Structure-function study of the enzymes of the cyanuric acid catabolic pathways

Up to now the degradation of atrazine by Pseudomonas sp. strain ADP1 bacterium was thought to involve six steps successively catalysed by enzymes: AtzA, AtzB, AtzC, degrading atrazine into cyanuric acid, and AtzD, AtzE and AtzF, successively mineralising cyanuric acid to ammonia and carbon dioxide. The genes atzD-aztE-atzF are arranged in an operon called the cyanuric acid degradation operon. The exploration of cyanuric acid degradation pathways in different bacteria showed that substantial differences exist in the cyanuric acid degradation pathways between microorganisms. In Rhizobium leguminasorum bv. viciae 3841, for example, a biuret hydrolase (BiuH) belonging to the isochorismatase family performs the deamination of biuret to produce allophanate (118); whereas, in the model s-triazine degrading bacterium Pseudomonas sp. strain ADP, it is an amidase, AtzE, that is thought to perform that step. The characterisation of AtzE revealed the existence of two new enzymes in the Pseudomonas sp. strain ADP1 cyanuric acid operon. The first part of this PhD, reports the structure-function study of the BiuH in Rhizobium leguminasorum bv. viciae 3841. The atomic structure of BiuH was solved and site-directed mutagenesis was used to gain a better understanding of the BiuH catalytic mechanism. Additionally, molecular dynamics simulations highlighted the presence of three channels from the active site to the enzyme surface forming a potential substrate channel, a co-product (ammonia) channel and a co-substrate (water) channel. Although the cyanuric acid degradation pathway in Pseudomonas sp. strain ADP1 has been known and studied for more than twenty years, no one had purified and characterised AtzE. The second part of this PhD reports the purification of the native AtzE from Pseudomonas sp. strain ADP, allowing its biochemical and structural characterisation. The structure revealed the presence of a small, essential protein (AtzG), with which AtzE forms a heterotetramer. Biochemical characterisation and molecular dynamics experiments revealed AtzE acts as a 1-carboxybiuret hydrolase, not as a biuret hydrolase as previously thought. Finally, this work suggests that AtzE might have evolved from the GatCAB transamidosome complex. The final part of my PhD presents the discovery and the study of AtzH, a previously unknown small protein encoded by a gene located in the Pseudomonas sp. strain ADP’s cyanuric acid degradation operon. The structural characterisation of AtzH determined it belonged to the versatile NFT2 protein superfamily. A combination of structural modelling and mutagenesis studies was used to provide evidence that AtzH is an allophanate forming, 1,3-dicarboxyurea amidohydrolase. Mutagenesis also indicated that Tyr22 and Arg46 may play an essential role in the catalysis of 1,3-dicarboxyurea. Finally, a comparison of the genomic context suggests AtzH might be involved more broadly in the catabolism of nitrogenous compounds in Proteobacteria. Moreover, this observation also suggests that the atzG-atzE-atzH cluster predates the formation of the cyanuric acid catabolism operon

Comment coupler observations et prédictions pour améliorer les prédictions d'épidémie de Septoriose sur le blé ?

Wheat Septoria leaf blotch disease is one of the most important in France. Fungicides are more efficient just before the appearance of symptomes, thug it is challenging to find the right timing. Nowadays, the decision of treatment is based on observations made in the fields and on modelling of the severity of the disease made with the SeptoLISO model. So far no one had ever tried to fuse the modelled and the observed data. We had two aims: one was to predict whether the contamination threshold of 4/20 is reached or not on a given leaf, of a given field on a given week, the other was to predict whether this threshold will be reached on the next week or not. Finally we compared our prediction performances to the current prediction performances in 4 different scenarios. Binomial regression models and classification trees were the most efficient to predict contaminations, when precise information from the field was scarce. Fusion of observed and modeled data seems a promising approach to improve epidemics prediction performances.La Septoriose est une des maladies du blé les plus répandues en France. Il est difficile de prédire le moment où les applications fongicides seront les plus efficaces ; pour ce faire, on se base sur des observations faites en champs et sur des modélisations statistiques réalisées à l'aide du modèle SeptoLISO. Jusque-là, aucun couplage formel de ces deux types de données n'avait jamais été réalisé. Les deux objectifs du stage étaient : d'une part, de prédire précisément si le seuil de contamination de 4/20 était atteint sur un étage foliaire, en un site donné ; d'autre part, de pouvoir prédire si ce seuil sera atteint la semaine suivante ou non. Nous avons ensuite testé la plus-value de nos modèles par rapport aux performances de prédictions actuelles dans 4 situations différentes. Les modèles de régression binomiale et les arbres de décisions ont atteint les meilleures performances dans les scénarios où l'on a peu d'information sur la parcelle à prédire. Le couplage entre les données observées et les modélisations paraît être une idée prometteuse pour améliorer les prédictions d'épidémies

Current Metabolic Engineering Strategies for Photosynthetic Bioproduction in Cyanobacteria

Cyanobacteria are photosynthetic microorganisms capable of using solar energy to convert CO2 and H2O into O2 and energy-rich organic compounds, thus enabling sustainable production of a wide range of bio-products. More and more strains of cyanobacteria are identified that show great promise as cell platforms for the generation of bioproducts. However, strain development is still required to optimize their biosynthesis and increase titers for industrial applications. This review describes the most well-known, newest and most promising strains available to the community and gives an overview of current cyanobacterial biotechnology and the latest innovative strategies used for engineering cyanobacteria. We summarize advanced synthetic biology tools for modulating gene expression and their use in metabolic pathway engineering to increase the production of value-added compounds, such as terpenoids, fatty acids and sugars, to provide a go-to source for scientists starting research in cyanobacterial metabolic engineering

A novel decarboxylating amidohydrolase involved in avoiding metabolic dead ends during cyanuric acid catabolism in Pseudomonas sp. strain ADP.

Cyanuric acid is a common environmental contaminant and a metabolic intermediate in the catabolism of s-triazine compounds, including atrazine and other herbicides. Cyanuric acid is catabolized via a number of bacterial pathways, including one first identified in Pseudomonas sp. strain ADP, which is encoded by a single, five-gene operon (atzDGEHF) found on a self-transmissible plasmid. The discovery of two of the five genes (atzG and atzH) was reported in 2018 and although the function of atzG was determined, the role of atzH was unclear. Here, we present the first in vitro reconstruction of the complete, five-protein cyanuric acid catabolism pathway, which indicates that AtzH may be an amidase responsible for converting 1,3-dicarboxyurea (the AtzE product) to allophanate (the AtzF substrate). We have solved the AtzH structure (a DUF3225 protein from the NTF2 superfamily) and used it to predict the substrate-binding pocket. Site-directed mutagenesis experiments suggest that two residues (Tyr22 and Arg46) are needed for catalysis. We also show that atzH homologs are commonly found in Proteobacteria associated with homologs of the atzG and atzE genes. The genetic context of these atzG-atzE-atzH clusters imply that they have a role in the catabolism of nitrogenous compounds. Moreover, their presence in many genomes in the absence of homologs of atzD and atzF suggests that the atzG-atzE-atzH cluster may pre-date the evolution of the cyanuric acid catabolism operon

Tunable In Vivo Co-localisation of Enzymes Within P22 Capsid-Based Nanoreactors

The spatial organisation of enzymatic pathways through compartmentalisation is a mechanism used in nature for the regulation of multi-step biocatalytic processes. Virus-like particles (VLPs) derived from Bacteriophage P22 have been explored as biomimetic catalytic compartments. The in vivo co-encapsulation of enzymes is typically achieved via sequential fusion to the scaffold protein (SP), which results in an equimolar ratio of enzyme monomers. However, control over enzyme stoichiometry, which has been shown to influence pathway flux, is key to realising the full potential of P22 VLPs as artificial metabolons. Here we present a strategy for the stoichiometrically controlled in vivo co-encapsulation of cargo proteins within P22-based VLPs. Co-encapsulation was achieved via co-expression of cargo proteins with individual SP fusions using a dual plasmid system and verified for fluorescent protein cargo by Förster resonance energy transfer. This strategy was subsequently applied to a two-enzyme reaction cascade. L-homoalanine, an unnatural amino acid and chiral precursor to several drugs, can be synthesised from the readily available L-threonine by the sequential activity of threonine dehydratase and glutamate dehydrogenase. We find that scaffolding by this system has a profound impact on the activity of each enzyme and, using a purification strategy designed to isolate the range of particle forms that exist in vivo, that scaffolding of multimeric enzymes can be at unexpectedly high densities. This work demonstrates the controlled co-localisation of multiple heterologous cargo proteins in a P22-based nanoreactor and shows that careful consideration of loading densities of individual enzymes in an enzymatic cascade is required for the optimal design of synthetic metabolons

Structural and biochemical characterization of the biuret hydrolase (BiuH) from the cyanuric acid catabolism pathway of Rhizobium leguminasorum bv. viciae 3841

Biuret deamination is an essential step in cyanuric acid mineralization. In the well-studied atrazine degrading bacterium Pseudomonas sp. strain ADP, the amidase AtzE catalyzes this step. However, Rhizobium leguminosarum bv. viciae 3841 uses an unrelated cysteine hydrolase, BiuH, instead. Herein, structures of BiuH, BiuH with bound inhibitor and variants of BiuH are reported. The substrate is bound in the active site by a hydrogen bonding network that imparts high substrate specificity. The structure of the inactive Cys175Ser BiuH variant with substrate bound in the active site revealed that an active site cysteine (Cys175), aspartic acid (Asp36) and lysine (Lys142) form a catalytic triad, which is consistent with biochemical studies of BiuH variants. Finally, molecular dynamics simulations highlighted the presence of three channels from the active site to the enzyme surface: a persistent tunnel gated by residues Val218 and Gln215 forming a potential substrate channel and two smaller channels formed by Val28 and a mobile loop (including residues Phe41, Tyr47 and Met51) that may serve as channels for co-product (ammonia) or co-substrate (water)