Enzymatic microreactors in chemical analysis and kinetic studies

The fields of application of microreactors are becoming wider every year. A considerable number of papers have been published recently reporting successful application of enzymatic microreactors in chemistry and biochemistry. Most are devices with enzymes immobilized on beads or walls of microfluidic channels, whilst some use dissolved enzymes to run a reaction in the microfluidic system. Apart from model systems, mostly with glucose oxidase, horseradish peroxidase and alkaline phosphatase, the principal fields of application of microreactors are tryptic digestion of proteins and polymerase chain reaction in automated analyses of proteomic and genetic material, respectively. Enzymatic microreactors also facilitate characterization of enzyme activity as a function of substrate concentration, and enable fast screening of new biocatalysts and their substrates. They may constitute key parts of lab-on-a-chip and muTAS, assisting the analysis of biomolecules. This review provides systematic coverage of examples of reports on enzymatic microreactors published recently, as well as relevant older papers.     

RNF4 is a poly-SUMO-specific E3 ubiquitin ligase required for arsenic-induced PML degradation

In acute promyelocytic leukaemia (APL), the promyelocytic leukaemia (PML) protein is fused to the retinoic acid receptor alpha (RAR). This disease can be treated effectively with arsenic, which induces PML modification by small ubiquitin-like modifiers (SUMO) and proteasomal degradation. Here we demonstrate that the RING-domain-containing ubiquitin E3 ligase, RNF4 (also known as SNURF), targets poly-SUMO-modified proteins for degradation mediated by ubiquitin. RNF4 depletion or proteasome inhibition led to accumulation of mixed, polyubiquitinated, poly-SUMO chains. PML protein accumulated in RNF4-depleted cells and was ubiquitinated by RNF4 in a SUMO-dependent fashion in vitro. In the absence of RNF4, arsenic failed to induce degradation of PML and SUMO-modified PML accumulated in the nucleus. These results demonstrate that poly-SUMO chains can act as discrete signals from mono-SUMOylation, in this case targeting a poly-SUMOylated substrate for ubiquitin-mediated proteolysis.     

Morphological analysis of three equivocal sibling species of Deprea (Solanaceae)

Taxonomic confusion among closely related and morphologically similar Deprea species has persisted in the literature and in the identification of species. Morphological variation among three closely related, monophyletic Deprea species was studied to determine if and how they can be distinguished. Their sympatric occurrence in Venezuela afforded an opportunity to couple field study with analysis of herbarium specimens representing their entire geographic range. An analysis of 94 morphological characters resulted in five vegetative and 13 reproductive taxonomically informative traits. Canonical variates analysis clearly separated the three species using six quantitative traits. We conclude that these taxa, although quite variable and similar morphologically, are taxonomically distinct. Results of character analysis indicated that D. orinocensis is morphologically more similar to D. bitteriana than either are to D. paneroi. In D. paneroi, small, sterile anthers on fruit-bearing plants and the absence of fruits on plants possessing large, plllen-bearing anthers, suggest cryptic dioecy. Based on these data, D. granulosa is considered to be a synonym of D. orinocensis: Athenaea bitteriana, a misapplied synonym, is the correct basionym and is applicable to many specimens identified as D. granulosa. We submit a new combination, D. bitteriana (Werderm.) Sawyer & Benítez, and designate a lectotype to accommodate these findings.     

The cytochromes c-550 of Paracoccus denitrificans and Thiosphaera pantotropha: a need for re-evaluation of the history of Paracoccus cultures

Abstract The c -type cytochrome and protein profiles were compared for a number of cultures of Paracoccus denitrificans obtained from a range of culture collections. The cultures fell into two groups corresponding to the two original isolates of this bacterial species. One group, which included NCIMB 8944, ATCC 13543, ATCC 17741, ATCC 19367, Pd 1222 and DSM 413, were similar or identical to LMD 22.21. The second group, including DSM 65 and LMG 4218, were similar or identical to LMD 52.44. These groupings were not compatible with the recorded history of culture deposition. Mass spectrometry and amino acid sequence comparisons showed that the cytochrome c -550 of the LMD 52.44 culture group differed by 16% from that of the LMD 22.21 group, and yet was only 1% different from the cytochrome c -550 of Thiosphaera pantotropha . These results suggest that consideration should be given to creation of a new species of Paracoccus pantotropha , which would include Thiosphaera pantotropha and Paracoccus denitrificans LMD 52.44.     

Crystal structure of Leishmania major oligopeptidase B gives insight into the enzymatic properties of a trypanosomatid virulence factor

Oligopeptidase B (OPB) is a serine peptidase with dibasic substrate specificity. It is found in bacteria, plants, and trypanosomatid pathogens, where it has been identified as a virulence factor and potential drug target. In this study we expressed active recombinant Leishmania major OPB and provide the first structure of an oligopeptidase B at high resolution. The crystallographic study reveals that OPB comprises two domains, a catalytic and a propeller domain, linked together by a hinge region. The structure has been determined in complex with the oligopeptide, protease-inhibitor antipain, giving detailed information on the enzyme active site and extended substrate binding pockets. It shows that Glu-621 plays a critical role in the S1 binding pocket and, along with Phe-603, is largely responsible for the enzyme substrate specificity in P1. In the S2 binding pocket, Tyr-499 was shown to be important for substrate stability. The structure also allowed an investigation into the function of residues highlighted in other studies including Glu-623, which was predicted to be involved in the S1 binding pocket but is found forming an inter-domain hydrogen bond. Additional important salt bridges/hydrogen bonds between the two domains were observed, highlighting the significance of the domain interface in OPB. This work provides a foundation for the study of the role of OPBs as virulence factors in trypanosomatids. It could facilitate the development of specific OPB inhibitors with therapeutic potential by exploiting its unique substrate recognition properties as well as providing a model for OPBs in general.     

Expression of NEP2, a soluble neprilysin-like endopeptidase, during embryogenesis in Drosophila melanogaster

Members of the neprilysin family of neutral endopeptidases (M13) are typically membrane-bound enzymes known to be involved in the extra-cellular metabolism of signalling peptides and have important roles during mammalian embryogenesis. In this study we show that membranes prepared from embryos of Drosophila melanogaster possess neprilysin-like activity that is inhibited by phosphoramidon and thiorphan, both inhibitors of mammalian neprilysin. Unexpectedly, we also found strong neprilysin-like neutral endopeptidase activity in a soluble embryo fraction, which we identify as NEP2 by Western blot and immunoprecipitation experiments using NEP2 specific antibodies. NEP2 is a soluble secreted member of the neprilysin family that has been shown previously to be expressed in larval and adult Malpighian tubules and in the testes of adult males. In situ hybridization studies reveal expression at stage 10-11 in a pattern similar to that previously described for stellate cell progenitors of the caudal visceral mesoderm. In later stages of embryogenesis, some of these cells appear to migrate into the growing Malpighian tubule. Recombinant NEP2 protein is N-glycosylated and displays optimum endopeptidase activity at neutral pH, consistent with a role as an extracellular peptidase. The recombinant enzyme hydrolyses Drosophila tachykinin peptides (DTK) at peptide bonds N-terminal to hydrophobic residues. DTK2, like Locusta tachykinin-1, was cleaved at the penultimate peptide bond (Gly(7)-Leu(8)), whereas the other Drosophila peptides were cleaved centrally at Xxx-Phe bonds. However, the rates of hydrolysis of the latter substrates were much slower than the hydrolysis rates of DTK2 and Locusta tachykinin-1, suggesting that the interaction of the bulky side-chain of phenylalanine at the S'(1) sub-site is less favorable for peptide bond hydrolysis. The secretion of NEP2 from tissues during embryogenesis suggests a possible developmental role for this endopeptidase in peptide signalling in D. melanogaster.     

Linkage of X-linked retinitis pigmentosa to the hypervariable DNA marker M27β (DXS255)

Summary A hypervariable DNA marker is closely linked to one of the most severe forms of night blindness, X-linked retinitis pigmentosa (RP). Affected individuals with X-linked RP, obligate carriers, and ophthalmologically identifiable carriers of the disease were included in a linkage study. The diagnosis was established in five sibships by funduscopic and electrophysiological investigations. When the X-linked probe M27 was used, 2 recombinants out of 29 informative meioses were detected (=0.07 at a maximum lod of 4.75). The hypervariable probe detected two different alleles in 38 of 39 females tested. M27 is therefore a potentially very useful probe for carrier detection and prenatal diagnosis, as well as for addressing the question of heterogeneity of X-linked RP.     

Whole genome sequencing of a natural recombinant Toxoplasma gondii strain reveals chromosome sorting and local allelic variants

Background  

Toxoplasma gondii is a zoonotic parasite of global importance. In common with many protozoan parasites it has the capacity for sexual recombination, but current evidence suggests this is rarely employed. The global population structure is dominated by a small number of clonal genotypes, which exhibit biallelic variation and limited intralineage divergence. Little is known of the genotypes present in Africa despite the importance of AIDS-associated toxoplasmosis.