Mutational and structural analysis of an ancestral fungal dye-decolorizing peroxidase.

Dye-decolorizing peroxidases (DyPs) constitute a superfamily of heme-containing peroxidases that are related neither to animal nor to plant peroxidase families. These are divided into four classes (types A, B, C, and D) based on sequence features. The active site of DyPs contains two highly conserved distal ligands, an aspartate and an arginine, the roles of which are still controversial. These ligands have mainly been studied in class A-C bacterial DyPs, largely because no effective recombinant expression systems have been developed for the fungal (D-type) DyPs. In this work, we employ ancestral sequence reconstruction (ASR) to resurrect a D-type DyP ancestor, AncDyPD-b1. Expression of AncDyPD-b1 in Escherichia coli results in large amounts of a heme-containing soluble protein and allows for the first mutagenesis study on the two distal ligands of a fungal DyP. UV-Vis and resonance Raman (RR) spectroscopic analyses, in combination with steady-state kinetics and the crystal structure, reveal fine pH-dependent details about the heme active site structure and show that both the aspartate (D222) and the arginine (R390) are crucial for hydrogen peroxide reduction. Moreover, the data indicate that these two residues play important but mechanistically different roles on the intraprotein long-range electron transfer process. DATABASE: Structural data are available in the PDB database under the accession number 7ANV.

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.

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.

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. To the best of our knowledge, this is the first report of a biocatalyst exhibiting opposite redox transformations of the carboxylic acid moiety in benzoic acid derivatives, namely oxidative decarboxylation and carboxyl reduction, in a structure-dependent fashion.

Cinnamic acid derivatives acting against Aspergillus fungi. Taq polymerase I a potential molecular target.

Some members of a series of cinnamic acid derivatives possess promising inhibitory activities in cellular assays against fungi of the Aspergillus genus. In order to search for a possible molecular target of such compounds, their role as Taq polymerase I inhibitors was studied. Four of the compounds studied displayed IC50 values within the range of those considered active as DNA polymerase inhibitors when searching for new cytotoxic molecules. The results obtained in our molecular modeling study appear to show that the inhibitory activity depends on the presence of a stabilizing interaction between the phenylpropanoid derivatives and the residues Asp610, Thr664, Phe667, Tyr671, and Asp785 located in the active site of Taq polymerase I. Also, it is possible to assert that the polymerization of DNA would be the molecular target of cinnamic acid derivatives with antifungal activity, which correlates with the inhibition of Taq polymerase I and the quantitative descriptor for the lipophilia (ClogP).