Sucrose phosphorylase from Alteromonas mediterranea: Structural insight into the regioselective α-glucosylation of (+)-catechin.

Sucrose phosphorylases, through transglycosylation reactions, are interesting enzymes that can transfer regioselectively glucose from sucrose, the donor substrate, onto acceptors like flavonoids to form glycoconjugates and hence modulate their solubility and bioactivity. Here, we report for the first time the structure of sucrose phosphorylase from the marine bacteria Alteromonas mediterranea (AmSP) and its enzymatic properties. Kinetics of sucrose hydrolysis and transglucosylation capacities on (+)-catechin were investigated. Wild-type enzyme (AmSP-WT) displayed high hydrolytic activity on sucrose and was devoid of transglucosylation activity on (+)-catechin. Two variants, AmSP-Q353F and AmSP-P140D catalysed the regiospecific transglucosylation of (+)-catechin: 89 % of a novel compound (+)-catechin-4'-O-α-d-glucopyranoside (CAT-4') for AmSP-P140D and 92 % of (+)-catechin-3'-O-α-d-glucopyranoside (CAT-3') for AmSP-Q353F. The compound CAT-4' was fully characterized by NMR and mass spectrometry. An explanation for this difference in regiospecificity was provided at atomic level by molecular docking simulations: AmSP-P140D was found to preferentially bind (+)-catechin in a mode that favours glucosylation on its hydroxyl group in position 4' while the binding mode in AmSP-Q353F favoured glucosylation on its hydroxyl group in position 3'.

Influence of pH on indole-dependent heterodimeric interactions between Anopheles gambiae odorant-binding proteins OBP1 and OBP4.

Insect Odorant Binding Proteins (OBPs) constitute important components of their olfactory apparatus, as they are essential for odor recognition. OBPs undergo conformational changes upon pH change, altering their interactions with odorants. Moreover, they can form heterodimers with novel binding characteristics. Anopheles gambiae OBP1 and OBP4 were found capable of forming heterodimers possibly involved in the specific perception of the attractant indole. In order to understand how these OBPs interact in the presence of indole and to investigate the likelihood of a pH-dependent heterodimerization mechanism, the crystal structures of OBP4 at pH 4.6 and 8.5 were determined. Structural comparison to each other and with the OBP4-indole complex (3Q8I, pH 6.85) revealed a flexible N-terminus and conformational changes in the α4-loop-α5 region at acidic pH. Fluorescence competition assays showed a weak binding of indole to OBP4 that becomes further impaired at acidic pH. Additional Molecular Dynamic and Differential Scanning Calorimetry studies displayed that the influence of pH on OBP4 stability is significant compared to the modest effect of indole. Furthermore, OBP1-OBP4 heterodimeric models were generated at pH 4.5, 6.5, and 8.5, and compared concerning their interface energy and cross-correlated motions in the absence and presence of indole. The results indicate that the increase in pH may induce the stabilization of OBP4 by increasing its helicity, thereby enabling indole binding at neutral pH that further stabilizes the protein and possibly promotes the creation of a binding site for OBP1. A decrease in interface stability and loss of correlated motions upon transition to acidic pH may provoke the heterodimeric dissociation allowing indole release. Finally, we propose a potential OBP1-OBP4 heterodimer formation/disruption mechanism induced by pH change and indole binding.

Regioselective glucosylation of (+)-catechin using a new variant of sucrose phosphorylase from Bifidobacterium adolescentis.

Mutation Q345F in sucrose phosphorylase from Bifidobacterium adolescentis (BaSP) has shown to allow efficient (+)-catechin glucosylation yielding a regioisomeric mixture: (+)-catechin-3'-O-α-D-glucopyranoside, (+)-catechin-5-O-α-D-glucopyranoside and (+)-catechin-3',5-O-α-D-diglucopyranoside with a ratio of 51 : 25 : 24. Here, we efficiently increased the control of (+)-catechin glucosylation regioselectivity with a new variant Q345F/P134D. The same products were obtained with a ratio of 82 : 9 : 9. Thanks to bioinformatics models, we successfully explained the glucosylation favoured at the OH-3' position due to the mutation P134D.

An Expanded Survey of the Moth PBP/GOBP Clade in Bombyx mori: New Insight into Expression and Functional Roles.

We studied the expression profile and ontogeny (from the egg stage through the larval stages and pupal stages, to the elderly adult age) of four OBPs from the silkworm moth Bombyx mori. We first showed that male responsiveness to female sex pheromone in the silkworm moth B. mori does not depend on age variation; whereas the expression of BmorPBP1, BmorPBP2, BmorGOBP1, and BmorGOBP2 varies with age. The expression profile analysis revealed that the studied OBPs are expressed in non-olfactory tissues at different developmental stages. In addition, we tested the effect of insecticide exposure on the expression of the four OBPs studied. Exposure to a toxic macrolide insecticide endectocide molecule (abamectin) led to the modulated expression of all four genes in different tissues. The higher expression of OBPs was detected in metabolic tissues, such as the thorax, gut, and fat body. All these data strongly suggest some alternative functions for these proteins other than olfaction. Finally, we carried out ligand docking studies and reported that PBP1 and GOBP2 have the capacity of binding vitamin K1 and multiple different vitamins.

Structural Design and Analysis of the RHOA-ARHGEF1 Binding Mode: Challenges and Applications for Protein-Protein Interface Prediction.

The interaction between two proteins may involve local movements, such as small side-chains re-positioning or more global allosteric movements, such as domain rearrangement. We studied how one can build a precise and detailed protein-protein interface using existing protein-protein docking methods, and how it can be possible to enhance the initial structures using molecular dynamics simulations and data-driven human inspection. We present how this strategy was applied to the modeling of RHOA-ARHGEF1 interaction using similar complexes of RHOA bound to other members of the Rho guanine nucleotide exchange factor family for comparative assessment. In parallel, a more crude approach based on structural superimposition and molecular replacement was also assessed. Both models were then successfully refined using molecular dynamics simulations leading to protein structures where the major data from scientific literature could be recovered. We expect that the detailed strategy used in this work will prove useful for other protein-protein interface design. The RHOA-ARHGEF1 interface modeled here will be extremely useful for the design of inhibitors targeting this protein-protein interaction (PPI).

A glance into the evolution of template-free protein structure prediction methodologies.

Prediction of protein structures using computational approaches has been explored for over two decades, paving a way for more focused research and development of algorithms in comparative modelling, ab intio modelling and structure refinement protocols. A tremendous success has been witnessed in template-based modelling protocols, whereas strategies that involve template-free modelling still lag behind, specifically for larger proteins (>150 a.a.). Various improvements have been observed in ab initio protein structure prediction methodologies overtime, with recent ones attributed to the usage of deep learning approaches to construct protein backbone structure from its amino acid sequence. This review highlights the major strategies undertaken for template-free modelling of protein structures while discussing few tools developed under each strategy. It will also briefly comment on the progress observed in the field of ab initio modelling of proteins over the course of time as seen through the evolution of CASP platform.

Comprehensive History of CSP Genes: Evolution, Phylogenetic Distribution and Functions.

In this review we present the developmental, histological, evolutionary and functional properties of insect chemosensory proteins (CSPs) in insect species. CSPs are small globular proteins folded like a prism and notoriously known for their complex and arguably obscure function(s), particularly in pheromone olfaction. Here, we focus on direct functional consequences on protein function depending on duplication, expression and RNA editing. The result of our analysis is important for understanding the significance of RNA-editing on functionality of CSP genes, particularly in the brain tissue.

One-shot optimization of multiple enzyme parameters: Tailoring glucose oxidase for pH and electron mediators.

Enzymes are biological catalysts with many industrial applications, but natural enzymes are usually unsuitable for industrial processes because they are not optimized for the process conditions. The properties of enzymes can be improved by directed evolution, which involves multiple rounds of mutagenesis and screening. By using mathematical models to predict the structure-activity relationship of an enzyme, and by defining the optimal combination of mutations in silico, we can significantly reduce the number of bench experiments needed, and hence the time and investment required to develop an optimized product. Here, we applied our innovative sequence-activity relationship methodology (innov'SAR) to improve glucose oxidase activity in the presence of different mediators across a range of pH values. Using this machine learning approach, a predictive model was developed and the optimal combination of mutations was determined, leading to a glucose oxidase mutant (P1) with greater specificity for the mediators ferrocene-methanol (12-fold) and nitrosoaniline (8-fold), compared to the wild-type enzyme, and better performance in three pH-adjusted buffers. The kcat /KM ratio of P1 increased by up to 121 folds compared to the wild type enzyme at pH 5.5 in the presence of ferrocene methanol.

Genetic background and immunological status influence B cell repertoire diversity in mice.

The relationship between the immune repertoire and the physiopathological status of individuals is essential to apprehend the genesis and the evolution of numerous pathologies. Nevertheless, the methodological approaches to understand these complex interactions are challenging. We performed a study evaluating the diversity harbored by different immune repertoires as a function of their physiopathological status. In this study, we base our analysis on a murine scFv library previously described and representing four different immune repertoires: i) healthy and naïve, ii) healthy and immunized, iii) autoimmune prone and naïve, and iv) autoimmune prone and immunized. This library, 2.6 × 109 in size, is submitted to high throughput sequencing (Next Generation Sequencing, NGS) in order to analyze the gene subgroups encoding for immunoglobulins. A comparative study of the distribution of immunoglobulin gene subgroups present in the four libraries has revealed shifts in the B cell repertoire originating from differences in genetic background and immunological status of mice.

Structural variations within proteins can be as large as variations observed across their homologues.

Understanding the structural plasticity of proteins is key to understanding the intricacies of their functions and mechanistic basis. In the current study, we analyzed the available multiple crystal structures of the same protein for the structural differences. For this purpose we used an abstraction of protein structures referred as Protein Blocks (PBs) that was previously established. We also characterized the nature of the structural variations for a few proteins using molecular dynamics simulations. In both the cases, the structural variations were summarized in the form of substitution matrices of PBs. We show that certain conformational states are preferably replaced by other specific conformational states. Interestingly, these structural variations are highly similar to those previously observed across structures of homologous proteins (r2 = 0.923) or across the ensemble of conformations from NMR data (r2 = 0.919). Thus our study quantitatively shows that overall trends of structural changes in a given protein are nearly identical to the trends of structural differences that occur in the topologically equivalent positions in homologous proteins. Specific case studies are used to illustrate the nature of these structural variations.

Flux prediction using artificial neural network (ANN) for the upper part of glycolysis.

The selection of optimal enzyme concentration in multienzyme cascade reactions for the highest product yield in practice is very expensive and time-consuming process. The modelling of biological pathways is a difficult process because of the complexity of the system. The mathematical modelling of the system using an analytical approach depends on the many parameters of enzymes which rely on tedious and expensive experiments. The artificial neural network (ANN) method has been successively applied in different fields of science to perform complex functions. In this study, ANN models were trained to predict the flux for the upper part of glycolysis as inferred by NADH consumption, using four enzyme concentrations i.e., phosphoglucoisomerase, phosphofructokinase, fructose-bisphosphate-aldolase, triose-phosphate-isomerase. Out of three ANN algorithms, the neuralnet package with two activation functions, "logistic" and "tanh" were implemented. The prediction of the flux was very efficient: RMSE and R2 were 0.847, 0.93 and 0.804, 0.94 respectively for logistic and tanh functions using a cross validation procedure. This study showed that a systemic approach such as ANN could be used for accurate prediction of the flux through the metabolic pathway. This could help to save a lot of time and costs, particularly from an industrial perspective. The R-code is available at: https://github.com/DSIMB/ANN-Glycolysis-Flux-Prediction.

EcRBPome: a comprehensive database of all known E. coli RNA-binding proteins.

The repertoire of RNA-binding proteins (RBPs) in bacteria play a crucial role in their survival, and interactions with the host machinery, but there is little information, record or characterisation in bacterial genomes. As a first step towards this, we have chosen the bacterial model system Escherichia coli, and organised all RBPs in this organism into a comprehensive database named EcRBPome. It contains RBPs recorded from 614 complete E. coli proteomes available in the RefSeq database (as of October 2018). The database provides various features related to the E. coli RBPs, like their domain architectures, PDB structures, GO and EC annotations etc. It provides the assembly, bioproject and biosample details of each strain, as well as cross-strain comparison of occurrences of various RNA-binding domains (RBDs). The percentage of RBPs, the abundance of the various RBDs harboured by each strain have been graphically represented in this database and available alongside other files for user download. To the best of our knowledge, this is the first database of its kind and we hope that it will be of great use to the biological community.

A machine learning approach for reliable prediction of amino acid interactions and its application in the directed evolution of enantioselective enzymes.

Directed evolution is an important research activity in synthetic biology and biotechnology. Numerous reports describe the application of tedious mutation/screening cycles for the improvement of proteins. Recently, knowledge-based approaches have facilitated the prediction of protein properties and the identification of improved mutants. However, epistatic phenomena constitute an obstacle which can impair the predictions in protein engineering. We present an innovative sequence-activity relationship (innov'SAR) methodology based on digital signal processing combining wet-lab experimentation and computational protein design. In our machine learning approach, a predictive model is developed to find the resulting property of the protein when the n single point mutations are permuted (2n combinations). The originality of our approach is that only sequence information and the fitness of mutants measured in the wet-lab are needed to build models. We illustrate the application of the approach in the case of improving the enantioselectivity of an epoxide hydrolase from Aspergillus niger. n = 9 single point mutants of the enzyme were experimentally assessed for their enantioselectivity and used as a learning dataset to build a model. Based on combinations of the 9 single point mutations (29), the enantioselectivity of these 512 variants were predicted, and candidates were experimentally checked: better mutants with higher enantioselectivity were indeed found.

Knowledge-based prediction of protein backbone conformation using a structural alphabet.

Libraries of structural prototypes that abstract protein local structures are known as structural alphabets and have proven to be very useful in various aspects of protein structure analyses and predictions. One such library, Protein Blocks, is composed of 16 standard 5-residues long structural prototypes. This form of analyzing proteins involves drafting its structure as a string of Protein Blocks. Predicting the local structure of a protein in terms of protein blocks is the general objective of this work. A new approach, PB-kPRED is proposed towards this aim. It involves (i) organizing the structural knowledge in the form of a database of pentapeptide fragments extracted from all protein structures in the PDB and (ii) applying a knowledge-based algorithm that does not rely on any secondary structure predictions and/or sequence alignment profiles, to scan this database and predict most probable backbone conformations for the protein local structures. Though PB-kPRED uses the structural information from homologues in preference, if available. The predictions were evaluated rigorously on 15,544 query proteins representing a non-redundant subset of the PDB filtered at 30% sequence identity cut-off. We have shown that the kPRED method was able to achieve mean accuracies ranging from 40.8% to 66.3% depending on the availability of homologues. The impact of the different strategies for scanning the database on the prediction was evaluated and is discussed. Our results highlight the usefulness of the method in the context of proteins without any known structural homologues. A scoring function that gives a good estimate of the accuracy of prediction was further developed. This score estimates very well the accuracy of the algorithm (R2 of 0.82). An online version of the tool is provided freely for non-commercial usage at http://www.bo-protscience.fr/kpred/.

Genotyping and Bio-Sensing Chemosensory Proteins in Insects.

Genotyping is the process of determining differences in the genetic make-up of an individual and comparing it to that of another individual. Focus on the family of chemosensory proteins (CSPs) in insects reveals differences at the genomic level across various strains and biotypes, but none at the level of individuals, which could be extremely useful in the biotyping of insect pest species necessary for the agricultural, medical and veterinary industries. Proposed methods of genotyping CSPs include not only restriction enzymatic cleavage and amplification of cleaved polymorphic sequences, but also detection of retroposons in some specific regions of the insect chromosome. Design of biosensors using CSPs addresses tissue-specific RNA mutations in a particular subtype of the protein, which could be used as a marker of specific physiological conditions. Additionally, we refer to the binding properties of CSP proteins tuned to lipids and xenobiotic insecticides for the development of a new generation of biosensor chips, monitoring lipid blood concentration and chemical environmental pollution.

The alternatively spliced LRRFIP1 Isoform-1 is a key regulator of the Wnt/ß-catenin transcription pathway.

The GC-rich Binding Factor 2/Leucine Rich Repeat in the Flightless 1 Interaction Protein 1 gene (GCF2/LRRFIP1) is predicted to be alternatively spliced in five different isoforms. Although important peptide sequence differences are expected to result from this alternative splicing, to date, only the gene transcription regulator properties of LRRFIP1-Iso5 were unveiled. Based on molecular, cellular and biochemical data, we show here that the five isoforms define two molecular entities with different expression profiles in human tissues, subcellular localizations, oligomerization properties and transcription enhancer properties of the canonical Wnt pathway. We demonstrated that LRRFIP1-Iso3, -4 and -5, which share over 80% sequence identity, are primarily located in the cell cytoplasm and form homo and hetero-multimers between each other. In contrast, LRRFIP1-Iso1 and -2 are primarily located in the cell nucleus in part thanks to their shared C-terminal domain. Furthermore, we showed that LRRFIP1-Iso1 is preferentially expressed in the myocardium and skeletal muscle. Using the in vitro Topflash reporter assay we revealed that among LRRFIP1 isoforms, LRRFIP1-Iso1 is the strongest enhancer of the ß-catenin Wnt canonical transcription pathway thanks to a specific N-terminal domain harboring two critical tryptophan residues (W76, 82). In addition, we showed that the Wnt enhancer properties of LRRFIP1-Iso1 depend on its homo-dimerisation which is governed by its specific coiled coil domain. Together our study identified LRRFIP1-Iso1 as a critical regulator of the Wnt canonical pathway with a potential role in myocyte differentiation and myogenesis.

Multitarget selection of catalytic antibodies with ß-lactamase activity using phage display.

ß-lactamase enzymes responsible for bacterial resistance to antibiotics are among the most important health threats to the human population today. Understanding the increasingly vast structural motifs responsible for the catalytic mechanism of ß-lactamases will help improve the future design of new generation antibiotics and mechanism-based inhibitors of these enzymes. Here we report the construction of a large murine single chain fragment variable (scFv) phage display library of size 2.7 × 109 with extended diversity by combining different mouse models. We have used two molecularly different inhibitors of the R-TEM ß-lactamase as targets for selection of catalytic antibodies with ß-lactamase activity. This novel methodology has led to the isolation of five antibody fragments, which are all capable of hydrolyzing the ß-lactam ring. Structural modeling of the selected scFv has revealed the presence of different motifs in each of the antibody fragments potentially responsible for their catalytic activity. Our results confirm (a) the validity of using our two target inhibitors for the in vitro selection of catalytic antibodies endowed with ß-lactamase activity, and (b) the plasticity of the ß-lactamase active site responsible for the wide resistance of these enzymes to clinically available inhibitors and antibiotics.

Biotype Characterization, Developmental Profiling, Insecticide Response and Binding Property of Bemisia tabaci Chemosensory Proteins: Role of CSP in Insect Defense.

Chemosensory proteins (CSPs) are believed to play a key role in the chemosensory process in insects. Sequencing genomic DNA and RNA encoding CSP1, CSP2 and CSP3 in the sweet potato whitefly Bemisia tabaci showed strong variation between B and Q biotypes. Analyzing CSP-RNA levels showed not only biotype, but also age and developmental stage-specific expression. Interestingly, applying neonicotinoid thiamethoxam insecticide using twenty-five different dose/time treatments in B and Q young adults showed that Bemisia CSP1, CSP2 and CSP3 were also differentially regulated over insecticide exposure. In our study one of the adult-specific gene (CSP1) was shown to be significantly up-regulated by the insecticide in Q, the most highly resistant form of B. tabaci. Correlatively, competitive binding assays using tryptophan fluorescence spectroscopy and molecular docking demonstrated that CSP1 protein preferentially bound to linoleic acid, while CSP2 and CSP3 proteins rather associated to another completely different type of chemical, i.e. α-pentyl-cinnamaldehyde (jasminaldehyde). This might indicate that some CSPs in whiteflies are crucial to facilitate the transport of fatty acids thus regulating some metabolic pathways of the insect immune response, while some others are tuned to much more volatile chemicals known not only for their pleasant odor scent, but also for their potent toxic insecticide activity.

Converting bulk sugars into prebiotics: semi-rational design of a transglucosylase with controlled selectivity.

Despite the growing importance of prebiotics in nutrition and gastroenterology, their structural variety is currently still very limited. The lack of straightforward procedures to gain new products in sufficient amounts often hampers application testing and further development. Although the enzyme sucrose phosphorylase can be used to produce the rare disaccharide kojibiose (α-1,2-glucobiose) from the bulk sugars sucrose and glucose, the target compound is only a side product that is difficult to isolate. Accordingly, for this biocatalyst to become economically attractive, the formation of other glucobioses should be avoided and therefore we applied semi-rational mutagenesis and low-throughput screening, which resulted in a double mutant (L341I_Q345S) with a selectivity of 95% for kojibiose. That way, an efficient and scalable production process with a yield of 74% could be established, and with a simple yeast treatment and crystallization step over a hundred grams of highly pure kojibiose (>99.5%) was obtained.