8 research outputs found
Flavoprotein monooxygenases for oxidative biocatalysis: recombinant expression in microbial hosts and applications
External flavoprotein monooxygenases comprise a group of flavin-dependent oxidoreductases that catalyze the insertion of one atom of molecular oxygen into an organic substrate and the second atom is reduced to water. These enzymes are involved in a great number of metabolic pathways both in prokaryotes and eukaryotes. Flavoprotein monooxygenases have attracted the attention of researchers for several decades and the advent of recombinant DNA technology caused a great progress in the field. These enzymes are subjected to detailed biochemical and structural characterization and some of them are also regarded as appealing oxidative biocatalysts for the production of fine chemicals and valuable intermediates toward active pharmaceutical ingredients due to their high chemo-, stereo-, and regioselectivity. Here, we review the most representative reactions catalyzed both in vivo and in vitro by prototype flavoprotein monooxygenases, highlighting the strategies employed to produce them recombinantly, to enhance the yield of soluble proteins, and to improve cofactor regeneration in order to obtain versatile biocatalysts. Although we describe the most outstanding features of flavoprotein monooxygenases, we mainly focus on enzymes that were cloned, expressed and used for biocatalysis during the last yearsFil: Ceccoli, Romina Denis. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario. Instituto de BiologĂa Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas. Instituto de BiologĂa Molecular y Celular de Rosario; ArgentinaFil: Bianchi, Dario Alejandro. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario. Instituto de QuĂmica Rosario. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas. Instituto de QuĂmica Rosario; ArgentinaFil: Rial, Daniela Veronica. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario. Instituto de BiologĂa Molecular y Celular de Rosario. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas. Instituto de BiologĂa Molecular y Celular de Rosario; Argentin
Environmental selection pressures related to iron utilization are involved in the loss of the Flavodoxin Gene from the plant genome
Oxidative stress and iron limitation represent the grim side of life in an oxygen-rich atmosphere. The versatile electron transfer shuttle ferredoxin,aniron-sulfurprotein,isparticularlysensitivetothesehardships,anditsdownregulationunderadverseconditionsseverely compromises survival of phototrophs. Replacement of ferredoxin by a stress-resistant isofunctional carrier, flavin-containing flavodoxin, is a widespread strategy employed by photosynthetic microorganisms to overcome environmental adversities. The flavodoxin gene was lostin the course ofplantevolution, but its reintroduction in transgenicplants confers increased tolerance to environmental stress and iron starvation, raising the question as to whya genetic asset with obvious adaptive value was not kept by natural selection. Phylogenetic analyses reveal that the evolutionary history of flavodoxin is intricate, with several horizontal gene transfer events between distant organisms, including Eukarya, Bacteria, and Archaea. The flavodoxin gene is unevenly distributed in most algal lineages, with flavodoxin-containing species being overrepresented in iron-limited regions and scarce or absent in iron-rich environments. Evaluation of cyanobacterial genomic and metagenomic data yielded essentially the same results, indicating that there was little selection pressure to retain flavodoxin in iron-rich coastal/freshwater phototrophs. Our results show a highly dynamic evolution pattern of flavodoxin tightly connected to the bioavailability of iron. Evidence presented here also indicates that the high concentration of iron in coastal and freshwater habitats may have facilitated the loss of flavodoxin in the freshwater ancestor of modern plants during the transition of photosynthetic organisms from the open oceans to the firm land.Fil: Pierella Karlusich, Juan JosĂ©. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Rosario. Instituto de BiologĂa Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario; ArgentinaFil: Ceccoli, Romina Denis. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Rosario. Instituto de BiologĂa Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas; ArgentinaFil: Graña, MartĂn. Instituto Pasteur de Montevideo; UruguayFil: Romero, HĂ©ctor. Universidad de la Republica; UruguayFil: Carrillo, Nestor Jose. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Rosario. Instituto de BiologĂa Molecular y Celular de Rosario; Argentina. Universidad Nacional de Rosario; Argentin
VisibilizaciĂłn del rol de las mujeres en la ciencia
La conmemoraciĂłn del DĂa Internacional de la Mujer y la Niña en la Ciencia tiene como propĂłsito mejorar el acceso de las niñas a las ciencias y visibilizar el papel de las mujeres en la comunidad cientĂfica. En lĂnea con las actividades que para esta fecha se realizan todos los años, en este artĂculo se abordan las acciones que en 2023 se llevaron a cabo en la Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas de la Universidad Nacional de Rosario (exposiciones grĂĄficas, conversatorios y experiencias cientĂficas) con el objetivo de resaltar la importancia de integrar la perspectiva de gĂ©nero en la educaciĂłn universitaria y de promover la equidad en el ĂĄmbito cientĂfico
Micro-Maravillas: IntroducciĂłn a los microorganismos
Realizando talleres experimentales en escuelas de nuestra ciudad vimos que en las aulas existĂa la oportunidad de acercar el conocimiento cientĂfico de manera lĂșdica y didĂĄctica. Fue por eso que nos propusimos diseñar cuadernillos que faciliten el proceso de aprendizaje de las Ciencias Naturales en los niveles inicial y primario. Como parte de este trayecto decidimos realizar un abordaje interdisciplinario incluyendo cuentos de autorĂa propia que puedan ser utilizados como disparador de debates y reflexiones sobre la temĂĄtica en estudio. AsĂ surge nuestra primera serie llamada Micro-Maravillas que invita a descubrir algunos aspectos del fascinante mundo de la MicrobiologĂa.Fil: Di Capua, Cecilia Beatriz. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas; ArgentinaFil: Bortolotti, Ana. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas; ArgentinaFil: Campero, MarĂa BelĂ©n. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Humanidades y Artes; ArgentinaFil: Tondo, Maria Laura. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas; ArgentinaFil: Ceccoli, Romina Denis. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas; ArgentinaFil: Balaban, Cecilia LucĂa. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas; Argentin
Expression of the minor isoform pea ferredoxin in tobacco alters photosynthetic electron partitioning and enhances cyclic electron flow
Ferredoxins (Fds) are ferrosulfoproteins that function as low-potential electron carriers in plants. The Fd family is composed of several isoforms that share high sequence homology but differ in functional characteristics. In leaves, at least two isoforms conduct linear and cyclic photosynthetic electron transport around photosystem I, and mounting evidence suggests the existence of at least partial division of duties between these isoforms. To evaluate the contribution of different kinds of Fds to the control of electron ïŹuxes along the photosynthetic electron transport chain, we overexpressed a minor pea (Pisum sativum) Fd isoform (PsFd1) in tobacco (Nicotiana tabacum) plants. The transplastomic OeFd1 plants exhibited variegated leaves and retarded growth and developmental rates. Photosynthetic studies of these plants indicated a reduction in carbon dioxide assimilation rates, photosystem II photochemistry, and linear electron ïŹow. However, the plants showed an increase in nonphotochemical quenching, better control of excitation pressure at photosystem II, and no evidence of photoinhibition, implying a better dynamic regulation to remove excess energy from the photosynthetic electron transport chain. Finally, analysis of P700 redox status during illumination conïŹrmed that the minor pea Fd isoform promotes enhanced cyclic ïŹow around photosystem I. The two novel features of this work are: (1) that Fd levels achieved in transplastomic plants promote an alternative electron partitioning even under greenhouse light growth conditions, a situation that is exacerbated at higher light intensity measurements; and (2) that an alternative, minor Fd isoform has been overexpressed in plants, giving new evidence of labor division among Fd isoforms.Fil: Blanco, NicolĂĄs Ernesto. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Rosario. Instituto de BiologĂa Molecular y Celular de Rosario; Argentina. Universidad de Umea; SueciaFil: Ceccoli, Romina Denis. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Rosario. Instituto de BiologĂa Molecular y Celular de Rosario; ArgentinaFil: Dalla Via, Maria Virginia. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Instituto de Investigaciones en IngenierĂa GenĂ©tica y BiologĂa Molecular; ArgentinaFil: Voss, Ingo. UniversitĂ€t OsnabrĂŒck; AlemaniaFil: Segretin, Maria Eugenia. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Instituto de Investigaciones En IngenierĂa GenĂ©tica y BiologĂa Molecular; ArgentinaFil: Bravo Almonacid, Fernando Felix. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Instituto de Investigaciones En IngenierĂa GenĂ©tica y BiologĂa Molecular; ArgentinaFil: Melzer, Michael. Leibniz-Institut fĂŒr Pflanzengenetik und Kulturpflanzenforschung, Physiologie und Zellbiologie; AlemaniaFil: Hajirezaei, Mohammad Reza. Leibniz-Institut fĂŒr Pflanzengenetik und Kulturpflanzenforschung, Physiologie und Zellbiologie; ArgentinaFil: Scheibe, Renate. UniversitĂ€t OsnabrĂŒck; AlemaniaFil: Hanke, Guy T.. UniversitĂ€t OsnabrĂŒck; Alemani
Genome mining reveals new bacterial type I Baeyer-Villiger monooxygenases with (bio)synthetic potential
Baeyer-Villiger monooxygenases (BVMOs) are oxidorreductases that catalyze the oxidation of ketones in a very selective manner. By genome mining we detected seven putative type I BVMOs in Bradyrhizobium diazoefficiens USDA 110. As we established the phylogenetic relationships among them and with other type I BVMOs, we found out that they belong to different clades of the phylogenetic tree. Thus, we decided to clone and heterologously express five of them. Three of them, each one from a divergent phylogenetic group, were obtained as soluble proteins, allowing us to proceed with their biocatalytic assessment and enzymatic characterization. As to substrate scope and selectivity, we observed a complementary behavior among the three BVMOs. BVMO2 was the more versatile biocatalyst in whole-cell systems while BVMO4 and BVMO5 showed a narrow substrate profile with preference for linear ketones and particular regioselectivity for (±)-cis-bicyclo[3.2.0]hept-2-en-6-one.Fil: Ceccoli, Romina Denis. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario; ArgentinaFil: Bianchi, Dario Alejandro. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario. Instituto de QuĂmica Rosario. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas. Instituto de QuĂmica Rosario; ArgentinaFil: Carabajal, MarĂa AyelĂ©n. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario; Argentina. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas; ArgentinaFil: Rial, Daniela Veronica. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas; Argentina. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario; Argentin
Cloning and characterization of the Type I BaeyerâVilliger monooxygenase from Leptospira biflexa
BaeyerâVilliger monooxygenases are recognized by their ability and high selectivity as oxidative biocatalysts for the generation of esters or lactones using ketones as starting materials. These enzymes represent valuable tools for biooxidative syntheses since they can catalyze reactions that otherwise involve strong oxidative reagents. In this work, we present a novel enzyme, the Type I BaeyerâVilliger monooxygenase from Leptospira biflexa. This protein is phylogenetically distant from other well-characterized BVMOs. In order to study this new enzyme, we cloned its gene, expressed it in Escherichia coli and characterized the substrate scope of the BaeyerâVilliger monooxygenase from L. biflexa as a whole-cell biocatalyst. For this purpose, we performed the screening of a collection of ketones with variable structures and sizes, namely acyclic ketones, aromatic ketones, cyclic ketones, and fused ketones. As a result, we observed that this biocatalyst readily oxidized linear- and branched- medium-chain ketones, alkyl levulinates and linear ketones with aromatic substituents with excellent regioselectivity. In addition, this enzyme catalyzed the oxidation of 2-substituted cycloketone derivatives but showed an unusual selection against substituents in positions 3 or 4 of the ring.Fil: Ceccoli, Romina Denis. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas Centro CientĂfico TecnolĂłgico - CONICET -Rosario. Instituto de Biologia Molecular y Celular de Rosario; ArgentinaFil: Bianchi, Dario Alejandro. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas. Centro CientĂfico TecnolĂłgico Conicet - Rosario. Instituto de QuĂmica Rosario. Universidad Nacional de Rosario. Facultad de Ciencias BioquĂmicas y FarmacĂ©uticas. Instituto de QuĂmica Rosario; ArgentinaFil: Fink, Michael J.. Vienna University of Technology; Austria. Harvard University; Estados UnidosFil: Mihovilovic, Marko D.. Vienna University of Technology; AustriaFil: Rial, Daniela Veronica. Consejo Nacional de Investigaciones CientĂficas y TĂ©cnicas Centro CientĂfico TecnolĂłgico - CONICET -Rosario. Instituto de Biologia Molecular y Celular de Rosario; Argentin