicoplanin and did not show haemolytic activity. The absence of the glycopeptide-resistant genotypes and the genes involved in the production of the lantibiotic cytolysin was confirmed by PCR. The enterocins were active towards Listeria innocua and other lactic acid bacteria. Their temperature stability was dependent on the pH and their activity was higher at acidic pH. A bactericidal and bacteriolytic effect was shown. PCR analyses revealed that the gene of enterocin A was present in the genome of Ent. faecium CCM 4231, Ent. faecium 306 I.2.20 and Ent. faecalis Y; both enterocin A and B genes were present in the genome of Ent. faecium LMG 11423T, Ent. faecium RZS C5 and Ent. faecium RZS C13. Enterocin P was detected in the genome of Ent. faecium RZS C5 and Ent. faecium RZS C13. No signal was found for Ent. faecium SF 68. Enterocins from Ent. faecium RZS C5, Ent. faecium RZS C13 and Ent. faecium SF 68 were purified to homogeneity. CONCLUSIONS: Ent. faecium RZS C5 and Ent. faecium RZS C13 produced an enterocin with a molecular mass of 5460 and 5477 Da, respectively, which was in the range of that of enterocin B. The amino acid sequence analysis of the enterocin from Ent. faecium RZS C13 revealed 24 N-terminal residues, which were identical to those of enterocin B. The enterocin from Ent. faecium SF 68 had a molecular mass of 4488 Da, which did not correspond to any enterocin known so far. SIGNIFICANCE AND IMPACT OF THE STUDY: The number of characterized enterocins is increasing. As this type of work is tedious and time-consuming, it may be interesting to include PCR as a first step to know if the Enterococcus strain in study produces either a known or a new enterocin. Also, it is important to check the absence of cytolysin and resistance to vancomycin for a further application of the Enterococcus strain in food or health applications.

Automated nanoflow liquid chromatography-tandem mass spectrometry for a differential display proteomic study on Xenopus laevis neuroendocrine cells.

Many proteomic projects based on a comparison of protein profiles displayed on two-dimensional polyacrylamide gel electrophoresis rely on the identification of these proteins using peptide mass fingerprinting on a matrix-assisted laser desorption/ionization mass spectrometer after tryptic digestion. However, this approach is limited to an organism of which genomic information is largely available, i.e. when the total genome sequence is known. For other organisms, mass spectrometric sequence analysis is necessary for protein identification. We established a nano-LC-MS-MS system based on a quadrupole time-of-flight mass spectrometer, which allows automated sequence analysis of tryptic digestion mixtures from single gel spots. This system is applied in a differential-display proteomic study to identify differentially expressed proteins in the neuroendocrine cells of the neurointermediate pituitary of black- and white-background adapted Xenopus laevis.

Mass spectrometric identification of in vivo carbamylation of the amino terminus of Ectothiorhodospira mobilis high-potential iron-sulfur protein, isozyme 1.

The complete amino acid sequence of a novel high-potential iron-sulfur protein (HiPIP) isozyme 1 from the moderately halophilic phototrophic bacterium Ectothiorhodospira mobilis was determined by a combined approach of chemical and mass spectrometric sequencing techniques. By mass analysis of the apo- and holo-protein in the positive electrospray ionization mode using different electrospray solvents, the protein was found to be post-translationally modified by a moiety of 43 Da. Further analysis showed the nature and location of this modification to be a carbamyl group at the N-terminus of the HiPIP. This rare type of modification has previously been reported to occur in the water-soluble human lens alphaB-crystallin, class D beta-lactamases and some prokaryotic ureases, albeit at an internal lysine residue. In this paper, we discuss the mass spectrometric features of a carbamylated residue at the N-terminus of a peptide or a lysine side-chain during sequence analysis by collision-induced dissociation tandem mass spectrometry. Our data provide evidence for the first case of a prokaryotic carbamylated electron transport protein occurring in vivo.