A component in seminal fluid elicits an ovulatory response and has been discovered in every species examined thus far. The existence of an ovulation-inducing factor (OIF) in seminal plasma has broad implications and evokes questions about identity, tissue sources, mechanism of action, role among species, and clinical relevance in infertility. Most of these questions remain unanswered. The goal of this study was to determine the identity of OIF in support of the hypothesis that it is a single distinct and widely conserved entity. Seminal plasma from llamas and bulls was used as representative of induced and spontaneous ovulators, respectively. A fraction isolated from llama seminal plasma by column chromatography was identified as OIF by eliciting luteinizing hormone (LH) release and ovulation in llamas. MALDI-TOF revealed a molecular mass of 13,221 Da, and 12-23 aa sequences of OIF had homology with human, porcine, bovine, and murine sequences of β nerve growth factor (β-NGF). X-ray diffraction data were used to solve the full sequence and structure of OIF as β-NGF. Neurite development and up-regulation of trkA in phaeochromocytoma (PC 12 ) cells in vitro confirmed NGF-like properties of OIF. Western blot analysis of llama and bull seminal plasma confirmed immunorecognition of OIF using polyclonal mouse anti-NGF, and administration of β-NGF from mouse submandibular glands induced ovulation in llamas. We conclude that OIF in seminal plasma is β-NGF and that it is highly conserved. An endocrine route of action of NGF elucidates a previously unknown pathway for the direct influence of the male on the hypothalamo-pituitary-gonadal axis of the inseminated female.neurotrophins | hypothalamus | fertility | neuroendocrine I n a monograph nearly 50 y ago, Thaddeus Mann summarized the natural properties of seminal plasma as a vehicle for sperm transport, a controller of sperm motility and capacitation, and as a stimulant of uterine contractility (1). Notwithstanding Mann's admonishment to resist the temptation "to assign to every newly discovered chemical constituent of semen a major role in the process of fertilization," recent isolation of a protein factor in seminal plasma (2-4) suggests an additional role of the ejaculateas an inducer of ovulation.The role of the fluid portion of the ejaculate, and the male accessory glands responsible for producing it, has been enigmatic. From an evolutionary perspective, it has been suggested that the male accessory glands likely originated as the machinery for producing a copulatory plug, which has the "chastity effect" of preventing the sperm of other males from entering the female tract, as well as minimizing sperm loss after insemination (5). If this is so, then the persistence of an elaborate accessory gland system in many species in which plug formation does not occur may be viewed as nothing more than an evolutionary vestige.The first reports of an ovulation-inducing factor (OIF) in semen resulted from the observation that ovulation occurred after intravaginal or intramuscula...

Lytic transglycosylases are bacterial enzymes involved in the maintenance and growth of the bacterial cell-wall peptidoglycan. They cleave the b-(1,4)-glycosidic bonds in peptidoglycan forming non-reducing 1,6-anhydro-muropeptides. The crystal structure of the lytic transglycosylase MltA from Escherichia coli without a membrane anchor was solved at 2.0 A ˚ resolution. The enzyme has a fold completely different from those of the other known lytic transglycosylases. It contains two domains, the largest of which has a double-psi b-barrel fold, similar to that of endoglucanase V from Humicola insolens. The smaller domain also has a b-barrel fold topology, which is weakly related to that of the RNA-binding domain of ribosomal proteins L25 and TL5. A large groove separates the two domains, which can accommodate a glycan strand, as shown by molecular modelling. Several conserved residues, one of which is in a position equivalent to that of the catalytic acid of the H. insolens endoglucanase, flank this putative substrate-binding groove. Mutation of this residue, Asp308, abolished all activity of the enzyme, supporting the direct participation of this residue in catalysis.

UDP-Galactopyranose mutase (UGM) is a flavin-containing enzyme that catalyses the reversible conversion of UDP-Galactopyranose (UDP-Galp) to UDP-Galactofuranose (UDPGalf) and plays a key role in the biosynthesis of the mycobacterial cell wall galactofuran. A soluble, active form of UGM from Mycobacterium tuberculosis (MtUGM) was obtained from a dual His 6 -MBP tagged MtUGM construct. We present the first complex structures of MtUGM with bound substrate UDP-Galp (both oxidized flavin and reduced flavin). In addition, we have determined the complex structures of MtUGM with inhibitors (UDP and the dideoxytetrafluorinated analogs of both UDP-Galp (UDP-F 4 -Galp) and UDP-Galf (UDP-F 4 -Galf)), which represent the first complex structures of UGM with an analogue in the furanose form, as well as the first structures of dideoxy-tetrafluorinated sugar analogs bound to a protein. These structures provide detailed insight into ligand recognition by MtUGM and show a similar overall binding mode as reported for other prokaryotic UGMs. The binding of the ligand induces conformational changes in the enzyme, allowing ligand binding and active site closure. In addition, the complex structure of MtUGM with UDP-F 4 -Galf reveals the first detailed insight into how the furanose moiety binds to UGM. In particular, this study confirmed that the furanoside adopts a high energy conformation ( 4 E) within the catalytic pocket. Moreover, these investigations provide structural insights to the enhanced binding of the dideoxy-tetrafluorinated sugars compared to unmodified analogs. These results will help in the design of carbohydrate mimetics and drug development, and show the enormous possibilities on the use of polyfluorination in the design of carbohydrate mimetics.

Background: UDP-galactopyranose mutase (UGM) is a critical enzyme for the proper formation of the cell wall of pathogenic microbes.Results: The structure of UGM in complex with substrate reveals novel features of substrate binding.Conclusion: Oxidation and reduction of the flavin cofactor causes rearrangements in the active site that affect substrate binding.Significance: These are the first structures of UGM from a eukaryotic pathogen.

Crystal structures of an inactive mutant (D308A) of the lytic transglycosylase MltA from Escherichia coli have been determined in two different apo-forms, as well as in complex with the substrate analogue chitohexaose. The chitohexaose binds with all six saccharide residues in the active site groove, with an intact glycosidic bond at the bond cleavage center. Its binding induces a large reorientation of the two structural domains in MltA, narrowing the active site groove and allowing tight interactions of the oligosaccharide with residues from both domains. The structures identify residues in MltA with key roles in the binding and recognition of peptidoglycan and confirm that Asp-308 is the single catalytic residue, acting as a general acid/base. Moreover, the structures suggest that catalysis involves a high energy conformation of the scissile glycosidic linkage and that the putative oxocarbenium ion intermediate is stabilized by the dipole moment of a nearby ␣-helix. Lytic transglycosylases (LTs)4 are bacterial muramidases that cleave the bacterial cell wall heteropolymer peptidoglycan (murein) for turnover and recycling to facilitate cell growth and division, as well as to allow local cell wall opening without loss of integrity (1, 2). The lytic activity of these enzymes is directed toward the ␤-1,4-glycosidic bonds between N-acetylmuramic acid (MurNAc) and GlcNAc residues, the two sugar units that make up the glycan strands of peptidoglycan. Concomitant to bond cleavage, they catalyze an intramolecular transglycosylation reaction resulting in the formation of muropeptides terminated with a non-reducing 1,6-anhydromuramic acid residue.LTs are found in a wide variety of bacteria, as well as in a few bacteriophages, and are often present as several distinct enzymes within the same species. For instance, in Escherichia coli, at least six different LTs have been identified: one soluble (Slt70) and five that are outer membrane-anchored (MltAMltD, EmtA) (1, 3, 4). A comparison of genes encoding LTs allowed their classification into four families based on the identification of different sets of consensus motifs (5). LT family 1 includes proteins with amino acid sequence similarity to Slt70, MltC, MltD, and EmtA, whereas MltA and MltB are representatives of families 2 and 3, respectively. The fourth LT family comprises mostly enzymes encoded by bacteriophages.Studies of the LTs have focused on understanding their precise cellular roles, as well as on explaining their reaction mechanism. Concerning the latter interest, important insights have been obtained from the study of crystal structures of these enzymes, both in unliganded forms and in complex with various murein-derived compounds (6 -11). These investigations showed that members from LT families 1, 3, and 4 have a catalytic domain that resembles the fold of goose-type lysozyme (12). The reaction catalyzed by these enzymes most likely takes place via a general acid/base mechanism that requires participation of a single catalytic residue, i.e. an invariant glutam...