Biochemical, structural and dynamical characterizations of the lactate dehydrogenase from Selenomonas ruminantium provide information about an intermediate evolutionary step prior to complete allosteric regulation acquisition in the super family of lactate and malate dehydrogenases.

In this work, we investigated the lactate dehydrogenase (LDH) from Selenomonas ruminantium (S. rum), an enzyme that differs at key amino acid positions from canonical allosteric LDHs. The wild type (Wt) of this enzyme recognises pyuvate as all LDHs. However, introducing a single point mutation in the active site loop (I85R) allows S. Rum LDH to recognize the oxaloacetate substrate as a typical malate dehydrogenase (MalDH), whilst maintaining homotropic activation as an LDH. We report the tertiary structure of the Wt and I85RLDH mutant. The Wt S. rum enzyme structure binds NADH and malonate, whilst also resembling the typical compact R-active state of canonical LDHs. The structure of the mutant with I85R was solved in the Apo State (without ligand), and shows no large conformational reorganization such as that observed with canonical allosteric LDHs in Apo state. This is due to a local structural feature typical of S. rum LDH that prevents large-scale conformational reorganization. The S. rum LDH was also studied using Molecular Dynamics simulations, probing specific local deformations of the active site that allow the S. rum LDH to sample the T-inactive state. We propose that, with respect to the LDH/MalDH superfamily, the S. rum enzyme possesses a specificstructural and dynamical way to ensure homotropic activation.

High-resolution crystal structure of the eukaryotic HMP-P synthase (THIC) from Arabidopsis thaliana.

Vitamin B1 is an essential compound in all organisms acting as a cofactor in key metabolic reactions. It is formed by the condensation of two independently biosynthesized molecules referred to as the pyrimidine and thiazole moieties. In bacteria and plants, the biosynthesis of the pyrimidine moiety, 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P), requires a single enzyme, THIC (HMP-P synthase). The enzyme uses an iron-sulfur cluster as well as a 5'-deoxyadenosyl radical as cofactors to rearrange the 5-amino-imidazole ribonucleotide (AIR) substrate to the pyrimidine ring. So far, the only structure reported is the one from the bacteria Caulobacter crescentus. In an attempt to structurally characterize an eukaryotic HMP-P synthase, we have determined the high-resolution crystal structure of THIC from Arabidopsis thaliana at 1.6 Å. The structure is highly similar to its bacterial counterpart although several loop regions show significant differences with potential implications for the enzymatic properties. Furthermore, we have found a metal ion with octahedral coordination at the same location as a zinc ion in the bacterial enzyme. Our high-resolution atomic model shows a metal ion with multiple coordinated water molecules in the close vicinity of the substrate binding sites and is an important step toward the full characterization of the chemical rearrangement occurring during HMP-P biosynthesis.

The last piece in the vitamin B1 biosynthesis puzzle: structural and functional insight into yeast 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P) synthase.

Vitamin B(1) is essential for all organisms being well recognized as a necessary cofactor for key metabolic pathways such as glycolysis, and was more recently implicated in DNA damage responses. Little is known about the enzyme responsible for the formation of the pyrimidine moiety (4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate (HMP-P) synthase). We report a structure-function study of the HMP-P synthase from yeast, THI5p. Our crystallographic structure shows that THI5p is a mix between periplasmic binding proteins and pyridoxal 5'-phosphate-dependent enzymes. Mutational and yeast complementation studies identify the key residues for HMP-P biosynthesis as well as the use of pyridoxal 5'-phosphate as a substrate rather than as a cofactor. Furthermore, we could show that iron binding to HMP-P synthase is essential for the reaction.

Design, Synthesis, and Pharmacological Evaluation of Second-Generation Soluble Adenylyl Cyclase (sAC, ADCY10) Inhibitors with Slow Dissociation Rates.

Soluble adenylyl cyclase (sAC: ADCY10) is an enzyme involved in intracellular signaling. Inhibition of sAC has potential therapeutic utility in a number of areas. For example, sAC is integral to successful male fertility: sAC activation is required for sperm motility and ability to undergo the acrosome reaction, two processes central to oocyte fertilization. Pharmacologic evaluation of existing sAC inhibitors for utility as on-demand, nonhormonal male contraceptives suggested that both high intrinsic potency, fast on and slow dissociation rates are essential design elements for successful male contraceptive applications. During the course of the medicinal chemistry campaign described here, we identified sAC inhibitors that fulfill these criteria and are suitable for in vivo evaluation of diverse sAC pharmacology.

Cairaella henrii gen. n., sp. n., a parasite of Norops trachyderma (Polychrotidae), and Ophiotaenia nicoleae sp. n. (Eucestoda: Proteocephalidea), a parasite of Thecadactylus rapicauda (Gekkonidae), in Ecuador.

Cairaella henrii gen. n., sp. n. (Proteocephalidea: Proteocephalinae) is described from the intestine of Norops trachyderma (Cope) (Polychrotidae) from San Pablo de Kantesiya in Ecuador. The new genus differs from the 12 other known genera of the Proteocephalinae (and all other proteocephalidean genera) by the presence of a dense network of osmoregulatory canals situated in the cortex and by the morphology of the scolex which is flattened dorsoventrally, with elongated deeply embedded suckers possessing a well-developed circular musculature situated in the anterolateral region, and by eggs with a three-layered embryophore possessing small outgrowths on its external surface. Ophiotaenia nicoleae sp. n. is described from the intestine of Thecadactylus rapicauda (Houttuyn) (Gekkonidae) from San Pablo de Kantesiya in Ecuador. This new species is characterised by the testes arranged in two fields, numbering 142-204, the cirrus-sac length representing 21-33% of proglottis width, the genital pore situated in the middle of the proglottis or slightly anteriorly, and the ovary width representing 68-88% of proglottis width. It differs from 20 of 27 Ophiotaenia species parasitic in New World reptiles by the presence of an apical organ and from the remaining species by one to several other morphological characters, such as the number of testes, diameter and shape of the scolex, position of ventral and dorsal osmoregulatory canals, or the presence of a vaginal sphincter. Both taxa represent the first record of proteocephalidean tapeworms in polychrotid and gekkonid lizards, respectively.

Ophiotaenia bonneti sp. n. (Eucestoda: Proteocephalidea), a parasite of Rana vaillanti (Anura: Ranidae) in Costa Rica.

Ophiotaenia bonneti sp. n. is described from the intestine of the frog Rana vaillanti Brocchi, 1877 (Anura: Ranidae) from San Gerardo, Guanacaste, Costa Rica. The new species is characterized by the testes 100-177 in number, the genital pores situated anteriorly, the osmoregulatory canals overlapping the testis field, the cirrus pouch length as 15-24% of proglottis width, and the uterus with 18-32 ramified diverticula on each side. It differs from the 23 known species of the genus Ophiotaenia La Rue, 1911, parasitic in amphibians, by one to several morphological characters. It differs from O. gracilis Jones, Cheng et Gillespie, 1958, the most morphologically similar species, in the sucker diameter in % of scolex diameter and in the morphology of the eggs - funnel-like depression and embryophore closely investing the oncosphere in O. gracilis. We generally observe a very low mean prevalence of the Proteocephalidea in Neotropical amphibians (about 0.41%-3%), but in the case of some host species, the prevalence can reach up to 25%. We conclude that these cestodes exhibit a strict host specificity of the oioxene type. Ophiotaenia junglensis Srivastava et Capoor, 1980 is considered a species inquirenda. Batrachotaenia hernandezi (Flores-Barroeta, 1955) becomes Ophiotaenia hernandezi (Flores-Barroeta, 1955) comb. n., B. tigrina (Woodland, 1925) becomes O. tigrina (Woodland, 1925) comb. n. and B. ceratophryos (Parodi et Widakowich, 1916) becomes O. ceratophryos (Parodi et Widakowich, 1916) comb. n.

Polyuridylation in Eukaryotes: A 3'-End Modification Regulating RNA Life.

In eukaryotes, mRNA polyadenylation is a well-known modification that is essential for many aspects of the protein-coding RNAs life cycle. However, modification of the 3' terminal nucleotide within various RNA molecules is a general and conserved process that broadly modulates RNA function in all kingdoms of life. Numerous types of modifications have been characterized, which are generally specific for a given type of RNA such as the CCA addition found in tRNAs. In recent years, the addition of nontemplated uridine nucleotides or uridylation has been shown to occur in various types of RNA molecules and in various cellular compartments with significantly different outcomes. Indeed, uridylation is able to alter RNA half-life both in positive and in negative ways, highlighting the importance of the enzymes in charge of performing this modification. The present review aims at summarizing the current knowledge on the various processes leading to RNA 3'-end uridylation and on their potential impacts in various diseases.

An artificial PPR scaffold for programmable RNA recognition.

Pentatricopeptide repeat (PPR) proteins control diverse aspects of RNA metabolism in eukaryotic cells. Although recent computational and structural studies have provided insights into RNA recognition by PPR proteins, their highly insoluble nature and inconsistencies between predicted and observed modes of RNA binding have restricted our understanding of their biological functions and their use as tools. Here we use a consensus design strategy to create artificial PPR domains that are structurally robust and can be programmed for sequence-specific RNA binding. The atomic structures of these artificial PPR domains elucidate the structural basis for their stability and modelling of RNA-protein interactions provides mechanistic insights into the importance of RNA-binding residues and suggests modes of PPR-RNA association. The modular mode of RNA binding by PPR proteins holds great promise for the engineering of new tools to target RNA and to understand the mechanisms of gene regulation by natural PPR proteins.

Redescription of Testudotaenia testudo (Magath, 1924) (Eucestoda: Proteocephalidea), a parasite of Apalone spinifera (Le Sueur) (Reptilia: Trionychidae) and Amia calva L. (Pisces: Amiidae) in North America and erection of the Testudotaeniinae n. subfam.

Testudotaenia testudo (Magath, 1924) is redescribed from the intestine of the softshell turtle Apalone spinifera (Le Sueur) (Trionychidae) and the bowfin Amia calva Linnaeus (Amiidae) from Reelfoot Lake, Tennessee, United States. A new subfamily, the Testudotaeniinae, is erected. The new taxon differs from all proteocephalidean subfamilies in the position of the genital organs in relation to the longitudinal internal musculature, i.e. the testes are cortical, rarely medullary; the ovary is partly medullary, with cortical lobes; the vitelline follicles are mainly medullary, with some follicles in the cortex; and the uterus is cortical. A key to the subfamilies of the order Proteocephalidea Mola, 1928 is provided. The most characteristic features of T. testudo are the precocious uterine aperture, the presence of internal uterine pores (as previously described for Proteocephalus paraguayensis (Rudin, 1917)), the eggs laid unripe, the very long strobila (up to 970 mm), and the presence of an anterior circular musculature in the suckers, which is considered as a good differential character. Three other species were found in Amia calva: Proteocephalus perplexus La Rue, 1911, P. ambloplitis (Leidy, 1887) and a new, undescribed form. Sequences of the partial nuclear 28S rRNA gene of specimens of T. testudo from Apalone spinulifera and Amia calva confirm the conspecificity of samples from these two very distinct hosts, which may represent a capture phenomenon. As the subfamily Adenobrechmoinae Bursey, Goldberg & Kraus, 2006 and the genus Adenobrechmos Bursey, Goldberg & Kraus, 2006 are based on the presence of an apical organ, a character which reflects a rather common convergence, we consider the Adenobrechmoinae to be a junior synonym of the Proteocephalinae La Rue, 1911 and Adenobrechmos a junior synonym of Ophiotaenia La Rue, 1911. Adenobrechmos greeri Bursey, Goldberg & Kraus, 2006 thus becomes Ophiotaenia greeri (Bursey, Goldberg & Kraus, 2006) n. comb.