Structure‐based design of novel Chlamydomonas reinhardtii D1‐D2 photosynthetic proteins for herbicide monitoring

Abstract

The D1-D2 heterodimer in the reaction center core of phototrophs binds the redox plastoquinone cofactors, Q A and Q B , the terminal acceptors of the photosynthetic electron transfer chain in the photosystem II (PSII). This complex is the target of the herbicide atrazine, an environmental pollutant competitive inhibitor of Q B binding, and consequently it represents an excellent biomediator to develop biosensors for pollutant monitoring in ecosystems. In this context, we have undertaken a study of the Chlamydomonas reinhardtii D1-D2 proteins aimed at designing site directed mutants with increased affinity for atrazine. The three-dimensional structure of the D1 and D2 proteins from C. reinhardtii has been homology modeled using the crystal structure of the highly homologous Thermosynechococcus elongatus proteins as templates. Mutants of D1 and D2 were then generated in silico and the atrazine binding affinity of the mutant proteins has been calculated to predict mutations able to increase PSII affinity for atrazine. The computational approach has been validated through comparison with available experimental data and production and characterization of one of the predicted mutants. The latter analyses indicated an increase of one order of magnitude of the mutant sensitivity and affinity for atrazine as compared to the control strain. Finally, D1-D2 heterodimer mutants were designed and selected which, according to our model, increase atrazine binding affinity by up to 20 kcal/mol, representing useful starting points for the development of high affinity biosensors for atrazine.

Mutations of Photosystem II D1 Protein That Empower Efficient Phenotypes of Chlamydomonas reinhardtii under Extreme Environment in Space

“…Replacement of highly light-labile D1 protein is a primary event of the PSII repair cycle [5] to sustain crop productivity. It is also known that mutations or genetic substitution of some amino acids in D1 can lead to either an increase or a decrease in photosynthetic activity [6], [7].…”

“…This unicellular green alga is widely used as a model in studies of oxygenic photosynthesis [25]–[27] and can adapt to environmental extremes on Earth [28]. Other factors that favoured this choice included the ease in making specific amino acid substitutions in the D1 protein [6] and its ancient origin [6], [7], [27]. The amino acid substitutions in D1 were made in the Q B binding pocket (Ala 250 and Ala 251), and close to the redox-active Tyr 161 (Val 160 and Ile 163) [1].…”

Computational Biology, Protein Engineering, and Biosensor Technology: a Close Cooperation for Herbicides Monitoring

“…Minimal media were used to select photosynthetically active colonies generated after the integration of the psbA variant produced both by random and site-directed PCR (Dauvillee et al, 2004). Selected mutants were then characterised by analysing their photosynthetic performance and the sensitivity and/or resistance to different classes of herbicides assessed (Tibuzzi et al, 2007;Rea et al, 2009;Giardi et al, 2009;Scognamiglio et al, 2009). After the characterization, the best performing mutants were immobilized on screen-printed electrodes and integrated in amperometric or potentiometric circuits.…”

A Powerful Molecular Engineering Tool Provided Efficient Chlamydomonas Mutants as Bio-Sensing Elements for Herbicides Detection

“…The research for biosensor specificity improvement concerning microalgae is focused on screening for sensitive microalgae species [9], [10], selection of microalgal resistant genotypes [5], or generation of genetically modified species [7], [11]. In this respect, the use of biodevices exploiting an array of several bio-recognition elements specifically sensitive or resistant to a certain toxic compound represents a promising strategy.…”

“…It offers the benefits of high growth rate and easy cultivation of the microorganisms, and the ability to perform post-transcriptional and post-translational modifications distinctive for the higher plants. All these advantages and the complete annotation of its genome [15] turned C. reinhardtii into a robust platform for synthesis of bioactive compounds, recombinant protein expression [16], [17], biomass and biofuels production [18], [19], and a main protagonist in bioremediation and biological sensing research fields [8], [11], [20], [21]. In particular, the rational design and ad hoc production of C. reinhardtii mutants for biosensor purposes has been truly facilitated by the advances in its chloroplast transformation and resolution of PSII crystal structure [7], [11], [20], [22].…”