Genome sequencing of Porostereum spadiceum to study the degradation of levofloxacin.

Despite various plans to rationalize antibiotic use, antibiotic resistance in environmental bacteria is increasing due to the accumulation of antibiotic residues in the environment. This study aimed to test the ability of basidiomycete fungal strains to biotransform the antibiotic levofloxacin, a widely-used third-generation broad-spectrum fluoroquinolone, and to propose enzyme targets potentially involved in this biotransformation. The biotransformation process was performed using fungal strains. Levofloxacin biotransformation reached 100% after 9 days of culture with Porostereum spadiceum BS34. Using genomics and proteomics analyses coupled with activity tests, we showed that P. spadiceum produces several heme-peroxidases together with H2O2-producing enzymes that could be involved in the antibiotic biotransformation process. Using UV and high-resolution mass spectrometry, we were able to detect five levofloxacin degradation products. Their putative identity based on their MS2 fragmentation patterns led to the conclusion that the piperazine moiety was the main target of oxidative modification of levofloxacin by P. spadiceum, leading to a decrease in antibiotic activity.

Beyond the coupled distortion model: structural analysis of the single domain cupredoxin AcoP, a green mononuclear copper centre with original features.

Cupredoxins are widely occurring copper-binding proteins with a typical Greek-key beta barrel fold. They are generally described as electron carriers that rely on a T1 copper centre coordinated by four ligands provided by the folded polypeptide. The discovery of novel cupredoxins demonstrates the high diversity of this family, with variations in terms of copper-binding ligands, copper centre geometry, redox potential, as well as biological function. AcoP is a periplasmic cupredoxin belonging to the iron respiratory chain of the acidophilic bacterium Acidithiobacillus ferrooxidans. AcoP presents original features, including high resistance to acidic pH and a constrained green-type copper centre of high redox potential. To understand the unique properties of AcoP, we undertook structural and biophysical characterization of wild-type AcoP and of two Cu-ligand mutants (H166A and M171A). The crystallographic structures, including native reduced AcoP at 1.65 Å resolution, unveil a typical cupredoxin fold. The presence of extended loops, never observed in previously characterized cupredoxins, might account for the interaction of AcoP with physiological partners. The Cu-ligand distances, determined by both X-ray diffraction and EXAFS, show that the AcoP metal centre seems to present both T1 and T1.5 features, in turn suggesting that AcoP might not fit well to the coupled distortion model. The crystal structures of two AcoP mutants confirm that the active centre of AcoP is highly constrained. Comparative analysis with other cupredoxins of known structures, suggests that in AcoP the second coordination sphere might be an important determinant of active centre rigidity due to the presence of an extensive hydrogen bond network. Finally, we show that other cupredoxins do not perfectly follow the coupled distortion model as well, raising the suspicion that further alternative models to describe copper centre geometries need to be developed, while the importance of rack-induced contributions should not be underestimated.

Modification of a Marine Pine Kraft Lignin Sample by Enzymatic Treatment with a Pycnoporus cinnabarinus Laccase.

Here, we report work on developing an enzymatic process to improve the functionalities of industrial lignin. A kraft lignin sample prepared from marine pine was treated with the high-redox-potential laccase from the basidiomycete fungus Pycnoporus cinnabarinus at three different concentrations and pH conditions, and with and without the chemical mediator 1-hydroxybenzotriazole (HBT). Laccase activity was tested in the presence and absence of kraft lignin. The optimum pH of PciLac was initially 4.0 in the presence and absence of lignin, but at incubation times over 6 h, higher activities were found at pH 4.5 in the presence of lignin. Structural changes in lignin were investigated by Fourier-transform infrared spectroscopy (FTIR) with differential scanning calorimetry (DSC), and solvent-extractable fractions were analyzed using high-performance size-exclusion chromatography (HPSEC) and gas chromatography-mass spectrometry (GC-MS). The FTIR spectral data were analyzed with two successive multivariate series using principal component analysis (PCA) and ANOVA statistical analysis to identify the best conditions for the largest range of chemical modifications. DSC combined with modulated DSC (MDSC) revealed that the greatest effect on glass transition temperature (Tg) was obtained at 130 U g cm-1 and pH 4.5, with the laccase alone or combined with HBT. HPSEC data suggested that the laccase treatments led to concomitant phenomena of oligomerization and depolymerization, and GC-MS revealed that the reactivity of the extractable phenolic monomers depended on the conditions tested. This study demonstrates that P. cinnabarinus laccase can be used to modify marine pine kraft lignin, and that the set of analytical methods implemented here provides a valuable tool for screening enzymatic treatment conditions.

The Maize Pathogen Ustilago maydis Secretes Glycoside Hydrolases and Carbohydrate Oxidases Directed toward Components of the Fungal Cell Wall.

Filamentous fungi are keystone microorganisms in the regulation of many processes occurring on Earth, such as plant biomass decay and pathogenesis as well as symbiotic associations. In many of these processes, fungi secrete carbohydrate-active enzymes (CAZymes) to modify and/or degrade carbohydrates. Ten years ago, while evaluating the potential of a secretome from the maize pathogen Ustilago maydis to supplement lignocellulolytic cocktails, we noticed it contained many unknown or poorly characterized CAZymes. Here, and after reannotation of this data set and detailed phylogenetic analyses, we observed that several CAZymes (including glycoside hydrolases and carbohydrate oxidases) are predicted to act on the fungal cell wall (FCW), notably on ß-1,3-glucans. We heterologously produced and biochemically characterized two new CAZymes, called UmGH16_1-A and UmAA3_2-A. We show that UmGH16_1-A displays ß-1,3-glucanase activity, with a preference for ß-1,3-glucans with short ß-1,6 substitutions, and UmAA3_2-A is a dehydrogenase catalyzing the oxidation of ß-1,3- and ß-1,6-gluco-oligosaccharides into the corresponding aldonic acids. Working on model ß-1,3-glucans, we show that the linear oligosaccharide products released by UmGH16_1-A are further oxidized by UmAA3_2-A, bringing to light a putative biocatalytic cascade. Interestingly, analysis of available transcriptomics data indicates that both UmGH16_1-A and UmAA3_2-A are coexpressed, only during early stages of U. maydis infection cycle. Altogether, our results suggest that both enzymes are connected and that additional accessory activities still need to be uncovered to fully understand the biocatalytic cascade at play and its physiological role. IMPORTANCE Filamentous fungi play a central regulatory role on Earth, notably in the global carbon cycle. Regardless of their lifestyle, filamentous fungi need to remodel their own cell wall (mostly composed of polysaccharides) to grow and proliferate. To do so, they must secrete a large arsenal of enzymes, most notably carbohydrate-active enzymes (CAZymes). However, research on fungal CAZymes over past decades has mainly focused on finding efficient plant biomass conversion processes while CAZymes directed at the fungus itself have remained little explored. In the present study, using the maize pathogen Ustilago maydis as model, we set off to evaluate the prevalence of CAZymes directed toward the fungal cell wall during growth of the fungus on plant biomass and characterized two new CAZymes active on fungal cell wall components. Our results suggest the existence of a biocatalytic cascade that remains to be fully understood.

Biotransformation of the Fluoroquinolone, Levofloxacin, by the White-Rot Fungus Coriolopsis gallica.

The wastewater from hospitals, pharmaceutical industries and more generally human and animal dejections leads to environmental releases of antibiotics that cause severe problems for all living organisms. The aim of this study was to investigate the capacity of three fungal strains to biotransform the fluoroquinolone levofloxacin. The degradation processes were analyzed in solid and liquid media. Among the three fungal strains tested, Coriolopsis gallica strain CLBE55 (BRFM 3473) showed the highest removal efficiency, with a 15% decrease in antibiogram zone of inhibition for Escherichia coli cultured in solid medium and 25% degradation of the antibiotic in liquid medium based on high-performance liquid chromatography (HPLC). Proteomic analysis suggested that laccases and dye-decolorizing peroxidases such as extracellular enzymes could be involved in levofloxacin degradation, with a putative major role for laccases. Degradation products were proposed based on mass spectrometry analysis, and annotation suggested that the main product of biotransformation of levofloxacin by Coriolopsis gallica is an N-oxidized derivative.

Optimization of the Decolorization of the Reactive Black 5 by a Laccase-like Active Cell-Free Supernatant from Coriolopsis gallica.

The textile industry generates huge volumes of colored wastewater that require multiple treatments to remove persistent toxic and carcinogenic dyes. Here we studied the decolorization of a recalcitrant azo dye, Reactive Black 5, using laccase-like active cell-free supernatant from Coriolopsis gallica. Decolorization was optimized in a 1 mL reaction mixture using the response surface methodology (RSM) to test the influence of five variables, i.e., laccase-like activity, dye concentration, redox mediator (HBT) concentration, pH, and temperature, on dye decolorization. Statistical tests were used to determine regression coefficients and the quality of the models used, as well as significant factors and/or factor interactions. Maximum decolorization was achieved at 120 min (82 ± 0.6%) with the optimized protocol, i.e., laccase-like activity at 0.5 U mL−1, dye at 25 mg L−1, HBT at 4.5 mM, pH at 4.2 and temperature at 55 °C. The model proved significant (ANOVA test with p < 0.001): coefficient of determination (R²) was 89.78%, adjusted coefficient of determination (R²A) was 87.85%, and root mean square error (RMSE) was 10.48%. The reaction conditions yielding maximum decolorization were tested in a larger volume of 500 mL reaction mixture. Under these conditions, the decolorization rate reached 77.6 ± 0.4%, which was in good agreement with the value found on the 1 mL scale. RB5 decolorization was further evaluated using the UV-visible spectra of the treated and untreated dyes.

Screening New Xylanase Biocatalysts from the Mangrove Soil Diversity.

Mangrove sediments from New Caledonia were screened for xylanase sequences. One enzyme was selected and characterized both biochemically and for its industrial potential. Using a specific cDNA amplification method coupled with a MiSeq sequencing approach, the diversity of expressed genes encoding GH11 xylanases was investigated beneath Avicenia marina and Rhizophora stylosa trees during the wet and dry seasons and at two different sediment depths. GH11 xylanase diversity varied more according to tree species and season, than with respect to depth. One complete cDNA was selected (OFU29) and expressed in Pichia pastoris. The corresponding enzyme (called Xyn11-29) was biochemically characterized, revealing an optimal activity at 40-50 °C and at a pH of 5.5. Xyn11-29 was stable for 48 h at 35 °C, with a half-life of 1 h at 40 °C and in the pH range of 5.5-6. Xyn11-29 exhibited a high hydrolysis capacity on destarched wheat bran, with 40% and 16% of xylose and arabinose released after 24 h hydrolysis. Its activity on wheat straw was lower, with a release of 2.8% and 6.9% of xylose and arabinose, respectively. As the protein was isolated from mangrove sediments, the effect of sea salt on its activity was studied and discussed.

Crystal structure and functional characterization of an oligosaccharide dehydrogenase from Pycnoporus cinnabarinus provides insights into fungal breakdown of lignocellulose.

BACKGROUND: Fungal glucose dehydrogenases (GDHs) are FAD-dependent enzymes belonging to the glucose-methanol-choline oxidoreductase superfamily. These enzymes are classified in the "Auxiliary Activity" family 3 (AA3) of the Carbohydrate-Active enZymes database, and more specifically in subfamily AA3_2, that also includes the closely related flavoenzymes aryl-alcohol oxidase and glucose 1-oxidase. Based on sequence similarity to known fungal GDHs, an AA3_2 enzyme active on glucose was identified in the genome of Pycnoporus cinnabarinus, a model Basidiomycete able to completely degrade lignin. RESULTS: In our work, substrate screening and functional characterization showed an unexpected preferential activity of this enzyme toward oligosaccharides containing a ß(1→3) glycosidic bond, with the highest efficiency observed for the disaccharide laminaribiose. Despite its sequence similarity to GDHs, we defined a novel enzymatic activity, namely oligosaccharide dehydrogenase (ODH), for this enzyme. The crystallographic structures of ODH in the sugar-free form and in complex with glucose and laminaribiose unveiled a peculiar saccharide recognition mechanism which is not shared with previously characterized AA3 oxidoreductases and accounts for ODH preferential activity toward oligosaccharides. The sugar molecules in the active site of ODH are mainly stabilized through CH-π interactions with aromatic residues rather than through hydrogen bonds with highly conserved residues, as observed instead for the fungal glucose dehydrogenases and oxidases characterized to date. Finally, three sugar-binding sites were identified on ODH external surface, which were not previously observed and might be of importance in the physiological scenario. CONCLUSIONS: Structure-function analysis of ODH is consistent with its role as an auxiliary enzyme in lignocellulose degradation and unveils yet another enzymatic function within the AA3 family of the Carbohydrate-Active enZymes database. Our findings allow deciphering the molecular determinants of substrate binding and provide insight into the physiological role of ODH, opening new perspectives to exploit biodiversity for lignocellulose transformation into fuels and chemicals.

Exploring the Diversity of Fungal DyPs in Mangrove Soils to Produce and Characterize Novel Biocatalysts.

The functional diversity of the New Caledonian mangrove sediments was examined, observing the distribution of fungal dye-decolorizing peroxidases (DyPs), together with the complete biochemical characterization of the main DyP. Using a functional metabarcoding approach, the diversity of expressed genes encoding fungal DyPs was investigated in surface and deeper sediments, collected beneath either Avicennia marina or Rhizophora stylosa trees, during either the wet or the dry seasons. The highest DyP diversity was observed in surface sediments beneath the R. stylosa area during the wet season, and one particular operational functional unit (OFU1) was detected as the most abundant DyP isoform. This OFU was found in all sediment samples, representing 51-100% of the total DyP-encoding sequences in 70% of the samples. The complete cDNA sequence corresponding to this abundant DyP (OFU 1) was retrieved by gene capture, cloned, and heterologously expressed in Pichia pastoris. The recombinant enzyme, called DyP1, was purified and characterized, leading to the description of its physical-chemical properties, its ability to oxidize diverse phenolic substrates, and its potential to decolorize textile dyes; DyP1 was more active at low pH, though moderately stable over a wide pH range. The enzyme was very stable at temperatures up to 50 °C, retaining 60% activity after 180 min incubation. Its ability to decolorize industrial dyes was also tested on Reactive Blue 19, Acid Black, Disperse Blue 79, and Reactive Black 5. The effect of hydrogen peroxide and sea salt on DyP1 activity was studied and compared to what is reported for previously characterized enzymes from terrestrial and marine-derived fungi.

Enzyme Properties of a Laccase Obtained from the Transcriptome of the Marine-Derived Fungus Stemphylium lucomagnoense.

Only a few studies have examined how marine-derived fungi and their enzymes adapt to salinity and plant biomass degradation. This work concerns the production and characterisation of an oxidative enzyme identified from the transcriptome of marine-derived fungus Stemphylium lucomagnoense. The laccase-encoding gene SlLac2 from S. lucomagnoense was cloned for heterologous expression in Aspergillus niger D15#26 for protein production in the extracellular medium of around 30 mg L-1. The extracellular recombinant enzyme SlLac2 was successfully produced and purified in three steps protocol: ultrafiltration, anion-exchange chromatography, and size exclusion chromatography, with a final recovery yield of 24%. SlLac2 was characterised by physicochemical properties, kinetic parameters, and ability to oxidise diverse phenolic substrates. We also studied its activity in the presence and absence of sea salt. The molecular mass of SlLac2 was about 75 kDa, consistent with that of most ascomycete fungal laccases. With syringaldazine as substrate, SlLac2 showed an optimal activity at pH 6 and retained nearly 100% of its activity when incubated at 50°C for 180 min. SlLac2 exhibited more than 50% of its activity with 5% wt/vol of sea salt.

Characterization of the CAZy Repertoire from the Marine-Derived Fungus Stemphylium lucomagnoense in Relation to Saline Conditions.

Even if the ocean represents a large part of Earth's surface, only a few studies describe marine-derived fungi compared to their terrestrial homologues. In this ecosystem, marine-derived fungi have had to adapt to the salinity and to the plant biomass composition. This articles studies the growth of five marine isolates and the tuning of lignocellulolytic activities under different conditions, including the salinity. A de novo transcriptome sequencing and assembly were used in combination with a proteomic approach to characterize the Carbohydrate Active Enzymes (CAZy) repertoire of one of these strains. Following these approaches, Stemphylium lucomagnoense was selected for its adapted growth on xylan in saline conditions, its high xylanase activity, and its improved laccase activities in seagrass-containing cultures with salt. De novo transcriptome sequencing and assembly indicated the presence of 51 putative lignocellulolytic enzymes. Its secretome composition was studied in detail when the fungus was grown on either a terrestrial or a marine substrate, under saline and non-saline conditions. Proteomic analysis of the four S. lucomagnoense secretomes revealed a minimal suite of extracellular enzymes for plant biomass degradation and highlighted potential enzyme targets to be further studied for their adaptation to salts and for potential biotechnological applications.

Screening of five marine-derived fungal strains for their potential to produce oxidases with laccase activities suitable for biotechnological applications.

BACKGROUND: Environmental pollution is one of the major problems that the world is facing today. Several approaches have been taken, from physical and chemical methods to biotechnological strategies (e.g. the use of oxidoreductases). Oxidative enzymes from microorganisms offer eco-friendly, cost-effective processes amenable to biotechnological applications, such as in industrial dye decolorization. The aim of this study was to screen marine-derived fungal strains isolated from three coastal areas in Tunisia to identify laccase-like activities, and to produce and characterize active cell-free supernatants of interest for dye decolorization. RESULTS: Following the screening of 20 fungal strains isolated from the harbors of Sfax and Monastir (Tunisia), five strains were identified that displayed laccase-like activities. Molecular-based taxonomic approaches identified these strains as belonging to the species Trichoderma asperellum, Stemphylium lucomagnoense and Aspergillus nidulans. Among these five isolates, one T. asperellum strain (T. asperellum 1) gave the highest level of secreted oxidative activities, and so was chosen for further studies. Optimization of the growth medium for liquid cultures was first undertaken to improve the level of laccase-like activity in culture supernatants. Finally, the culture supernatant of T. asperellum 1 decolorized different synthetic dyes belonging to diverse dye families, in the presence or absence of 1-hydroxybenzotriazole (HBT) as a mediator. CONCLUSIONS: The optimal growth conditions to produce laccase-like active cell-free supernatants from T. asperellum 1 were 1.8 mM CuSO4 as an inducer, 1% NaCl to mimic a seawater environment and 3% sucrose as a carbon source. The culture supernatant of T. asperellum 1 effectively decolorized different synthetic dyes belonging to diverse chemical classes, and the presence of HBT as a mediator improved the decolorization process.

Characterization and Dye Decolorization Potential of Two Laccases from the Marine-Derived Fungus Pestalotiopsis sp.

: Two laccase-encoding genes from the marine-derived fungus Pestalotiopsis sp. have been cloned in Aspergillus niger for heterologous production, and the recombinant enzymes have been characterized to study their physicochemical properties, their ability to decolorize textile dyes for potential biotechnological applications, and their activity in the presence of sea salt. The optimal pH and temperature of PsLac1 and PsLac2 differed in relation to the substrates tested, and both enzymes were shown to be extremely stable at temperatures up to 50 °C, retaining 100% activity after 3 h at 50 °C. Both enzymes were stable between pH 4-6. Different substrate specificities were exhibited, and the lowest Km and highest catalytic efficiency values were obtained against syringaldazine and 2,6-dimethoxyphenol (DMP) for PsLac1 and PsLac2, respectively. The industrially important dyes-Acid Yellow, Bromo Cresol Purple, Nitrosulfonazo III, and Reactive Black 5-were more efficiently decolorized by PsLac1 in the presence of the redox mediator 1-hydroxybenzotriazole (HBT). Activities were compared in saline conditions, and PsLac2 seemed more adapted to the presence of sea salt than PsLac1. The overall surface charges of the predicted PsLac three-dimensional models showed large negatively charged surfaces for PsLac2, as found in proteins for marine organisms, and more balanced solvent exposed charges for PsLac1, as seen in proteins from terrestrial organisms.

Impact of copper ligand mutations on a cupredoxin with a green copper center.

Mononuclear cupredoxins contain a type 1 copper center with a trigonal or tetragonal geometry usually maintained by four ligands, a cystein, two histidines and a methionine. The recent discovery of new members of this family with unusual properties demonstrates, however, the versatility of this class of proteins. Changes in their ligand set lead to drastic variation in their metal site geometry and in the resulting spectroscopic and redox features. In our work, we report the identification of the copper ligands in the recently discovered cupredoxin AcoP. We show that even though AcoP possesses a classical copper ligand set, it has a highly perturbed copper center. In depth studies of mutant's properties suggest a high degree of constraint existing in the copper center of the wild type protein and even the addition of exogenous ligands does not lead to the reconstitution of the initial copper center. Not only the chemical nature of the axial ligand but also constraints brought by its covalent binding to the protein backbone might be critical to maintain a green copper site with high redox potential. This work illustrates the importance of experimentally dissecting the molecular diversity of cupredoxins to determine the molecular determinants responsible for their copper center geometry and redox potential.

Functional analysis and crystallographic structure of clotrimazole bound OleP, a cytochrome P450 epoxidase from Streptomyces antibioticus involved in oleandomycin biosynthesis.

BACKGROUND: OleP is a cyt P450 from Streptomyces antibioticus carrying out epoxigenation of the antibiotic oleandomycin during its biosynthesis. The timing of its reaction has not been fully clarified, doubts remain regarding its substrate and catalytic mechanism. METHODS: The crystal structure of OleP in complex with clotrimazole, an inhibitor of P450s used in therapy, was solved and the complex formation dynamics was characterized by equilibrium and kinetic binding studies and compared to ketoconazole, another azole differing for the N1-substituent. RESULTS: Clotrimazole coordinates the heme and occupies the active site. Most of the residues interacting with clotrimazole are conserved and involved in substrate binding in MycG, the P450 epoxigenase with the highest homology with OleP. Kinetic characterization of inhibitor binding revealed OleP to follow a simple bimolecular reaction, without detectable intermediates. CONCLUSIONS: Clotrimazole-bound OleP adopts an open form, held by a π-π stacking chain that fastens helices F and G and the FG loop. Affinity is affected by the interactions of the N1 substituent within the active site, given the one order of magnitude difference of the off-rate constants between clotrimazole and ketoconazole. Based on structural similarities with MycG, we propose a binding mode for both oleandomycin intermediates, that are the candidate substrates of OleP. GENERAL SIGNIFICANCE: Among P450 epoxigenases OleP is the only one that introduces an epoxide on a non-activated C­C bond. The data here presented are necessary to understand the rare chemistry carried out by OleP, to engineer it and to design more selective and potent P450-targeted drugs.

Spectroscopic characterization of a green copper site in a single-domain cupredoxin.

Cupredoxins are widespread copper-binding proteins, mainly involved in electron transfer pathways. They display a typical rigid greek key motif consisting of an eight stranded ß-sandwich. A fascinating feature of cupredoxins is the natural diversity of their copper center geometry. These geometry variations give rise to drastic changes in their color, such as blue, green, red or purple. Based on several spectroscopic and structural analyses, a connection between the geometry of their copper-binding site and their color has been proposed. However, little is known about the relationship between such diversity of copper center geometry in cupredoxins and possible implications for function. This has been difficult to assess, as only a few naturally occurring green and red copper sites have been described so far. We report herein the spectrocopic characterization of a novel kind of single domain cupredoxin of green color, involved in a respiratory pathway of the acidophilic organism Acidithiobacillus ferrooxidans. Biochemical and spectroscopic characterization coupled to bioinformatics analysis reveal the existence of some unusual features for this novel member of the green cupredoxin sub-family. This protein has the highest redox potential reported to date for a green-type cupredoxin. It has a constrained green copper site insensitive to pH or temperature variations. It is a green-type cupredoxin found for the first time in a respiratory pathway. These unique properties might be explained by a region of unknown function never found in other cupredoxins, and by an unusual length of the loop between the second and the fourth copper ligands. These discoveries will impact our knowledge on non-engineered green copper sites, whose involvement in respiratory chains seems more widespread than initially thought.

Structural basis for catalysis in a CDP-alcohol phosphotransferase.

The CDP-alcohol phosphotransferase (CDP-AP) family of integral membrane enzymes catalyses the transfer of a substituted phosphate group from a CDP-linked donor to an alcohol acceptor. This is an essential reaction for phospholipid biosynthesis across all kingdoms of life, and it is catalysed solely by CDP-APs. Here we report the 2.0 Å resolution crystal structure of a representative CDP-AP from Archaeoglobus fulgidus. The enzyme (AF2299) is a homodimer, with each protomer consisting of six transmembrane helices and an N-terminal cytosolic domain. A polar cavity within the membrane accommodates the active site, lined with the residues from an absolutely conserved CDP-AP signature motif (D(1)xxD(2)G(1)xxAR...G(2)xxxD(3)xxxD(4)). Structures in the apo, CMP-bound, CDP-bound and CDP-glycerol-bound states define functional roles for each of these eight conserved residues and allow us to propose a sequential, base-catalysed mechanism universal for CDP-APs, in which the fourth aspartate (D4) acts as the catalytic base.

Highlights from recently determined structures of membrane proteins: a focus on channels and transporters.

After decades of absent or lackluster growth, recent years have at long last witnessed an exponential growth in the number of novel membrane protein structures determined. Every single achievement has had a tremendous impact on the scientific community, providing an unprecedented wealth of information that typically only an atomic resolution structure can contribute to our molecular understanding of how a protein functions. Presented here is a review of some of the most exciting novel structures of channels and transporters determined by X-ray crystallography in the last two years, and a discussion of their analogies, differences and mechanistic implications.

Structure of lactococcal phage p2 baseplate and its mechanism of activation.

Siphoviridae is the most abundant viral family on earth which infects bacteria as well as archaea. All known siphophages infecting gram+ Lactococcus lactis possess a baseplate at the tip of their tail involved in host recognition and attachment. Here, we report analysis of the p2 phage baseplate structure by X-ray crystallography and electron microscopy and propose a mechanism for the baseplate activation during attachment to the host cell. This approximately 1 MDa, Escherichia coli-expressed baseplate is composed of three protein species, including six trimers of the receptor-binding protein (RBP). RBPs host-recognition domains point upwards, towards the capsid, in agreement with the electron-microscopy map of the free virion. In the presence of Ca(2+), a cation mandatory for infection, the RBPs rotated 200 degrees downwards, presenting their binding sites to the host, and a channel opens at the bottom of the baseplate for DNA passage. These conformational changes reveal a novel siphophage activation and host-recognition mechanism leading ultimately to DNA ejection.