Discovery of novel pathways of microbial arginine biosynthesis

The amino acid L-arginine is an essential component of all living organisms. Its importance resides in the variety of functions that arginine itself, along with some intermediary metabolites involved in its de novo synthesis in the cell. In many prokaryotes, fungi, and plants the de novo biosynthesis of arginine proceeds from glutamate in eight enzymatic steps (Figure 2). The first committed step of this pathway is the N-acetylation of glutamate. Acetylation of the early precursors of arginine distinguishes them from the analogous intermediates in the biosynthesis of proline. Although each and every step of the pathway is essential for its completion, transcarbamylation of ornithine to produce citrulline is one of the crucial steps in the pathway. This reaction is catalyzed by the enzyme ornithine transcarbamylase (OTC). The transcarbamylases family of enzymes belongs to the carboxyl- and carbamoyltransferases group, EC 2.1.3, a family that comprises enzymes that catalyze the transfer of a carbamoyl group from carbamylphosphate (CP) to an amino or oxygen group of a second substrate. Members of the transcarbamylase family can be identified based on sequence identities in the N-terminal or CP-binding domain as all the members of this family share common residues involved in the binding of CP to the enzyme. All the evolutionary conserved motifs present in the transcarbamylase family have led to the erroneous annotation of a large, yet increasing, number of genes as OTCases without experimental confirmation of such activity. The case of the transcarbamylase-like gene found in B. fragilis illustrates this statement. The present thesis work is focused in the case of two families of misannotated microbial transcarbamylases that have been identified as essential for arginine biosynthesis, but which lacked the ability to catalyze the conversion of ornithine into citrulline. The objectives of the present thesis work are the biochemical and molecular characterization of the N-acetylornithine transcarbamylase (AOTC) activity of XcArgF’ as well as the elucidation of the enzymatic activity of BfArgF’ and its biochemical and molecular characterization. We show that Xanthomonads and Bacteroidetes use novel pathways for the de novo arginine biosynthesis mediated by acetyl- and succinyl-ornithine transcarbamylases, we identify a recognition motif for these enzymes and we provide evidence supporting the view that the existence of succinyl-ornithine transcarbamylase requires that the first five steps of the arginine biosynthesis pathway use succinylated rather than acetylated intermediates

Plasma fibroblast growth factor-21 levels in patients with inborn errors of metabolism

Fibroblast growth factor-21 (FGF21) levels are elevated in patients with primary mitochondrial disorders but have not been studied in patients with inborn errors of metabolism (IEM) known to have secondary mitochondrial dysfunction. We measured plasma FGF21 by ELISA in patients with and without IEM. FGF21 levels were higher in patients with IEM compared to without IEM (370 pg/dL vs. 0–65 pg/dL). Further study of FGF21 as a biomarker in IEM is warranted

A Novel N-Acetylglutamate Synthase Architecture Revealed by the Crystal Structure of the Bifunctional Enzyme from Maricaulis maris

Novel bifunctional N-acetylglutamate synthase/kinases (NAGS/K) that catalyze the first two steps of arginine biosynthesis and are homologous to vertebrate N-acetylglutamate synthase (NAGS), an essential cofactor-producing enzyme in the urea cycle, were identified in Maricaulis maris and several other bacteria. Arginine is an allosteric inhibitor of NAGS but not NAGK activity. The crystal structure of M. maris NAGS/K (mmNAGS/K) at 2.7 Å resolution indicates that it is a tetramer, in contrast to the hexameric structure of Neisseria gonorrhoeae NAGS. The quaternary structure of crystalline NAGS/K from Xanthomonas campestris (xcNAGS/K) is similar, and cross-linking experiments indicate that both mmNAGS/K and xcNAGS are tetramers in solution. Each subunit has an amino acid kinase (AAK) domain, which is likely responsible for N-acetylglutamate kinase (NAGK) activity and has a putative arginine binding site, and an N-acetyltransferase (NAT) domain that contains the putative NAGS active site. These structures and sequence comparisons suggest that the linker residue 291 may determine whether arginine acts as an allosteric inhibitor or activator in homologous enzymes in microorganisms and vertebrates. In addition, the angle of rotation between AAK and NAT domains varies among crystal forms and subunits within the tetramer. A rotation of 26° is sufficient to close the predicted AcCoA binding site, thus reducing enzymatic activity. Since mmNAGS/K has the highest degree of sequence homology to vertebrate NAGS of NAGS and NAGK enzymes whose structures have been determined, the mmNAGS/K structure was used to develop a structural model of human NAGS that is fully consistent with the functional effects of the 14 missense mutations that were identified in NAGS-deficient patients

Sirt1 regulates glial progenitor proliferation and regeneration in white matter after neonatal brain injury.

Regenerative processes in brain pathologies require the production of distinct neural cell populations from endogenous progenitor cells. We have previously demonstrated that oligodendrocyte progenitor cell (OPC) proliferation is crucial for oligodendrocyte (OL) regeneration in a mouse model of neonatal hypoxia (HX) that reproduces diffuse white matter injury (DWMI) of premature infants. Here we identify the histone deacetylase Sirt1 as a Cdk2 regulator in OPC proliferation and response to HX. HX enhances Sirt1 and Sirt1/Cdk2 complex formation through HIF1α activation. Sirt1 deacetylates retinoblastoma (Rb) in the Rb/E2F1 complex, leading to dissociation of E2F1 and enhanced OPC proliferation. Sirt1 knockdown in culture and its targeted ablation in vivo suppresses basal and HX-induced OPC proliferation. Inhibition of Sirt1 also promotes OPC differentiation after HX. Our results indicate that Sirt1 is an essential regulator of OPC proliferation and OL regeneration after neonatal brain injury. Therefore, enhancing Sirt1 activity may promote OL recovery after DWMI

An integrated biochemical system for nitrate assimilation and nitric oxide detoxification in Bradyrhizobium japonicum

Rhizobia are recognized to establish N(2)-fixing symbiotic interactions with legume plants. Bradyrhizobium japonicum, the symbiont of soybeans, can denitrify and grow under free-living conditions with nitrate (NO(3)(−)) or nitrite (NO(2)(−)) as sole nitrogen source. Unlike related bacteria that assimilate NO(3)(−), genes encoding the assimilatory NO(3)(−) reductase (nasC) and NO(2)(−) reductase (nirA) in B. japonicum are located at distinct chromosomal loci. The nasC gene is located with genes encoding an ABC-type NO(3)(−) transporter, a major facilitator family NO(3)(−)/NO(2)(−) transporter (NarK), flavoprotein (Flp) and single-domain haemoglobin (termed Bjgb). However, nirA clusters with genes for a NO(3)(−)/NO(2)(−)-responsive regulator (NasS-NasT). In the present study, we demonstrate NasC and NirA are both key for NO(3)(−) assimilation and that growth with NO(3)(−), but not NO(2)(−) requires flp, implying Flp may function as electron donor to NasC. In addition, bjgb and flp encode a nitric oxide (NO) detoxification system that functions to mitigate cytotoxic NO formed as a by-product of NO(3)(−) assimilation. Additional experiments reveal NasT is required for NO(3)(−)-responsive expression of the narK-bjgb-flp-nasC transcriptional unit and the nirA gene and that NasS is also involved in the regulatory control of this novel bipartite assimilatory NO(3)(−)/NO(2)(−) reductase pathway

Evolution of Metabolic Phenotypes of Obesity in Coronary Patients after 5 Years of Dietary Intervention: From the CORDIOPREV Study

Background: Obesity phenotypes with different metabolic status have been described previously. We analyzed metabolic phenotypes in obese coronary patients during a 5-year follow-up, and examined the factors influencing this evolution. Methods: The CORDIOPREV study is a randomized, long-term secondary prevention study with two healthy diets: Mediterranean and low-fat. All obese patients were classified as either metabolically healthy obese (MHO) or metabolically unhealthy obese (MUO). We evaluated the changes in the metabolic phenotypes and related variables after 5 years of dietary intervention. Results: Initially, 562 out of the 1002 CORDIOPREV patients were obese. After 5 years, 476 obese patients maintained their clinical and dietary visits; 71.8% of MHO patients changed to unhealthy phenotypes (MHO-Progressors), whereas the MHO patients who maintained healthy phenotypes (MHO-Non-Progressors) lost more in terms of their body mass index (BMI) and had a lower fatty liver index (FLI-score) (p < 0.05). Most of the MUO (92%) patients maintained unhealthy phenotypes (MUO-Non-Responders), but 8% became metabolically healthy (MUO-Responders) after a significant decrease in their BMI and FLI-score, with improvement in all metabolic criteria. No differences were found among dietary groups. Conclusions: A greater loss of weight and liver fat is associated with a lower progression of the MHO phenotype to unhealthy phenotypes. Likewise, a marked improvement in these parameters is associated with regression from MUO to healthy phenotypes

Angiotensin-Converting Enzyme Inhibitors and Angiotensin Receptor Blockers Withdrawal Is Associated with Higher Mortality in Hospitalized Patients with COVID-19

Our main aim was to describe the effect on the severity of ACEI (angiotensin-converting enzyme inhibitor) and ARB (angiotensin II receptor blocker) during COVID-19 hospitalization. A retrospective, observational, multicenter study evaluating hospitalized patients with COVID-19 treated with ACEI/ARB. The primary endpoint was the incidence of the composite outcome of prognosis (IMV (invasive mechanical ventilation), NIMV (non-invasive mechanical ventilation), ICU admission (intensive care unit), and/or all-cause mortality). We evaluated both outcomes in patients whose treatment with ACEI/ARB was continued or withdrawn. Between February and June 2020, 11,205 patients were included, mean age 67 years (SD = 16.3) and 43.1% female; 2162 patients received ACEI/ARB treatment. ACEI/ARB treatment showed lower all-cause mortality (p < 0.0001). Hypertensive patients in the ACEI/ARB group had better results in IMV, ICU admission, and the composite outcome of prognosis (p < 0.0001 for all). No differences were found in the incidence of major adverse cardiovascular events. Patients previously treated with ACEI/ARB continuing treatment during hospitalization had a lower incidence of the composite outcome of prognosis than those whose treatment was withdrawn (RR 0.67, 95%CI 0.63-0.76). ARB was associated with better survival than ACEI (HR 0.77, 95%CI 0.62-0.96). ACEI/ARB treatment during COVID-19 hospitalization was associated with protection on mortality. The benefits were greater in hypertensive, those who continue

A Radial Velocity Study of the Planetary System of pi Mensae: Improved Planet Parameters for pi Mensae c and a Third Planet on a 125 Day Orbit

π Men hosts a transiting planet detected by the Transiting Exoplanet Survey Satellite space mission and an outer planet in a 5.7 yr orbit discovered by radial velocity (RV) surveys. We studied this system using new RV measurements taken with the HARPS spectrograph on ESO's 3.6 m telescope, as well as archival data. We constrain the stellar RV semiamplitude due to the transiting planet, π Men c, as Kc = 1.21 ± 0.12 m s^{−1}, resulting in a planet mass of M_{c} = 3.63 ± 0.38 M_{⊕}. A planet radius of R_{c} = 2.145 ± 0.015 R_{⊕} yields a bulk density of ρc = 2.03 ± 0.22 g cm^{−3}. The precisely determined density of this planet and the brightness of the host star make π Men c an excellent laboratory for internal structure and atmospheric characterization studies. Our HARPS RV measurements also reveal compelling evidence for a third body, π Men d, with a minimum mass M_{d} sin i_{d} = 13.38 ± 1.35 M_{⊕} orbiting with a period of Porb,d = 125 days on an eccentric orbit (e_{d} = 0.22). A simple dynamical analysis indicates that the orbit of π Men d is stable on timescales of at least 20 Myr. Given the mutual inclination between the outer gaseous giant and the inner rocky planet and the presence of a third body at 125 days, π Men is an important planetary system for dynamical and formation studies