The testosterone binding mechanism of an antibody derived from a naïve human scFv library

A testosterone binding scFv antibody was isolated from a naïve human library with a modest size of 10⁸ clones. The crystal structure of the Fab fragment form of the 5F2 antibody clone complexed with testosterone determined at 1.5 Å resolution shows that the hapten is bound deeply in the antibody binding pocket. In addition to the interactions with framework residues only CDR‐L3 and CDR‐H3 loops interact with testosterone and the heavy chain forms the majority of the contacts with the hapten. The testosterone binding site of the 5F2 antibody with a high abundance of aromatic amino acid residues shows similarity with an in vitro affinity matured antibody having around 300 times higher affinity. The moderate affinity of the 5F2 antibody originates from the different orientation of the hapten and few light chain contacts. This is the first three‐dimensional structure of a human steroid hormone binding antibody that has been isolated from a naïve human repertoire. Copyright © 2010 John Wiley & Sons, Ltd.     

Crystal structures of two aromatic hydroxylases involved in the early tailoring steps of angucycline biosynthesis

Angucyclines are aromatic polyketides produced in Streptomycetes via complex enzymatic biosynthetic pathways. PgaE and CabE from S. sp PGA64 and S. sp. H021 are two related homo-dimeric FAD and NADPH dependent aromatic hydroxylases involved in the early steps of the angucycline core modification. Here we report the three-dimensional structures of these two enzymes determined by X-ray crystallography using multiple anomalous diffraction and molecular replacement, respectively, to resolutions of 1.8 A and 2.7 A. The enzyme subunits are built up of three domains, a FAD binding domain, a domain involved in substrate binding and a C-terminal thioredoxin-like domain of unknown function. The structure analysis identifies PgaE and CabE as members of the para-hydroxybenzoate hydroxylase (pHBH) fold family of aromatic hydroxylases. In contrast to phenol hydroxylase and 3-hydroxybenzoate hydroxylase that utilize the C-terminal domain for dimer formation, this domain is not part of the subunit-subunit interface in PgaE and CabE. Instead, dimer assembly occurs through interactions of their FAD binding domains. FAD is bound non-covalently in the "in"-conformation. The active sites in the two enzymes differ significantly from those of other aromatic hydroxylases. The volumes of the active site are significantly larger, as expected in view of the voluminous tetracyclic angucycline substrates. The structures further suggest that substrate binding and catalysis may involve dynamic rearrangements of the middle domain relative to the other two domains. Site-directed mutagenesis studies of putative catalytic groups in the active site of PgaE argue against enzyme-catalyzed substrate deprotonation as a step in catalysis. This is in contrast to pHBH, where deprotonation/protonation of the substrate has been suggested as an essential part of the enzymatic mechanism.     

Cytotype regulation by telomeric P elements in Drosophila melanogaster: interactions with P elements from M' strains

P strains of Drosophila are distinguished from M strains by having P elements in their genomes and also by having the P cytotype, a maternally inherited condition that strongly represses P-element-induced hybrid dysgenesis. The P cytotype is associated with P elements inserted near the left telomere of the X chromosome. Repression by the telomeric P elements TP5 and TP6 is significantly enhanced when these elements are crossed into M′ strains, which, like P strains, carry P elements, but have little or no ability to repress dysgenesis. The telomeric and M′ P elements must coexist in females for this enhanced repression ability to develop. However, once established, it is transmitted maternally to the immediate offspring independently of the telomeric P elements themselves. Females that carry a telomeric P element but that do not carry M′ P elements may also transmit an ability to repress dysgenesis to their offspring independently of the telomeric P element. Cytotype regulation therefore involves a maternally transmissible product of telomeric P elements that can interact synergistically with products from paternally inherited M′ P elements. This synergism between TP and M′ P elements also appears to persist for at least one generation after the TP has been removed from the genotype.     

Crystal structures of SnoaL2 and AclR: two putative hydroxylases in the biosynthesis of aromatic polyketide antibiotics

SnoaL2 and AclR are homologous enzymes in the biosynthesis of the aromatic polyketides nogalamycin in Streptomyces nogalater and cinerubin in Streptomyces galilaeus, respectively. Evidence obtained from gene transfer experiments suggested that SnoaL2 catalyzes the hydroxylation of the C-1 carbon atom of the polyketide chain. Here we show that AclR is also involved in the production of 1-hydroxylated anthracyclines in vivo. The three-dimensional structure of SnoaL2 has been determined by multi-wavelength anomalous diffraction to 2.5A resolution, and that of AclR to 1.8A resolution using molecular replacement. Both enzymes are dimers in solution and in the crystal. The fold of the enzyme subunits consists of an alpha+beta barrel. The dimer interface is formed by packing of the beta-sheets from the two subunits against each other. In the interior of the alpha+beta barrel a hydrophobic cavity is formed that most likely binds the substrate and harbors the active site. The subunit fold and the architecture of the active site in SnoaL2 and AclR are similar to that of the polyketide cyclases SnoaL and AknH; however, they show completely different quaternary structures. A comparison of the active site pockets of the putative hydroxylases AclR and SnoaL2 with those of bona fide polyketide cyclases reveals distinct differences in amino acids lining the cavity that might be responsible for the switch in chemistry. The moderate degree of sequence similarity and the preservation of the three-dimensional fold of the polypeptide chain suggest that these enzymes are evolutionary related. Members of this enzyme family appear to have evolved from a common protein scaffold by divergent evolution to catalyze reactions chemically as diverse as aldol condensation and hydroxylation.     

Potential risk of biologic pollution associated to the introduction of <i>Pinus radiata</i> in grassland areas

Afforestation is a recommended practice to mitigate global warming. However, their implementation may generate undesirable impacts, mostly if exotic species are used. Plantations of Pinus radiata D Don in Ventania (Bs. As., Argentina) soils showed notorious increments of extractable P (Pe), which could affect the dynamic of this element as well as the degree of phosphorus saturation (GSP_Bray). The objectives of this study were: i) to quantify the GSP_Bray in Mollisols afforested with P. radiata comparing the results with those coming from adjacent, natural grassland areas (base line); ii) to evaluate the potential environmental risk induced by afforestation through the identification of a change point (PC) in the GSP_Bray indicative of a phosphate leaching increment. Treatments included mature stands of P. radiata (TB) and adjacent areas with natural grassland vegetation (TP). Samples were taken at 0-15; 15-30 and 30-45 cm soil depth, and texture, pH, total organic carbon (COT), Pe, soluble reactive phosphorus (PSR), phosphorus sorption index (ISP) and GSP_Bray were determined. The results showed a significant acidification in TB and an increase in the COT stock, indicating an additional atmospheric CO₂ sequestration by the trees. The Pe and PSR values were notoriously higher in TB, and they were reflected in a significant increment in the GSP_Bray with respect to TP. The detection of a significant PC in the GSP_Bray-PSR regression indicates higher chances of phosphate leaching in the forest stands, which could reach water courses, lakes and artificial reservoirs promoting their eutrophication. Because of the potential environmental pollution risk of biologic origin derived from the afforestation with P. radiata in Mollisols areas, their inclusion in clean development practices must be reconsidered.     

Spermidine and methylglyoxal bis(guanylhydrazone) affect maturation and endogenous polyamine content of Scots pine embryogenic cultures

Exogenous spermidine (Spd) and methylglyoxal bis(guanylhydrazone) (MGBG), a putative inhibitor of Spd synthesis, improved somatic embryo formation of Scots pine (Pinus sylvestris L.). The induced maturation due to MGBG and Spd was accompanied by significantly retarded proliferation growth and by reduction in the concentration of free polyamines compared to the control cultures. The action of MGBG revealed that it has a non-specific effect on the whole polyamine metabolism of Scots pine. Furthermore, at certain concentrations it may induce plant differentiation as well.     

Actinomycetes and Fungi in Surface Waters and in Potable Water

In Finnish lakes and rivers used as water supplies, mesophilic fungi and actinomycetes were common, whereas thermophilic fungi and actinomycetes were present only in low concentrations. Fungi and actinomycetes were more abundant in eutrophic and mesotrophic lakes than in oligotrophic lakes. River water contained more thermophilic actinomycetes and fungi and mesophilic actinomycetes than did lake water. Runoff from soil seemed to be an important factor contributing to the incidence of these microbes in water. Chemical coagulation removed actinomycetes and fungi efficiently, but sand filtration allowed their passage. Disinfection could not prevent actinomycetes and fungi from reaching the distribution system. During infiltration in the production of recharged groundwater, mesophilic actinomycetes could even multiply appreciably.     

Stability of the fluorogenic enzyme substrates and pH optima of enzyme activities in different Finnish soils

Fluorogenic artificial substrates facilitate sensitive enzyme activity measurements for a variety of processes in soil and other environmental samples. It is possible to use in situ pH for measurements on condition that the substrates are chemically stable. We studied the stability of 12 different methyl umbellipherone (MUF) and amino methyl coumarine (AMC) derivatives used as substrates for arylsulphatase, alpha-glucosidase, beta-glucosidase, beta-xylosidase, cellobiosidase, chitinase, phosphomonoesterase (PME), phoshodiesterase (PDE), esterase, lipase and alanine- and leucine aminopeptidases (AP) over the pH range from 4.0 to 8.0 in modified universal buffer (MUB). Stability of the substrates for lipase (4-MUF-heptanoate) and esterase (4-MUF-acetate) measurements was poor, especially at the higher pH values. Chitinase substrate, 4-MUF-N-acetyl-beta-D-glucosamide, was unstable at high pH values whereas the substrate for PME activity measurement (4-MUF-phosphate) disintegrated at low pH. The other substrates and MUF and AMC standard solutions were stable over the pH range studied. The optima between pH 4 and 8 of the 11 different enzyme activities were measured in three forest and two agricultural soil samples and in one activated sludge sample. In soil, for alanine and leucine AP the pH optima were usually 7.5 or higher, for arylsulphatase, beta-glucosidase, beta-xylosidase, esterase and PDE between 4 and 5.5, and for cellobiosidase between 4 and 5. alpha-Glucosidase had an optimum below 5.5 but also exhibited high activity at pH 7. Soil-dependent variation in pH optima were observed for chitinase, esterase, PDE and PME. Enzyme activities were also measured in 0.5 M acetate buffer at pH 5.5. This buffer yielded the highest activities in all soil samples for arylsulphatase, PDE and PME.