Structure of the complex I-like molecule NDH of oxygenic photosynthesis.

Cyclic electron flow around photosystem I (PSI) is a mechanism by which photosynthetic organisms balance the levels of ATP and NADPH necessary for efficient photosynthesis1,2. NAD(P)H dehydrogenase-like complex (NDH) is a key component of this pathway in most oxygenic photosynthetic organisms3,4 and is the last large photosynthetic membrane-protein complex for which the structure remains unknown. Related to the respiratory NADH dehydrogenase complex (complex I), NDH transfers electrons originating from PSI to the plastoquinone pool while pumping protons across the thylakoid membrane, thereby increasing the amount of ATP produced per NADP+ molecule reduced4,5. NDH possesses 11 of the 14 core complex I subunits, as well as several oxygenic-photosynthesis-specific (OPS) subunits that are conserved from cyanobacteria to plants3,6. However, the three core complex I subunits that are involved in accepting electrons from NAD(P)H are notably absent in NDH3,5,6, and it is therefore not clear how NDH acquires and transfers electrons to plastoquinone. It is proposed that the OPS subunits-specifically NdhS-enable NDH to accept electrons from its electron donor, ferredoxin3-5,7. Here we report a 3.1 Å structure of the 0.42-MDa NDH complex from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1, obtained by single-particle cryo-electron microscopy. Our maps reveal the structure and arrangement of the principal OPS subunits in the NDH complex, as well as an unexpected cofactor close to the plastoquinone-binding site in the peripheral arm. The location of the OPS subunits supports a role in electron transfer and defines two potential ferredoxin-binding sites at the apex of the peripheral arm. These results suggest that NDH could possess several electron transfer routes, which would serve to maximize plastoquinone reduction and avoid deleterious off-target chemistry of the semi-plastoquinone radical.

Structural and functional characterization of a new thermophilic-like OYE from Aspergillus flavus.

Old yellow enzymes (OYEs) have been proven as powerful biocatalysts for the asymmetric reduction of activated alkenes. Fungi appear to be valuable sources of OYEs, but most of the fungal OYEs are unexplored. To expand the OYEs toolbox, a new thermophilic-like OYE (AfOYE1) was identified from Aspergillus flavus strain NRRL3357. The thermal stability analysis showed that the T1/2 of AfOYE1 was 60 °C, and it had the optimal temperature at 45 °C. Moreover, AfOYE1 exhibited high reduction activity in a wide pH range (pH 5.5-8.0). AfOYE1 could accept cyclic enones, acrylamide, nitroalkenes, and α, ß-unsaturated aldehydes as substrates and had excellent enantioselectivity toward prochiral alkenes (> 99% ee). Interestingly, an unexpected (S)-stereoselectivity bioreduction toward 2-methylcyclohexenone was observed. The further crystal structure of AfOYE1 revealed that the "cap" region from Ala132 to Thr182, the loop of Ser316 to Gly325, α short helix of Arg371 to Gln375, and the C-terminal "finger" structure endow the catalytic cavity of AfOYE1 quite deep and narrow, and flavin mononucleotide (FMN) heavily buried at the bottom of the active site tunnel. Furthermore, the catalytic mechanism of AfOYE1 was also investigated, and the results confirmed that the residues His211, His214, and Tyr216 compose its catalytic triad. This newly identified thermophilic-like OYE would thus be valuable for asymmetric alkene hydrogenation in industrial processes. KEY POINTS: A new thermophilic-like OYE AfOYE1 was identified from Aspergillus flavus, and the T1/2 of AfOYE1 was 60 °C AfOYE1 catalyzed the reduction of 2-methylcyclohexenone with (S)-stereoselectivity The crystal structure of AfOYE1 was revealedv.

Asymmetric Ene-Reduction of α,ß-Unsaturated Compounds by F420-Dependent Oxidoreductases A Enzymes from Mycobacterium smegmatis.

The stereoselective reduction of alkenes conjugated to electron-withdrawing groups by ene-reductases has been extensively applied to the commercial preparation of fine chemicals. Although several different enzyme families are known to possess ene-reductase activity, the old yellow enzyme (OYE) family has been the most thoroughly investigated. Recently, it was shown that a subset of ene-reductases belonging to the flavin/deazaflavin oxidoreductase (FDOR) superfamily exhibit enantioselectivity that is generally complementary to that seen in the OYE family. These enzymes belong to one of several FDOR subgroups that use the unusual deazaflavin cofactor F420. Here, we explore several enzymes of the FDOR-A subgroup, characterizing their substrate range and enantioselectivity with 20 different compounds, identifying enzymes (MSMEG_2027 and MSMEG_2850) that could reduce a wide range of compounds stereoselectively. For example, MSMEG_2027 catalyzed the complete conversion of both isomers of citral to (R)-citronellal with 99% ee, while MSMEG_2850 catalyzed complete conversion of ketoisophorone to (S)-levodione with 99% ee. Protein crystallography combined with computational docking has allowed the observed stereoselectivity to be mechanistically rationalized for two enzymes. These findings add further support for the FDOR and OYE families of ene-reductases displaying general stereocomplementarity to each other and highlight their potential value in asymmetric ene-reduction.

NamiRNA-enhancer network of miR-492 activates the NR2C1-TGF-ß/Smad3 pathway to promote epithelial-mesenchymal transition of pancreatic cancer.

Pancreatic cancer (PaCa) is one of the most fatal malignancies of the digestive system, and most patients are diagnosed at advanced stages due to the lack of specific and effective tumor-related biomarkers for the early detection of PaCa. miR-492 has been found to be upregulated in PaCa tumor tissue and may serve as a potential therapeutic target. However, the molecular mechanisms by which miR-492 promotes PaCa tumor growth and progression are unclear. In this study, we first found that miR-492 in enhancer loci activated neighboring genes (NR2C1/NDUFA12/TMCC3) and promoted PaCa cell proliferation, migration, and invasion in vitro. We also observed that miR-492-activating genes significantly enriched the TGF-ß/Smad3 signaling pathway in PaCa to promote epithelial-mesenchymal transition (EMT) during tumorigenesis and development. Using CRISPR-Cas9 and ChIP assays, we further observed that miR-492 acted as an enhancer trigger, and that antagomiR-492 repressed PaCa tumorigenesis in vivo, decreased the expression levels of serum TGF-ß, and suppressed the EMT process by downregulating the expression of NR2C1. Our results demonstrate that miR-492, as an enhancer trigger, facilitates PaCa progression via the NR2C1-TGF-ß/Smad3 pathway.

Ascorbate peroxidase postcold regulation of chloroplast NADPH dehydrogenase activity controls cold memory.

Exposure of Arabidopsis (Arabidopsis thaliana) to 4°C imprints a cold memory that modulates gene expression in response to a second (triggering) stress stimulus applied several days later. Comparison of plastid transcriptomes of cold-primed and control plants directly before they were exposed to the triggering stimulus showed downregulation of several subunits of chloroplast NADPH dehydrogenase (NDH) and regulatory subunits of ATP synthase. NDH is, like proton gradient 5 (PGR5)-PGR5-like1 (PGRL1), a thylakoid-embedded, ferredoxin-dependent plastoquinone reductase that protects photosystem I and stabilizes ATP synthesis by cyclic electron transport (CET). Like PGRL1A and PGRL1B transcript levels, ndhA and ndhD transcript levels decreased during the 24-h long priming cold treatment. PGRL1 transcript levels were quickly reset in the postcold phase, but expression of ndhA remained low. The transcript abundances of other ndh genes decreased within the next days. Comparison of thylakoid-bound ascorbate peroxidase (tAPX)-free and transiently tAPX-overexpressing or tAPX-downregulating Arabidopsis lines demonstrated that ndh expression is suppressed by postcold induction of tAPX. Four days after cold priming, when tAPX protein accumulation was maximal, NDH activity was almost fully lost. Lack of the NdhH-folding chaperonin Crr27 (Cpn60ß4), but not lack of the NDH activity modulating subunits NdhM, NdhO, or photosynthetic NDH subcomplex B2 (PnsB2), strengthened priming regulation of zinc finger of A. thaliana 10, which is a nuclear-localized target gene of the tAPX-dependent cold-priming pathway. We conclude that cold-priming modifies chloroplast-to-nucleus stress signaling by tAPX-mediated suppression of NDH-dependent CET and that plastid-encoded NdhH, which controls subcomplex A assembly, is of special importance for memory stabilization.

Membrane skeleton modulates erythroid proteome remodeling and organelle clearance.

The final stages of mammalian erythropoiesis involve enucleation, membrane and proteome remodeling, and organelle clearance. Concomitantly, the erythroid membrane skeleton establishes a unique pseudohexagonal spectrin meshwork that is connected to the membrane through junctional complexes. The mechanism and signaling pathways involved in the coordination of these processes are unclear. The results of our study revealed an unexpected role of the membrane skeleton in the modulation of proteome remodeling and organelle clearance during the final stages of erythropoiesis. We found that diaphanous-related formin mDia2 is a master regulator of the integrity of the membrane skeleton through polymerization of actin protofilament in the junctional complex. The mDia2-deficient terminal erythroid cell contained a disorganized and rigid membrane skeleton that was ineffective in detaching the extruded nucleus. In addition, the disrupted skeleton failed to activate the endosomal sorting complex required for transport-III (ESCRT-III) complex, which led to a global defect in proteome remodeling, endolysosomal trafficking, and autophagic organelle clearance. Chmp5, a component of the ESCRT-III complex, is regulated by mDia2-dependent activation of the serum response factor and is essential for membrane remodeling and autophagosome-lysosome fusion. Mice with loss of Chmp5 in hematopoietic cells in vivo resembled the phenotypes in mDia2-knockout mice. Furthermore, overexpression of Chmp5 in mDia2-deficient hematopoietic stem and progenitor cells significantly restored terminal erythropoiesis in vivo. These findings reveal a formin-regulated signaling pathway that connects the membrane skeleton to proteome remodeling, enucleation, and organelle clearance during terminal erythropoiesis.

The Glycolysis Inhibitor 2-Deoxy-D-Glucose Exerts Different Neuronal Effects at Circuit and Cellular Levels, Partially Reverses Behavioral Alterations and does not Prevent NADPH Diaphorase Activity Reduction in the Intrahippocampal Kainic Acid Model of Temporal Lobe Epilepsy.

Temporal lobe epilepsy is the most drug-resistant type with the highest incidence among the other focal epilepsies. Metabolic manipulations are of great interest among others, glycolysis inhibitors like 2-deoxy D-glucose (2-DG) being the most promising intervention. Here, we sought to investigate the effects of 2-DG treatment on cellular and circuit level electrophysiological properties using patch-clamp and local field potentials recordings and behavioral alterations such as depression and anxiety behaviors, and changes in nitric oxide signaling in the intrahippocampal kainic acid model. We found that epileptic animals were less anxious, more depressed, with more locomotion activity. Interestingly, by masking the effect of increased locomotor activity on the parameters of the zero-maze test, no altered anxiety behavior was noted in epileptic animals. However, 2-DG could partially reverse the behavioral changes induced by kainic acid. The findings also showed that 2-DG treatment partially suppresses cellular level alterations while failing to reverse circuit-level changes resulting from kainic acid injection. Analysis of NADPH-diaphorase positive neurons in the CA1 area of the hippocampus revealed that the number of positive neurons was significantly reduced in dorsal CA1 of the epileptic animals and 2-DG treatment did not affect the diminishing effect of kainic acid on NADPH-d+ neurons in the CA1 area. In the control group receiving 2-DG, however, an augmented NADPH-d+ cell number was noted. These data suggest that 2-DG cannot suppress epileptiform activity at the circuit-level in this model of epilepsy and therefore, may fail to control the seizures in temporal lobe epilepsy cases.

Modulation of the catalytic performance of OYE3 by engineering key residues at the entrance of the catalytic pocket.

The amino acid residues at the entrance of the catalytic pocket may impose steric hindrance on the substrate to enter the active center of the enzyme. Based on the analysis of the three-dimensional structure of the Saccharomyces cerevisiae old yellow enzyme 3 (OYE3), four bulky residues were chosen and mutated to small amino acids. The results showed that mutation of the W116 residue had interesting impacts on the catalytic performance. All four variants became inactive for the reduction of (R)-carvone and (S)-carvone, but inverted the stereoselectivity for the reduction of (E/Z)-citral. The mutation of the F250 residue had a more positive effect on the activity and stereoselectivity. Two variants, F250A and F250S, showed excellent diastereoselectivity and activity for the reduction of (R)-carvone (de > 99%, c > 99%) and increased diastereoselectivity and activity for the reduction of (S)-carvone (de > 96%, c > 80%). One variant of the P295 residue, P295G, displayed excellent diastereoselectivity and activity only for the reduction of (R)-carvone (de > 99%, c > 99%). Mutation of the Y375 residue had a negative impact on the activity of the enzyme. These findings provide some solutions for rational enzyme engineering of OYE3.

Ene-Reductase: A Multifaceted Biocatalyst in Organic Synthesis.

Biocatalysis integrate microbiologists, enzymologists, and organic chemists to access the repertoire of pharmaceutical and agrochemicals with high chemoselectivity, regioselectivity, and enantioselectivity. The saturation of carbon-carbon double bonds by biocatalysts challenges the conventional chemical methodology as it bypasses the use of precious metals (in combination with chiral ligands and molecular hydrogen) or organocatalysts. In this line, Ene-reductases (ERs) from the Old Yellow Enzymes (OYEs) family are found to be a prominent asymmetric biocatalyst that is increasingly used in academia and industries towards unparalleled stereoselective trans-hydrogenations of activated C=C bonds. ERs gained prominence as they were used as individual catalysts, multi-enzyme cascades, and in conjugation with chemical reagents (chemoenzymatic approach). Besides, ERs' participation in the photoelectrochemical and radical-mediated process helps to unlock many scopes outside traditional biocatalysis. These up-and-coming methodologies entice the enzymologists and chemists to explore, expand and harness the chemistries displayed by ERs for industrial settings. Herein, we reviewed the last five year's exploration of organic transformations using ERs.

NADP+-dependent l-arginine dehydrogenase from Pseudomonas velonii: Purification, characterization and application to an l-arginine assay.

l-Arginine dehydrogenase (L-ArgDH) is an amino acid dehydrogenase which catalyzes the reversible oxidative deamination of l-arginine to the oxo analog in the presence of NAD(P)+. We here found the gene homolog of L-ArgDH in genome data of Pseudomonas veronii and succeeded in expression of P. veronii JCM11942 gene in E. coli. The gene product exhibited strong NADP+-dependent L-ArgDH activity. The enzyme was unstable, but markedly stabilized by the addition of 10% glycerol. The enzyme first purified to homogeneity consisted of a homodimeric protein with a molecular mass of about 65 kDa. The enzyme selectively catalyzed NADP+-dependent l-arginine oxidation with maximal activity at pH 9.5. The apparent Km values for l-arginine and NADP+ were 2.5 and 0.21 mM, respectively. The nucleotide sequence coding the enzyme gene was determined and the amino acid sequence was deduced from the nucleotide sequence. The simple colorimetric microassay for l-arginine using the enzyme was achieved.

Microelectrode implants, inflammatory response and long-lasting effects on NADPH diaphorase neurons in the rat frontal cortex.

At present, one of the main therapeutic challenges comprises the development of technologies to improve the life quality of people suffering from different types of body paralysis, through the reestablishment of sensory and motor functions. In this regard, brain-machine interfaces (BMI) offer hope to effectively mitigate body paralysis through the control of paralyzed body parts by brain activity. Invasive BMI use chronic multielectrode implants to record neural activity directly from the brain tissue. While such invasive devices provide the highest amount of usable neural activity for BMI control, they also involve direct damage to the nervous tissue. In the cerebral cortex, high levels of the enzyme NADPH diaphorase (NADPH-d) characterize a particular class of interneurons that regulates neuronal excitability and blood supply. To gain insight into the biocompatibility of invasive BMI, we assessed the impact of chronic implanted tungsten multielectrode bundles on the distribution and morphology of NADPH-d-reactive neurons in the rat frontal cortex. NADPH-d neuronal labeling was correlated with glial response markers and with indices of healthy neuronal activity measured by electrophysiological recordings performed up to 3 months after multielectrode implantation. Chronic electrode arrays caused a small and quite localized structural disturbance on the implanted site, with neuronal loss and glial activation circumscribed to the site of implant. Electrodes remained viable during the entire period of implantation. Moreover, neither the distribution nor the morphology of NADPH-d neurons was altered. Overall, our findings provide additional evidence that tungsten multielectrodes can be employed as a viable element for long-lasting therapeutic BMI applications.

Combined active pocket and hinge region engineering to develop an NADPH-dependent phenylglycine dehydrogenase.

NADPH-dependent amino acid dehydrogenases (AADHs) are favorable enzymes to construct artificial biosynthetic pathways in whole-cell for high-value noncanonical amino acids (NcAAs) production. Glutamate dehydrogenases (GluDHs) represent attractive candidates for the development of novel NADPH-dependent AADHs. Here, we report the development of a novel NADPH-dependent phenylglycine dehydrogenase by combining active pocket engineering and hinge region engineering of a GluDH from Pseudomonas putida (PpGluDH). The active pocket of PpGluDH was firstly tailored to optimize its binding mode with bulky substrate α-oxobenzeneacetic acid (α-OA), and then, the hinge region was further engineered to tune the protein conformational dynamics, which finally resulted in a mutant M3 (T196A/T121I/L123D) with a 103-fold increase of catalytic efficiency (kcat/Km) toward α-OA. The M3 mutant exhibited high catalytic performance in both in vitro biocatalysis preparation and in vivo biosynthesis of l-phenylglycine, indicating its promising practical applications. Our results demonstrated that co-engineering of the active pocket and hinge region is an effective strategy for developing novel NADPH-dependent AADHs from GluDHs for NcAAs production.

The Family Keeps on Growing: Four Novel Fungal OYEs Characterized.

Aiming at expanding the portfolio of Old Yellow Enzymes (OYEs), which have been systematically studied to be employed in the chemical and pharmaceutical industries as useful biocatalysts, we decided to explore the immense reservoir of filamentous fungi. We drew from the genome of the two Ascomycetes Aspergillus niger and Botryotinia fuckeliana four new members of the OYE superfamily belonging to the classical and thermophilic-like subfamilies. The two BfOYEs show wider substrate spectra than the AnOYE homologues, which appear as more specialized biocatalysts. According to their mesophilic origins, the new enzymes neither show high thermostability nor extreme pH optimums. The crystal structures of BfOYE4 and AnOYE8 have been determined, revealing the conserved features of the thermophilic-like subclass as well as unique properties, such as a peculiar N-terminal loop involved in dimer surface interactions. For the classical representatives BfOYE1 and AnOYE2, model structures were built and analyzed, showing surprisingly wide open access to the active site cavities due to a shorter ß6-loop and a disordered capping subdomain.

Knockdown of formin mDia2 alters lamin B1 levels and increases osteogenesis in stem cells.

Nuclear actin plays a critical role in mediating mesenchymal stem cell (MSC) fate commitment. In marrow-derived MSCs, the principal diaphanous-related formin Diaph3 (mDia2) is present in the nucleus and regulates intranuclear actin polymerization, whereas Diaph1 (mDia1) is localized to the cytoplasm and controls cytoplasmic actin polymerization. We here show that mDia2 can be used as a tool to query actin-lamin nucleoskeletal structure. Silencing mDia2 affected the nucleoskeletal lamin scaffold, altering nuclear morphology without affecting cytoplasmic actin cytoskeleton, and promoted MSC differentiation. Attempting to target intranuclear actin polymerization by silencing mDia2 led to a profound loss in lamin B1 nuclear envelope structure and integrity, increased nuclear height, and reduced nuclear stiffness without compensatory changes in other actin nucleation factors. Loss of mDia2 with the associated loss in lamin B1 promoted Runx2 transcription and robust osteogenic differentiation and suppressed adipogenic differentiation. Hence, mDia2 is a potent tool to query intranuclear actin-lamin nucleoskeletal structure, and its presence serves to retain multipotent stromal cells in an undifferentiated state.

Multi-Omics Approach to Mitochondrial DNA Damage in Human Muscle Fibers.

Mitochondrial DNA deletions affect energy metabolism at tissue-specific and cell-specific threshold levels, but the pathophysiological mechanisms determining cell fate remain poorly understood. Chronic progressive external ophthalmoplegia (CPEO) is caused by mtDNA deletions and characterized by a mosaic distribution of muscle fibers with defective cytochrome oxidase (COX) activity, interspersed among fibers with retained functional respiratory chain. We used diagnostic histochemistry to distinguish COX-negative from COX-positive fibers in nine muscle biopsies from CPEO patients and performed laser capture microdissection (LCM) coupled to genome-wide gene expression analysis. To gain molecular insight into the pathogenesis, we applied network and pathway analysis to highlight molecular differences of the COX-positive and COX-negative fiber transcriptome. We then integrated our results with proteomics data that we previously obtained comparing COX-positive and COX-negative fiber sections from three other patients. By virtue of the combination of LCM and a multi-omics approach, we here provide a comprehensive resource to tackle the pathogenic changes leading to progressive respiratory chain deficiency and disease in mitochondrial deletion syndromes. Our data show that COX-negative fibers upregulate transcripts involved in translational elongation and protein synthesis. Furthermore, based on functional annotation analysis, we find that mitochondrial transcripts are the most enriched among those with significantly different expression between COX-positive and COX-negative fibers, indicating that our unbiased large-scale approach resolves the core of the pathogenic changes. Further enrichments include transcripts encoding LIM domain proteins, ubiquitin ligases, proteins involved in RNA turnover, and, interestingly, cell cycle arrest and cell death. These pathways may thus have a functional association to the molecular pathogenesis of the disease. Overall, the transcriptome and proteome show a low degree of correlation in CPEO patients, suggesting a relevant contribution of post-transcriptional mechanisms in shaping this disease phenotype.

Molecular Analysis Uncovers the Mechanism of Fertility Restoration in Temperature-Sensitive Polima Cytoplasmic Male-Sterile Brassica napus.

Temperature-sensitive male sterility is a heritable agronomic trait affected by genotype-environment interactions. In rapeseed (Brassica napus), Polima (pol) temperature-sensitive cytoplasmic male sterility (TCMS) is commonly used for two-line breeding, as the fertility of pol TCMS lines can be partially restored at certain temperatures. However, little is known about the underlying molecular mechanism that controls fertility restoration. Therefore, we aimed to investigate the fertility conversion mechanism of the pol TCMS line at two different ambient temperatures (16 °C and 25 °C). Our results showed that the anthers developed and produced vigorous pollen at 16 °C but not at 25 °C. In addition, we identified a novel co-transcript of orf224-atp6 in the mitochondria that might lead to fertility conversion of the pol TCMS line. RNA-seq analysis showed that 1637 genes were significantly differentially expressed in the fertile flowers of 596-L when compared to the sterile flower of 1318 and 596-H. Detailed analysis revealed that differentially expressed genes were involved in temperature response, ROS accumulation, anther development, and mitochondrial function. Single-molecule long-read isoform sequencing combined with RNA sequencing revealed numerous genes produce alternative splicing transcripts at high temperatures. Here, we also found that alternative oxidase, type II NAD(P)H dehydrogenases, and transcription factor Hsfs might play a crucial role in male fertility under the low-temperature condition. RNA sequencing and bulked segregant analysis coupled with whole-genome sequencing identified the candidate genes involved in the post-transcriptional modification of orf224. Overall, our study described a putative mechanism of fertility restoration in a pol TCMS line controlled by ambient temperature that might help utilise TCMS in the two-line breeding of Brassica crops.

Engineering of Yeast Old Yellow Enzyme OYE3 Enables Its Capability Discriminating of (E)-Citral and (Z)-Citral.

The importance of yeast old yellow enzymes is increasingly recognized for direct asymmetric reduction of (E/Z)-citral to (R)-citronellal. As one of the most performing old yellow enzymes, the enzyme OYE3 from Saccharomyces cerevisiae S288C exhibited complementary enantioselectivity for the reduction of (E)-citral and (Z)-citral, resulting in lower e.e. value of (R)-citronellal in the reduction of (E/Z)-citral. To develop a novel approach for the direct synthesis of enantio-pure (R)-citronellal from the reduction of (E/Z)-citral, the enzyme OYE3 was firstly modified by semi-rational design to improve its (R)-enantioselectivity. The OYE3 variants W116A and S296F showed strict (R)-enantioselectivity in the reduction of (E)-citral, and significantly reversed the (S)-enantioselectivity in the reduction of (Z)-citral. Next, the double substitution of OYE3 led to the unique variant S296F/W116G, which exhibited strict (R)-enantioselectivity in the reduction of (E)-citral and (E/Z)-citral, but was not active on (Z)-citral. Relying on its capability discriminating (E)-citral and (Z)-citral, a new cascade reaction catalyzed by the OYE3 variant S296F/W116G and glucose dehydrogenase was developed, providing the enantio-pure (R)-citronellal and the retained (Z)-citral after complete reduction of (E)-citral.

Metals in Biotechnology: Cr-Driven Stereoselective Reduction of Conjugated C=C Double Bonds.

Elemental metals are shown to be suitable sacrificial electron donors to drive the stereoselective reduction of conjugated C=C double bonds using Old Yellow Enzymes as catalysts. Both direct electron transfer from the metal to the enzyme as well as mediated electron transfer is feasible, although the latter excels by higher reaction rates. The general applicability of this new chemoenzymatic reduction method is demonstrated, and current limitations are outlined.

A multiscale biophysical model for the recruitment of actin nucleating proteins at the membrane interface.

The dynamics and organization of the actin cytoskeleton are crucial to many cellular events such as motility, polarization, cell shaping, and cell division. The intracellular and extracellular signaling associated with this cytoskeletal network is communicated through cell membranes. Hence the organization of membrane macromolecules and actin filament assembly are highly interdependent. Although the actin-membrane linkage is known to happen through many routes, the major class of interactions is through the direct interaction of actin-binding proteins with the lipid class containing poly-phosphatidylinositols (PPIs). Among the PPIs, phosphatidylinositol bisphosphate (PI(4,5)P2) acts as a significant factor controlling actin polymerization in the proximity of the membrane by binding to actin-associated proteins. The molecular interactions between these actin-binding proteins and the membrane lipids remain elusive. Here, using molecular modeling, analytical theory, and experimental methods, we investigate the binding of three different actin-binding proteins, mDia2, NWASP, and gelsolin, to membranes containing PI(4,5)P2 lipids. We perform molecular dynamics simulations on the protein-bilayer system and analyze the membrane binding in the form of hydrogen bonds and salt bridges at various PI(4,5)P2 and cholesterol concentrations. Our experimental study with PI(4,5)P2-containing large unilamellar vesicles mimics the computational experiments. Using the multivalencies of the proteins obtained in molecular simulations and the cooperative binding mechanisms of the proteins, we also propose a multivalent binding model that predicts the actin filament distributions at various PI(4,5)P2 and protein concentrations.