Recombinant S. cerevisiae expressing Old Yellow Enzymes from non-conventional yeasts: an easy system for selective reduction of activated alkenes

Background: Old Yellow Enzymes (OYEs) are flavin-dependent enoate reductases (EC 1.6.99.1) that catalyze the stereoselective hydrogenation of electron-poor alkenes. Their ability to generate up to two stereocenters by the trans-hydrogenation of the C = C double bond is highly demanded in asymmetric synthesis. Isolated redox enzymes utilization require the addition of cofactors and systems for their regeneration. Microbial whole-cells may represent a valid alternative combining desired enzymatic activity and efficient cofactor regeneration. Considerable efforts were addressed at developing novel whole-cell OYE biocatalysts, based on recombinant Saccharomyces cerevisiae expressing OYE genes.Results: Recombinant S. cerevisiae BY4741{increment}Oye2 strains, lacking endogenous OYE and expressing nine separate OYE genes from non-conventional yeasts, were used as whole-cell biocatalysts to reduce substrates with an electron-poor double bond activated by different electron-withdrawing groups. Ketoisophorone, \u3b1-methyl-trans-cinnamaldehyde, and trans-\u3b2-methyl-\u3b2-nitrostyrene were successfully reduced with high rates and selectivity. A series of four alkyl-substituted cyclohex-2-enones was tested to check the versatility and efficiency of the biocatalysts. Reduction of double bond occurred with high rates and enantioselectivity, except for 3,5,5-trimethyl-2-cyclohexenone. DFT (density functional theory) computational studies were performed to investigate whether the steric hindrance and/or the electronic properties of the substrates were crucial for reactivity. The three-dimensional structure of enoate reductases from Kluyveromyces lodderae and Candida castellii, predicted through comparative modeling, resulted similar to that of S. cerevisiae OYE2 and revealed the key role of Trp116 both in substrate specificity and stereocontrol. All the modeling studies indicate that steric hindrance was a major determinant in the enzyme reactivity.Conclusions: The OYE biocatalysts, based on recombinant S. cerevisiae expressing OYE genes from non-conventional yeasts, were able to differently reduce the activated double bond of enones, enals and nitro-olefins, exhibiting a wide range of substrate specificity. Moreover whole-cells biocatalysts bypassed the necessity of the cofactor recycling and, tuning reaction parameters, allowed the synthetic exploitation of endogenous carbonyl reductases. Molecular modeling studies highlighted key structural features for further improvement of catalytic properties of OYE enzymes

Recombinant S. cerevisiae expressing Old Yellow Enzymes from non-conventional yeasts: an easy system for selective reduction of activated alkenes

Background: Old Yellow Enzymes (OYEs) are flavin-dependent enoate reductases (EC 1.6.99.1) that catalyze the stereoselective hydrogenation of electron-poor alkenes. Their ability to generate up to two stereocenters by the trans-hydrogenation of the C = C double bond is highly demanded in asymmetric synthesis. Isolated redox enzymes utilization require the addition of cofactors and systems for their regeneration. Microbial whole-cells may represent a valid alternative combining desired enzymatic activity and efficient cofactor regeneration. Considerable efforts were addressed at developing novel whole-cell OYE biocatalysts, based on recombinant Saccharomyces cerevisiae expressing OYE genes.Results: Recombinant S. cerevisiae BY4741{increment}Oye2 strains, lacking endogenous OYE and expressing nine separate OYE genes from non-conventional yeasts, were used as whole-cell biocatalysts to reduce substrates with an electron-poor double bond activated by different electron-withdrawing groups. Ketoisophorone, α-methyl-trans-cinnamaldehyde, and trans-β-methyl-β-nitrostyrene were successfully reduced with high rates and selectivity. A series of four alkyl-substituted cyclohex-2-enones was tested to check the versatility and efficiency of the biocatalysts. Reduction of double bond occurred with high rates and enantioselectivity, except for 3,5,5-trimethyl-2-cyclohexenone. DFT (density functional theory) computational studies were performed to investigate whether the steric hindrance and/or the electronic properties of the substrates were crucial for reactivity. The three-dimensional structure of enoate reductases from Kluyveromyces lodderae and Candida castellii, predicted through comparative modeling, resulted similar to that of S. cerevisiae OYE2 and revealed the key role of Trp116 both in substrate specificity and stereocontrol. All the modeling studies indicate that steric hindrance was a major determinant in the enzyme reactivity.Conclusions: The OYE biocatalysts, based on recombinant S. cerevisiae expressing OYE genes from non-conventional yeasts, were able to differently reduce the activated double bond of enones, enals and nitro-olefins, exhibiting a wide range of substrate specificity. Moreover whole-cells biocatalysts bypassed the necessity of the cofactor recycling and, tuning reaction parameters, allowed the synthetic exploitation of endogenous carbonyl reductases. Molecular modeling studies highlighted key structural features for further improvement of catalytic properties of OYE enzymes

Literature search – Exploring in silico protein toxicity prediction methods to support the food and feed risk assessment

This report is the outcome of an EFSA procurement (NP/EFSA/GMO/2018/01) reviewing relevant scientific information on in silico prediction methods for protein toxicity, that could support the food and feed risk assessment. Several proteins are associated with adverse (toxic) effects in humans and animals, by a variety of mechanisms. These are produced by plants, animals and bacteria to prevail in hostile environments. In the present report, we present an integrated pipeline to perform a comprehensive literature and database search applied to proteins with toxic effects. \u201cToxin activity\u201d and \u201ctoxin-antitoxin system\u201d strings were used as inputs for this pipeline. UniProtKB was considered as the reference database, and only the UniProtKB curator-reviewed proteins were considered in the pipeline. Experimentally- determined structures and homology-based in silico 3D models were retrieved from protein structures repositories; family-, domain-, motif- and other molecular signature-related information was also obtained from specific databases which are part of the InterPro consortium. Protein aggregation associated with adverse effects was also investigated using different search strategies. This work can serve as the basis for further exploring novel risk assessment strategies for new proteins using in silico predictive methods

Hemolymph proteins: An overview across marine arthropods and molluscs

In this compilation we collect information about the main protein components in hemolymph and stress the continued interest in their study. The reasons for such an attention span several areas of biological, veterinarian and medical applications: from the notions for better dealing with the species – belonging to phylum Arthropoda, subphylum Crustacea, and to phylum Mollusca – of economic interest, to the development of ‘marine drugs’ from the peptides that, in invertebrates, act as antimicrobial, antifungal, antiprotozoal, and/or antiviral agents. Overall, the topic most often on focus is that of innate immunity operated by classes of pattern-recognition proteins. Significance: The immune response in invertebrates relies on innate rather than on adaptive/acquired effectors. At a difference from the soluble and membrane-bound immunoglobulins and receptors in vertebrates, the antimi- crobial, antifungal, antiprotozoal and/or antiviral agents in invertebrates interact with non-self material by targeting some common (rather than some highly specific) structural motifs. Developing this paradigm into (semi) synthetic pharmaceuticals, possibly optimized through the modeling opportunities offered by computa- tional biochemistry, is one of the lessons today's science may learn from the study of marine invertebrates, and specifically of the proteins and peptides in their hemolymph

Deciphering the ligand/xenobiotic molecular recognition mechanism in SLC transporters via a new structural binding site analysis

The Solute Carrier (SLC) superfamily of transporters is the second largest group of transmembrane proteins, only second to GPCRs [1]. Overall, they are able to transport a wide variety of solutes across membranes, from sugars, to nucleotides, to amino acids, to exogenous compounds, such as drugs. Despite being ubiquitous and playing many important roles in human physiology and disease, there is still much to learn about the structure and function of these proteins [2]. Their heterogeneity in both function and overall folding is one of the main factors that complicate their study and classification. More than 400 SLCs are currently known, divided into 66 subfamilies according to amino acid sequence similarity. Attempts to further group these subfamilies have been made according to different parameters such as their folding, but none were actually able to fully overcome their heterogeneity and classify them all [3]. Additionally, even inside of the same subfamily, individual members are differently selective for the transported solutes. In this work, we used a novel computational 3D approach [4] for the analysis of the binding sites to get potentially useful information to further our understanding of the SLCs recognition mechanism, to help in their classification, and to choose the best template to perform homology modelling of transporters whenever an experimentally solved structure is not available. Interesting insights have emerged on the relationship between primary structures and recognition sites from the analysis of a relatively small SLC database. Further in-depth analyses of larger SLC databases are in progress to confirm such preliminary and promising results

Glatiramer acetate : a complex drug beyond biologics

Complex drugs may be either biological, if the active ingredients are derived from a biological source, or non-biological, if obtained by chemical synthesis. In both cases, their quality depends considerably on the manufacturing process. In the case of Non Biological Complex Drugs (NBCDs), complexity may arise either from the active substance, as in the case of glatiramer acetate, or from other sources, such as the formulation, as in the case of liposomes. In this paper, the case of glatiramer acetate (GA) - a NBCD relevant for clinical and economic reasons - is considered and the differences between US and EU regulatory approaches to GA marketing authorization are highlighted. Indeed, though US and EU regulatory agencies have chosen a generic approach integrated with additional data the implementation is different in the two jurisdictions. In the US, the additional data required are listed in a product specific guideline and copies of Copaxone\uae have been approved as generics. In the EU, instead regulatory agencies followed a hybrid approach requiring an additional comparative study, and interchangeability policies and substitution schemes have been left to national agencies

The G Protein-Coupled Receptor GPR17: Overview and Update

The GPR17 receptor is a G protein-coupled receptor (GPCR) that seems to respond to two unrelated families of endogenous ligands: nucleotide sugars (UDP, UDP-galactose, and UDP-glucose) and cysteinyl leukotrienes (LTD4 , LTC4 , and LTE4 ), with significant affinity at micromolar and nanomolar concentrations, respectively. This receptor has a broad distribution at the level of the central nervous system (CNS) and is found in neurons and in a subset of oligodendrocyte precursor cells (OPCs). Unfortunately, disparate results emerging from different laboratories have resulted in a lack of clarity with regard to the role of GPR17-targeting ligands in OPC differentiation and in myelination. GPR17 is also highly expressed in organs typically undergoing ischemic damage and has various roles in specific phases of adaptations that follow a stroke. Under such conditions, GPR17 plays a crucial role; in fact, its inhibition decreases the progression of ischemic damage. This review summarizes some important features of this receptor that could be a novel therapeutic target for the treatment of demyelinating diseases and for repairing traumatic injury