Mechanism of the chorismate dehydratase MqnA, first enzyme of the futalosine pathway

MqnA, the only chorismate dehydratase known so far, catalyzes the initial step in the biosynthesis of menaquinone via the futalosine pathway. Here we present crystal structures of Streptomyces coelicolor MqnA and its active site variants in complex with chorismate and the product 3-enolpyruvyl-benzoate. Together with activity studies, our data are in line with dehydration proceeding via substrate assisted catalysis, with chorismate acting as catalytic base. Surprisingly, structures of the variant Asn17Asp with co-purified ligand suggest that the enzyme converts to a hydrolase by serendipitous positioning of the carboxyl group. All complex structures presented here exhibit a closed Venus flytrap fold, with the enzyme exploiting the characteristic ligand binding properties of the fold for specific substrate binding and catalysis. The conformational rearrangements that facilitate complete burial of substrate/product, with accompanying topological changes to the enzyme surface, could foster substrate channeling within the biosynthetic pathway

Structural basis of substrate specificity of plant 12-oxophytodienoate reductases

12-Oxophytodienoate reductase 3 (OPR3) is a FMN-dependent oxidoreductase that catalyzes the reduction of the cyclopentenone (9S,13S)-12-oxophytodienoate [(9S,13S)-OPDA] to the corresponding cyclopentanone in the biosynthesis of the plant hormone jasmonic acid. In vitro, however, OPR3 reduces the jasmonic acid precursor (9S,13S)-OPDA as well as the enantiomeric (9R,13R)-OPDA, while its isozyme OPR1 is highly selective, accepting only (9R,13R)-OPDA as a substrate. To uncover the molecular determinants of this remarkable enantioselectivity, we determined the crystal structures of OPR1 and OPR3 in complex with the ligand p-hydroxybenzaldehyde. Structural comparison with the OPR1:(9R,13R)-OPDA complex and further biochemical and mutational analyses revealed that two active-site residues, Tyr78 and Tyr246 in OPR1 and Phe74 and His244 in OPR3, are critical for substrate filtering. The relatively smaller OPR3 residues allow formation of a wider substrate binding pocket that is less enantio-restrictive. Substitution of Phe74 and His244 by the corresponding OPR1 tyrosines resulted in an OPR3 mutant showing enhanced, OPR1-like substrate selectivity. Moreover, sequence analysis of the OPR family supports the filtering function of Tyr78 and Tyr246 and allows predictions with respect to substrate specificity and biological function of thus far uncharacterized OPR isozymes. The discovered structural features may also be relevant for other stereoselective proteins and guide the rational design of stereospecific enzymes for biotechnological applications

Chimeric newcastle disease virus protects chickens against avian influenza in the presence of maternally derived NDV immunity.

Newcastle disease virus (NDV), an avian paramyxovirus type 1, is a promising vector for expression of heterologous proteins from a variety of unrelated viruses including highly pathogenic avian influenza virus (HPAIV). However, pre-existing NDV antibodies may impair vector virus replication, resulting in an inefficient immune response against the foreign antigen. A chimeric NDV-based vector with functional surface glycoproteins unrelated to NDV could overcome this problem. Therefore, an NDV vector was constructed which carries the fusion (F) and hemagglutinin-neuraminidase (HN) proteins of avian paramyxovirus type 8 (APMV-8) instead of the corresponding NDV proteins in an NDV backbone derived from the lentogenic NDV Clone 30 and a gene expressing HPAIV H5 inserted between the F and HN genes. After successful virus rescue by reverse genetics, the resulting chNDVFHN PMV8H5 was characterized in vitro and in vivo. Expression and virion incorporation of the heterologous proteins was verified by Western blot and electron microscopy. Replication of the newly generated recombinant virus was comparable to parental NDV in embryonated chicken eggs. Immunization with chNDVFHN PMV8H5 stimulated full protection against lethal HPAIV infection in chickens without as well as with maternally derived NDV antibodies. Thus, tailored NDV vector vaccines can be provided for use in the presence or absence of routine NDV vaccination

Distinction and temporal stability of conformational epitopes on myelin oligodendrocyte glycoprotein recognized by patients with different inflammatory central nervous system diseases

Autoantibodies targeting conformationally intact myelin oligodendrocyte glycoprotein (MOG) are found in different inflammatory diseases of the CNS, but their antigenic epitopes have not been mapped. We expressed mutants of MOG on human HeLa cells and analyzed sera from 111 patients (104 children, 7 adults) who recognized cell-bound human MOG, but had different diseases, including acute disseminated encephalomyelitis (ADEM), one episode of transverse myelitis or optic neuritis, multiple sclerosis (MS), anti-aquaporin-4 (AQP4)-negative neuromyelitis optica (NMO), and chronic relapsing inflammatory optic neuritis (CRION). We obtained insight into the recognition of epitopes in 98 patients. All epitopes identified were located at loops connecting the β strands of MOG. The most frequently recognized MOG epitope was revealed by the P42S mutation positioned in the CC'-loop. Overall, we distinguished seven epitope patterns, including the one mainly recognized by mouse mAbs. In half of the patients, the anti-MOG response was directed to a single epitope. The epitope specificity was not linked to certain disease entities. Longitudinal analysis of 11 patients for up to 5 y indicated constant epitope recognition without evidence for intramolecular epitope spreading. Patients who rapidly lost their anti-MOG IgG still generated a long-lasting IgG response to vaccines, indicating that their loss of anti-MOG reactivity did not reflect a general lack of capacity for long-standing IgG responses. The majority of human anti-MOG Abs did not recognize rodent MOG, which has implications for animal studies. Our findings might assist in future detection of potential mimotopes and pave the way to Ag-specific depletion

HI assay.

<p>Serum samples from immunized chickens before challenge collected at day 20 (MDA-) or 21 (MDA+) after vaccination with chNDVFHN _PMV8H5 were tested for NDV (A), APMV-8 (B) and AIV H5 (C) specific antibodies by HI test. Furthermore, serum samples of MDA- chickens and MDA+ chickens vaccinated at day one (MDA+a) or day 7 (MDA+b) after hatch, taken 14 dpch were tested for AIV H5 (D) specific antibodies by HI assay. The number of animals considered seropositive (HI titer > 2 log₂) is given below box plots. Significant differences (P < 0.05) between C and D are marked by *.</p

AIV NP-ELISA.

<p>Differentiation between vaccinated and infected animals (DIVA) based on antibodies against AIV NP. Sera of chickens collected 20 dpv (MDA-) or 21 dpv (MDA+) (A) and 14 dpch (B) were investigated by a competitive ELISA. Values ≤ 55% are indicative for the presence of AIV NP-specific antibodies.</p

Immunoelectron microscopy of purified virions.

<p>Immunolabeling of chNDVFHN _PMV8H5 was performed using a serum against APMV-8 F (A), a monoclonal antibody against NDV NP (B), or a serum against AIV H5 (C), followed by gold-tagged secondary antibodies. Scale bars: 150 nm.</p

Virus shedding after vaccination.

<p>Swab samples taken on the indicated days from oropharynx of MDA- chickens (A) immunized with chNDVFHN _PMV8H5 at three weeks of age and swabs combined from oropharynx and cloaca of MDA+ chickens (B) immunized on day 1 (group a) or day 7 (group b) after hatch were analyzed for the presence of NDV NP gene-specific RNA by RT-qPCR. Values were transformed to genome equivalents (GEQ) using calibration curves of defined RNA standards that were included with each RT-qPCR run. The number of positive swabs by RT-qPCR is given below the box plots. Significant differences (P < 0.0125, Bonferroni correction) between vaccinated groups and controls are indicated (*).</p

Construction of chNDVFHN _PMV8H5.

<p>Schematic representation of plasmid construction: The full length plasmid NDVH5Vlp was cleaved by <i>Apa</i>I and <i>BsiW</i>I and the resulting 8,568 nt fragment was inserted into a pUC18 derivative. This plasmid was altered by Phusion PCR using megaprimers coding for the F orf, HN orf of APMV-8, respectively to substitute the F- and HN orfs of NDV by those of APMV-8. Afterwards, the resulting plasmid DNA was cut with <i>Apa</i>I and <i>BsiW</i>I and the 8,568nt fragment of the full length genome NDVH5Vlp was substituted by the modified <i>Apa</i>I and <i>BsiW</i>I-fragment (8,538 nt) sites, resulting in chNDVFHN _PMV8H5.</p

Clinical course after HPAIV H5N1 challenge infection.

<p>MDA+ chickens which were vaccinated with NDVH5Vm (A), and MDA- (B) as well as MDA+ birds (group a vaccinated on day 1 after hatch (C), group b vaccinated on day 7 after hatch (D)) which were vaccinated with chNDVFHN _PMV8H5, and naive controls were challenged at day 21 after vaccination with A/duck/Vietnam/TG24-01/05 and daily classified as healthy (0), sick (1), or dead (2) over a period of 8 days. The average scores of all animals of each group are indicated.</p