Crystal structure of Escherichia coli ketopantoate reductase at 1.7 A resolution and insight into the enzyme mechanism.

Ketopantoate reductase (KPR, EC 1.1.1.169) catalyzes the NADPH-dependent reduction of ketopantoate to pantoate on the pantothenate (vitamin B(5)) biosynthetic pathway. The Escherichia coli panE gene encoding KPR was cloned and expressed at high levels as the native and selenomethionine-substituted (SeMet) proteins. Both native and SeMet recombinant proteins were purified by three chromatographic steps, to yield pure proteins. The wild-type enzyme was found to have a K(M)(NADPH) of 20 microM, a K(M)(ketopantoate) of 60 microM, and a k(cat) of 40 s(-1). Regular prismatic KPR crystals were prepared using the hanging drop technique. They belonged to the tetragonal space group P4(2)2(1)2, with cell parameters: a = b = 103.7 A and c = 55.7 A, accommodating one enzyme molecule per asymmetric unit. The structure of KPR was determined by the multiwavelength anomalous dispersion method using the SeMet protein, for which data were collected to 2.3 A resolution. The native data were collected to 1.7 A resolution and used to refine the final structure. The secondary structure comprises 12 alpha-helices, three 3(10)-helices, and 11 beta-strands. The enzyme is monomeric and has two domains separated by a cleft. The N-terminal domain has an alphabeta-fold of the Rossmann type. The C-terminal domain (residues 170-291) is composed of eight alpha-helices. KPR is shown to be a member of the 6-phosphogluconate dehydrogenase C-terminal domain-like superfamily. A model for the ternary enzyme-NADPH-ketopantoate ternary complex provides a rationale for kinetic data reported for specific site-directed mutants.     

Dispersal ability rather than ecological tolerance drives differences in range size between lentic and lotic water beetles (Coleoptera: Hydrophilidae)

Aim In aquatic ecosystems, standing (lentic) and running (lotic) waters differ fundamentally in their stability and persistence, shaping the comparative population genetic structure, geographical range size and speciation rates of lentic versus lotic lineages. While the drivers of this pattern remain incompletely understood, the suite of traits making up the ability of a species to establish new populations is instrumental in determining such differences. Here we explore the degree to which the association between habitat type and geographical range size results from differences in dispersal ability or fundamental niche breadth in the members of the Enochrus bicolor complex, an aquatic beetle clade with species across the lentic–lotic divide. Location Western Mediterranean, with a special focus on North Africa, the Iberian Peninsula and Sicily. Methods DNA sequences for four loci were obtained from species of the E. bicolor complex and analysed using phylogenetic inference. Dispersal and establishment abilities were assessed in lentic–lotic species pairs of the complex, using flight wing morphometrics and thermal tolerance ranges as surrogates, respectively. Results There were clear differences in range size between the lotic and lentic taxa of the complex, which appears to have had a lotic origin with two transitions to standing waters. Only small differences were observed in temperature tolerance and acclimation ability between the two lotic–lentic sister species studied. By contrast, wing morphometrics revealed clear, consistent differences between lotic and lentic Enochrus species pairs, the latter having a higher dispersal capacity. Main conclusions We hypothesize that there have been two habitat shifts from lotic to lentic waters, which have allowed marked expansions in geographical range size in western Mediterranean species of the E. bicolor complex. Differences in dispersal rather than in establishment ability appear to underlie differences in geographical range extent, as transitions to lentic waters were associated with changes in wing morphology, but not in thermal tolerance range. In this lineage of water beetles, selection for dispersal in geologically short‐lived lentic systems has driven the evolution of larger range sizes in lentic taxa compared with those of their lotic relatives.     

Cloning and functional expression of the small subunit of acetolactate synthase from Nicotiana plumbaginifolia

Acetolactate synthase (ALS) is the first committed step of branched-chain amino acid biosynthesis in plants and bacteria. The bacterial holoenzyme has been well characterized and is a tetramer of two identical large subunits (LSUs) of 60 kDa and two identical small subunits (SSUs) ranging in molecular mass from 9 to 17 kDa depending on the isozyme. The enzyme from plants is much less well characterized. Attempts to purify the protein have yielded an enzyme which appears to be an oligomer of LSUs, with the potential existence of a SSU for the plant enzyme remaining a matter of considerable speculation. We report here the discovery of a cDNA clone that encodes a SSU of plant ALS based upon the homology of the encoded peptide with various bacterial ALS SSUs. The plant ALS SSU is more than twice as large as any of its prokaryotic homologues and contains two domains that each encode a full-length copy of the prokaryotic SSU polypeptide. The cDNA clone was used to express Nicotiana plumbaginifolia SSU in Escherichia coli. Mixing a partially purified preparation of this SSU with the LSU of ALS from either N. plumbaginifolia or Arabidopsis thaliana results in both increased specific activity and increased stability of the enzymic activity. These results are consistent with those observed for the bacterial enzyme in similar experiments and represent the first functional demonstration of the existence of a SSU for plant ALS.     

Phylogeography of the European spiny lobster (Palinurus elephas): Influence of current oceanographical features and historical processes

The European spiny lobster (Palinurus elephas) is a suitable model organism to study the effects of past history and current oceanographic processes on the genetic diversity and population structure of marine species with a long-lived larval phase. A portion of the COI gene was sequenced in 227 individuals from 11 localities, covering most of the present distribution of the species. Divergence was found between Atlantic and Mediterranean regions, which could be explained by restricted gene flow between populations. Moreover, a principal component analysis detected differences within basins. The existence of genetic differentiation between Brittany and Ireland–Scotland populations could be accounted for by the large effect of the Gulf Stream, while mesoscale processes suffered by the incoming Atlantic waters could be responsible of genetic differentiation within the Mediterranean. Furthermore, historical processes could be responsible for a reduction on the overall genetic variability of P. elephas. The haplotypic distribution found in P. elephas, with the presence of one abundant haplotype and a large number of closely related haplotypes, is typical of species experiencing reduction in variability and subsequent expansions. Climatic fluctuations related to glacial cycles could explain the present level of variability and nucleotide diversity found. Interestingly, these glacial events do not seem to have the same impact in other species of the same genus. Our results indicate that recent glacial events could have had a lower impact on Palinurus mauritanicus, a congeneric species that presents an overlapping distribution area but is found in cooler waters than P. elephas.     

Synthesis, biological evaluation and molecular modelling of N-heterocyclic dipeptide aldehydes as selective calpain inhibitors

A series of N-heterocyclic dipeptide aldehydes 4-13 have been synthesised and evaluated as inhibitors of ovine calpain 1 (o-CAPN1) and ovine calpain 2 (o-CAPN2). 5-Formyl-pyrrole 9 (IC(50) values of 290 and 25nM against o-CAPN1 and o-CAPN2, respectively) was the most potent and selective o-CAPN2 inhibitor, displaying >11-fold selectivity. The amino acid sequences of o-CAPN1 and o-CAPN2 have been determined. Because of the lack of available structural information on the ovine calpains, in silico homology models of the active site cleft of o-CAPN1 and o-CAPN2 were developed based on human calpain 1 (h-CAPN1) X-ray crystal structure (PDB code 1ZCM). These models were used to rationalise the observed SAR for compounds 4-13 and the selectivity observed for 9. The o-CAPN2 selective inhibitor 9 (CAT0059) was assayed in an in vitro ovine lens culture system and shown to successfully protect the lens from calcium-induced opacification.     

Metagenomic analysis of the viral communities in fermented foods

Viruses are recognized as the most abundant biological components on Earth, and they regulate the structure of microbial communities in many environments. In soil and marine environments, microorganism-infecting phages are the most common type of virus. Although several types of bacteriophage have been isolated from fermented foods, little is known about the overall viral assemblages (viromes) of these environments. In this study, metagenomic analyses were performed on the uncultivated viral communities from three fermented foods, fermented shrimp, kimchi, and sauerkraut. Using a high-throughput pyrosequencing technique, a total of 81,831, 70,591 and 69,464 viral sequences were obtained from fermented shrimp, kimchi and sauerkraut, respectively. Moreover, 37 to 50% of these sequences showed no significant hit against sequences in public databases. There were some discrepancies between the prediction of bacteriophages hosts via homology comparison and bacterial distribution, as determined from 16S rRNA gene sequencing. These discrepancies likely reflect the fact that the viral genomes of fermented foods are poorly represented in public databases. Double-stranded DNA viral communities were amplified from fermented foods by using a linker-amplified shotgun library. These communities were dominated by bacteriophages belonging to the viral order Caudovirales (i.e., Myoviridae, Podoviridae, and Siphoviridae). This study indicates that fermented foods contain less complex viral communities than many other environmental habitats, such as seawater, human feces, marine sediment, and soil.