FIND-IT: Accelerated trait development for a green evolution.

Improved agricultural and industrial production organisms are required to meet the future global food demands and minimize the effects of climate change. A new resource for crop and microbe improvement, designated FIND-IT (Fast Identification of Nucleotide variants by droplet DigITal PCR), provides ultrafast identification and isolation of predetermined, targeted genetic variants in a screening cycle of less than 10 days. Using large-scale sample pooling in combination with droplet digital PCR (ddPCR) greatly increases the size of low-mutation density and screenable variant libraries and the probability of identifying the variant of interest. The method is validated by screening variant libraries totaling 500,000 barley (Hordeum vulgare) individuals and isolating more than 125 targeted barley gene knockout lines and miRNA or promoter variants enabling functional gene analysis. FIND-IT variants are directly applicable to elite breeding pipelines and minimize time-consuming technical steps to accelerate the evolution of germplasm.

Trifluorosubstrates as mechanistic probes for an FMN-dependent l-2-hydroxy acid-oxidizing enzyme.

A controversy exists with respect to the mechanism of l-2-hydroxy acid oxidation by members of a family of FMN-dependent enzymes. A so-called carbanion mechanism was initially proposed, in which the active site histidine abstracts the substrate α-hydrogen as a proton, followed by electron transfer from the carbanion to the flavin. But an alternative mechanism was not incompatible with some results, a mechanism in which the active site histidine instead picks up the substrate hydroxyl proton and a hydride transfer occurs. Even though more recent experiments ruling out such a mechanism were published (Rao & Lederer (1999) Protein Science 7, 1531-1537), a few authors have subsequently interpreted their results with variant enzymes in terms of a hydride transfer. In the present work, we analyse the reactivity of trifluorolactate, a substrate analogue, with the flavocytochrome b2 (Fcb2) flavodehydrogenase domain, compared to its reactivity with an NAD-dependent lactate dehydrogenase (LDH), for which this compound is known to be an inhibitor (Pogolotti & Rupley (1973) Biochem. Biophys. Res. Commun, 55, 1214-1219). Indeed, electron attraction by the three fluorine atoms should make difficult the removal of the α-H as a hydride. We also analyse the reactivity of trifluoropyruvate with the FMN- and NAD-dependent enzymes. The results substantiate a different effect of the fluorine substituents on the two enzymes compared to their normal substrates. In the discussion we analyse the conclusions of recent papers advocating a hydride transfer mechanism for the family of l-2-hydroxy acid oxidizing FMN-dependent enzymes.

Properties of the bubble protein, a defensin and an abundant component of a fungal exudate.

Colonies of the ascomycete fungus Penicillium brevicompactum Dierckx produce bright yellow-green fluorescent exudate bubbles on its surface when grown on standard plant cell culture medium. According to SDS-PAGE analysis, the exudate is enriched in one protein, named bubble protein (BP). Detailed characteristics of BP are described, and also its corresponding genomic promoter and terminator sequences that flank sequences encoding signal peptide and a precursor sequence upstream of that of the mature protein. Following on previous work, the protein is now biochemically characterized. BP, the structure of which mainly consists of beta sheets, has four very stable disulfide bridges that resist standard procedures for reduction. With such traits, BP can now be categorized as a new member of the ever growing class of defensins. Indeed, the protein revealed anti-fungal effects as it inhibits growth of the yeast Saccharomyces cerevisiae in a dose-dependent manner. Structural classification places BP into the group of proteins with a knottin fold, founding the BP superfamily. Based on genomic alignments that revealed very high homology to four proteins of related fungi, a 3D structure prediction of the corresponding proteins was made. In addition, it was discovered that the closely related fungus Penicillium chrysogenum encodes a BP homolog - in addition to its PAF protein, which also is similar to BP - further suggesting that fungi may possess more than one defensin.

Structural basis of oligosaccharide receptor recognition by human papillomavirus.

High risk human papillomavirus types 16 (HPV16) and 18 (HPV18) can cause cervical cancer. Efficient infection by HPV16 and HPV18 pseudovirions requires interactions of particles with cell-surface receptor heparan sulfate oligosaccharide. To understand the virus-receptor interactions for HPV infection, we determined the crystal structures of HPV16 and HPV18 capsids bound to the oligosaccharide receptor fragment using oligomeric heparin. The HPV-heparin structures revealed multiple binding sites for the highly negatively charged oligosaccharide fragment on the capsid surface, which is different from previously reported virus-receptor interactions in which a single type of binding pocket is present for a particular receptor. We performed structure-guided mutagenesis to generate mutant viruses, and cell binding and infectivity assays demonstrated the functional role of viral residues involved in heparin binding. These results provide a basis for understanding virus-heparan sulfate receptor interactions critical for HPV infection and for the potential development of inhibitors against HPV infection.

Chondroitin sulphate A (CSA)-binding of single recombinant Duffy-binding-like domains is not restricted to Plasmodium falciparum Erythrocyte Membrane Protein 1 expressed by CSA-binding parasites.

Individuals living in areas with high Plasmodium falciparum transmission acquire immunity to malaria over time and adults have a markedly reduced risk of contracting severe disease. However, pregnant women constitute an important exception. Pregnancy-associated malaria is a major cause of mother and offspring morbidity, such as severe maternal anaemia and low birth-weight, and is characterised by selective accumulation of parasite-infected erythrocytes (IE) in the placenta. A P. falciparum protein named VAR2CSA, which belongs to the large P. falciparum Erythrocyte Membrane Protein 1 (PfEMP1) family, enables the IE to bind chondroitin sulphate A (CSA) in the placenta. Knock-out studies have demonstrated the exclusive capacity of VAR2CSA to mediate IE binding to CSA, and it has been shown that four of the six Duffy-binding-like (DBL) domains of VAR2CSA have the ability to bind CSA in vitro. In this study, we confirm the CSA-binding of these DBL domains, however, the analysis of a number of DBL domains of a non-VAR2CSA origin shows that CSA-binding is not exclusively restricted to VAR2CSA DBL domains. Furthermore, we show that the VAR2CSA DBL domains as well as other DBL domains also bind heparan sulphate. These data explain a number of publications describing CSA-binding domains derived from PfEMP1 antigens not involved in placental adhesion. The data suggest that the ability of single domains to bind CSA does not predict the functional capacity of the whole PfEMP1 and raises doubt whether the CSA-binding domains of native VAR2CSA have been correctly identified.

Molecular structure of heparan sulfate from Spalax. Implications of heparanase and hypoxia.

Spalax, a subterranean blind mole rat, is well adapted to live in an extreme hypoxic environment through up-regulated expression of growth factors and enzymes for ensuring sufficient oxygen supply. One of the overexpressed enzymes is heparanase, an endoglucuronidase that selectively cleaves heparan sulfate (HS) and is implicated in angiogenesis. To assess the implications of the heparanase in Spalax, we have characterized the structure of HS isolated from various organs of the animal. The oligosaccharides obtained after deaminative cleavage of HS samples from the tissues show an overall higher sulfation degree, distinct from that of murine tissues. Of particular significance was the appearance of a trisaccharide moiety in the tissues examined, apart of the even numbered oligosaccharide fractions typically found in HS from human and mouse tissues. The formation of this odd-numbered saccharide is a consequence of heparanase action, in agreement with the notion of high expression of the enzyme in this species. Analysis of HS extracted from human embryonic kidney cells (HEK293) after exposure to hypoxic condition revealed a structural change in the distribution of oligosaccharides similar to HS derived from Spalax organs. The alterations are likely due to up-regulated activity of heparanase, as real-time RT-PCR showed a 2-fold increase in heparanase mRNA expression in the hypoxia treated cells. HEK293 cells stably overexpressing Spalax heparanase produced HS sharing similarity with that from the Spalax organs, and exhibited enhanced MAPK activity in comparison with HEK293 cells, indicating a regulation role of the heparanase in the activity of growth factors.

Surface-exposed amino acid residues of HPV16 L1 protein mediating interaction with cell surface heparan sulfate.

Efficient infection of cells by human papillomaviruses (HPVs) and pseudovirions requires primary interaction with cell surface proteoglycans with apparent preference for species carrying heparan sulfate (HS) side chains. To identify residues contributing to virus/cell interaction, we performed point mutational analysis of the HPV16 major capsid protein, L1, targeting surface-exposed amino acid residues. Replacement of lysine residues 278, 356, or 361 for alanine reduced cell binding and infectivity of pseudovirions. Various combinations of these amino acid exchanges further decreased cell attachment and infectivity with residual infectivity of less than 5% for the triple mutant, suggesting that these lysine residues cooperate in HS binding. Single, double, or triple exchanges for arginine did not impair infectivity, demonstrating that interaction is dependent on charge distribution rather than sequence-specific. The lysine residues are located within a pocket on the capsomere surface, which was previously proposed as the putative receptor binding site. Fab fragments of binding-neutralizing antibody H16.56E that recognize an epitope directly adjacent to lysine residues strongly reduced HS-mediated cell binding, further corroborating our findings. In contrast, mutation of basic surface residues located in the cleft between capsomeres outside this pocket did not significantly reduce interaction with HS or resulted in assembly-deficient proteins. Computer-simulated heparin docking suggested that all three lysine residues can form hydrogen bonds with 2-O-, 6-O-, and N-sulfate groups of a single HS molecule with a minimal saccharide domain length of eight monomer units. This prediction was experimentally confirmed in binding experiments using capsid protein, heparin molecules of defined length, and sulfate group modifications.

A metal-binding site in the catalytic subunit of anaerobic ribonucleotide reductase.

A Zn(Cys)(4) center has been found in the C-terminal region of the crystal structure of the anaerobic class III ribonucleotide reductase (RNR) from bacteriophage T4. The metal center is structurally related to the zinc ribbon motif and to rubredoxin and rubrerythrin. Mutant enzymes of the homologous RNR from Escherichia coli, in which the coordinating cysteines, conserved in almost all known class III RNR sequences, have been mutated into alanines, are shown to be inactive as the result of their inability to generate the catalytically essential glycyl radical. The possible roles of the metal center are discussed in relationship to the currently proposed reaction mechanism for generation of the glycyl radical in class III RNRs.