Whitefly genomes contain ribotoxin coding genes acquired from plants

Ribosome inactivating proteins (RIPs) are RNA N-glycosidases that depurinate a specific adenine residue in the conserved sarcin/ricin loop of 28S rRNA. These enzymes are widely distributed among plants and bacteria. Previously, we have described for the first time RIP genes in mosquitoes belonging to the Culicidae family. We showed that these genes are derived from a single event of horizontal gene transfer (HGT) from a prokaryotic donor. Mosquito RIP genes are evolving under purifying selection, strongly suggesting that these toxins have acquired a functional role. In this work, we show the existence of two RIP encoding genes in the genome of the whitefly Bemisia tabaci, a hemiptera species belonging to the Aleyrodidae family distantly related to mosquitoes. Contamination artifacts were ruled out analyzing three independent B. tabaci genome databases. In contrast to mosquito RIPs, whitefly genes harbor introns and according to transcriptomic evidence are transcribed and spliced. Phylogeny and the taxonomic distribution strongly support that whitefly RIP genes are derived from an independent HGT event from a plant source. These results, along with our previous description of RIPs in Diptera, suggest that the acquired genes are functional in these insects and confer some fitness advantage.Fil: Lapadula, Walter Jesús. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; ArgentinaFil: Mascotti, María Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; ArgentinaFil: Juri Ayub, Maximiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentin

On the diversity of F420 -dependent oxidoreductases:A sequence- and structure-based classification

The F(420) deazaflavin cofactor is an intriguing molecule as it structurally resembles the canonical flavin cofactor, although behaves as a nicotinamide cofactor due to its obligate hydride‐transfer reactivity and similar low redox potential. Since its discovery, numerous enzymes relying on it have been described. The known deazaflavoproteins are taxonomically restricted to Archaea and Bacteria. The biochemistry of the deazaflavoenzymes is diverse and they exhibit great structural variability. In this study a thorough sequence and structural homology evolutionary analysis was performed in order to generate an overarching classification of the F(420)‐dependent oxidoreductases. Five different deazaflavoenzyme Classes (I–V) are described according to their structural folds as follows: Class I encompassing the TIM‐barrel F(420)‐dependent enzymes; Class II including the Rossmann fold F(420)‐dependent enzymes; Class III comprising the β‐roll F(420)‐dependent enzymes; Class IV which exclusively gathers the SH3 barrel F(420)‐dependent enzymes and Class V including the three layer ββα sandwich F(420)‐dependent enzymes. This classification provides a framework for the identification and biochemical characterization of novel deazaflavoenzymes

Ancestral reconstruction of mammalian FMO1 enables structural determination, revealing unique features that explain its catalytic properties

Mammals rely on the oxidative flavin-containing monooxygenases (FMOs) to detoxify numerous and potentially deleterious xenobiotics; this activity extends to many drugs, giving FMOs high pharmacological relevance. However, our knowledge regarding these membrane-bound enzymes has been greatly impeded by the lack of structural information. We anticipated that ancestral-sequence reconstruction could help us identify protein sequences that are more amenable to structural analysis. As such, we hereby reconstructed the mammalian ancestral protein sequences of both FMO1 and FMO4, denoted as ancestral flavin-containing monooxygenase (AncFMO)1 and AncFMO4, respectively. AncFMO1, sharing 89.5% sequence identity with human FMO1, was successfully expressed as a functional enzyme. It displayed typical FMO activities as demonstrated by oxygenating benzydamine, tamoxifen, and thioanisole, drug-related compounds known to be also accepted by human FMO1, and both NADH and NADPH cofactors could act as electron donors, a feature only described for the FMO1 paralogs. AncFMO1 crystallized as a dimer and was structurally resolved at 3.0 Å resolution. The structure harbors typical FMO aspects with the flavin adenine dinucleotide and NAD(P)H binding domains and a C-terminal transmembrane helix. Intriguingly, AncFMO1 also contains some unique features, including a significantly porous and exposed active site, and NADPH adopting a new conformation with the 2'-phosphate being pushed inside the NADP+ binding domain instead of being stretched out in the solvent. Overall, the ancestrally reconstructed mammalian AncFMO1 serves as the first structural model to corroborate and rationalize the catalytic properties of FMO1

A conserved sequence motif in the Escherichia coli soluble FAD-containing pyridine nucleotide transhydrogenase is important for reaction efficiency

Soluble pyridine nucleotide transhydrogenases (STHs) are flavoenzymes involved in the redox homeostasis of the essential cofactors NAD(H) and NADP(H). They catalyze the reversible transfer of reducing equivalents between the two nicotinamide cofactors. The soluble transhydrogenase from Escherichia coli (SthA) has found wide use in both in vivo and in vitro applications to steer reducing equivalents toward NADPH-requiring reactions. However, mechanistic insight into SthA function is still lacking. In this work, we present a biochemical characterization of SthA, focusing for the first time on the reactivity of the flavoenzyme with molecular oxygen. We report on oxidase activity of SthA that takes place both during transhydrogenation and in the absence of an oxidized nicotinamide cofactor as an electron acceptor. We find that this reaction produces the reactive oxygen species hydrogen peroxide and superoxide anion. Furthermore, we explore the evolutionary significance of the well-conserved CXXXXT motif that distinguishes STHs from the related family of flavoprotein disulfide reductases in which a CXXXXC motif is conserved. Our mutational analysis revealed the cysteine and threonine combination in SthA leads to better coupling efficiency of transhydrogenation and reduced reactive oxygen species release compared to enzyme variants with mutated motifs. These results expand our mechanistic understanding of SthA by highlighting reactivity with molecular oxygen and the importance of the evolutionarily conserved sequence motif.Fil: Partipilo, Michele. University of Groningen; Países BajosFil: Yang, Guang. University of Groningen; Países BajosFil: Mascotti, María Laura. University of Groningen; Países Bajos. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; ArgentinaFil: Wijma, Hein J.. University of Groningen; Países BajosFil: Slotboom, Dirk Jan. University of Groningen; Países BajosFil: Fraaije, Marco Wilhelmus. University of Groningen; Países Bajo

In vitro construction of the COQ metabolon unveils the molecular determinants of coenzyme Q biosynthesis

Metabolons are protein assemblies that perform a series of reactions in a metabolic pathway. However, the general importance and aptitude of metabolons for enzyme catalysis remain poorly understood. In animals, biosynthesis of coenzyme Q is currently attributed to ten different proteins, with COQ3, COQ4, COQ5, COQ6, COQ7 and COQ9 forming the iconic COQ metabolon. Yet several reaction steps conducted by the metabolon remain enigmatic. To elucidate the prerequisites for animal coenzyme Q biosynthesis, we sought to construct the entire metabolon in vitro. Here we show that this approach, rooted in ancestral sequence reconstruction, reveals the enzymes responsible for the uncharacterized steps and captures the biosynthetic pathway in vitro. We demonstrate that COQ8, a kinase, increases and streamlines coenzyme Q production. Our findings provide crucial insight into how biocatalytic efficiency is regulated and enhanced by these biosynthetic engines in the context of the cell. (Figure presented.).</p

Biotransformation of dehydro-epi-androsterone by Aspergillus parasiticus: Metabolic evidences of BVMO activity

The research on the synthesis of steroids and its derivatives is of high interest due to their clinical applications. A particular focus is given to molecules bearing a D-ring lactone like testolactone because of its bioactivity. The Aspergillus genus has been used to perform steroid biotransformations since it offers a toolbox of redox enzymes. In this work, the use of growing cells of Aspergillus parasiticus to study the bioconversion of dehydro-epi-androsterone (DHEA) is described, emphasizing the metabolic steps leading to D-ring lactonization products. It was observed that A. parasiticus is not only capable of transforming bicyclo[3.2.0]hept-2-en-6-one, the standard Baeyer-Villiger monooxygenase (BVMO) substrate, but also yielded testololactone and the homo-lactone 3β-hydroxy-17a-oxa-d-homoandrost-5-en-17-one from DHEA. Moreover, the biocatalyst degraded the lateral chain of cortisone by an oxidative route suggesting the action of a BVMO, thus providing enough metabolic evidences denoting the presence of BVMO activity in A. parasiticus. Furthermore, since excellent biotransformation rates were observed, A. parasiticus is a promising candidate for the production of bioactive lactone-based compounds of steroidal origin in larger scales.Fil: Mascotti, María Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Investigaciones en Tecnología Química. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Instituto de Investigaciones en Tecnología Química; ArgentinaFil: Palazzolo, Martín Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Investigaciones en Tecnología Química. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Instituto de Investigaciones en Tecnología Química; ArgentinaFil: Bisogno, Fabricio Román. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones en Físico-química de Córdoba. Universidad Nacional de Córdoba. Facultad de Ciencias Químicas. Instituto de Investigaciones en Físico-química de Córdoba; ArgentinaFil: Kurina Sanz, Marcela Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Investigaciones en Tecnología Química. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Instituto de Investigaciones en Tecnología Química; Argentin

Metazoan Ribosome Inactivating Protein encoding genes acquired by Horizontal Gene Transfer

Ribosome inactivating proteins (RIPs) are RNA N-glycosidases that depurinate a specific adenine residue in the conserved sarcin/ricin loop of 28S rRNA. These enzymes are widely distributed among plants and their presence has also been confirmed in several bacterial species. Recently, we reported for the first time in silico evidence of RIP encoding genes in metazoans, in two closely related species of insects: Aedes aegypti and Culex quinquefasciatus. Here, we have experimentally confirmed the presence of these genes in mosquitoes and attempted to unveil their evolutionary history. A detailed study was conducted, including evaluation of taxonomic distribution, phylogenetic inferences and microsynteny analyses, indicating that mosquito RIP genes derived from a single Horizontal Gene Transfer (HGT) event, probably from a cyanobacterial donor species. Moreover, evolutionary analyses show that, after the HGT event, these genes evolved under purifying selection, strongly suggesting they play functional roles in these organisms.Fil: Lapadula, Walter Jesús. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; ArgentinaFil: Marcet, Paula Lorena. Centers for Disease Control and Prevention; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Mascotti, María Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; ArgentinaFil: Sánchez Puerta, María Virginia. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Biología Agrícola de Mendoza. Universidad Nacional de Cuyo. Facultad de Ciencias Agrarias. Instituto de Biología Agrícola de Mendoza; ArgentinaFil: Juri Ayub, Maximiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentin

Self-sufficient redox biotransformation of lignin-related benzoic acids with Aspergillus flavus

Aromatic carboxylic acids are readily obtained from lignin in biomass processing facilities. However, efficient technologies for lignin valorization are missing. In this work, a microbial screening was conducted to find versatile biocatalysts capable of transforming several benzoic acids structurally related to lignin, employing vanillic acid as model substrate. The wild-type Aspergillus flavus growing cells exhibited exquisite selectivity towards the oxidative decarboxylation product, 2-methoxybenzene-1,4-diol. Interestingly, when assaying a set of structurally-related substrates, the biocatalyst displayed the oxidative removal of the carboxyl moiety or its reduction to the primary alcohol whether electron withdrawing or donating groups were present in the aromatic ring, respectively. Additionally, A. flavus proved to be highly tolerant to vanillic acid increasing concentrations (up to 8 g/L), demonstrating its potential application in chemical synthesis. A. flavus growing cells were found to be efficient biotechnological tools to perform self-sufficient, structure-dependent redox reactions. All the biotransformations described herein are reported for the first time.Fil: Palazzolo, Martín Alejandro. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico San Luis. Instituto de Investigaciones En Tecnología Química; ArgentinaFil: Mascotti, María Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico San Luis. Instituto de Investigaciones en Tecnología Química; ArgentinaFil: Lewkowicz, Elizabeth Sandra. Universidad Nacional de Quilmes. Departamento de Ciencia y Tecnologia; ArgentinaFil: Kurina Sanz, Marcela Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico San Luis. Instituto de Investigaciones En Tecnología Química; Argentin

Insights in the kinetic mechanism of the eukaryotic Baeyer-Villiger monooxygenase BVMOAf1 from Aspergillus fumigatus Af293

This work reports a detailed kinetic study of the recently discovered BVMOAf1 from Aspergillus fumigatus Af293. By performing steady state and pre-steady state kinetic analyses, it was demonstrated that the rate of catalysis is partially limited by the NADPH-mediated reduction of the flavin cofactor, a unique hallmark of BVMOAf1. In addition, the oxygenating C4a-(hydro)peroxyflavin intermediate could be spectrophotometrically detected and it was found to be the most stable among all analyzed BVMOs. To assess the possible influence of some residues on the kinetic features, model-inspired site-directed mutagenesis was performed. Among the mutants, the Q436A variant showed a slightly broader substrate scope and a better catalytic efficiency. In summary, this study describes for the first time the kinetic parameters for an eukaryotic BVMO.Fil: Mascotti, María Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Investigaciones en Tecnología Química. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Instituto de Investigaciones en Tecnología Química; ArgentinaFil: Kurina Sanz, Marcela Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto de Investigaciones en Tecnología Química. Universidad Nacional de San Luis. Facultad de Química, Bioquímica y Farmacia. Instituto de Investigaciones en Tecnología Química; ArgentinaFil: Juri Ayub, Maximiliano. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - San Luis. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis. Universidad Nacional de San Luis. Facultad de Ciencias Físico Matemáticas y Naturales. Instituto Multidisciplinario de Investigaciones Biológicas de San Luis; ArgentinaFil: Fraaije, Marco Wilhelmus. University of Groningen; Países Bajo