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Microbial Biotechnology May 2022Bioproduction of optical pure (R)-citronellal from (E/Z)-citral at high substrate loading remains challenging. Low catalytic efficiency of (R)-stereoselective ene...
Bioproduction of optical pure (R)-citronellal from (E/Z)-citral at high substrate loading remains challenging. Low catalytic efficiency of (R)-stereoselective ene reductases towards crude citral mixture is one of the major bottlenecks. Herein, a structure-based engineering strategy was adopted to enhance the catalytic efficiency and stereoselectivity of an ene reductase (OYE2p) from Saccharomyces cerevisiae YJM1341 towards (E/Z)-citral. On basis of homologous modelling, molecular docking analysis and alanine scanning at the binding pocket of OYE2p, a mutant Y84A was obtained with simultaneous increase in catalytic efficiency and stereoselectivity. Furthermore, site-saturation mutagenesis of Y84 yielded seven mutants with improved activity and stereoselectivity in the (E/Z)-citral reduction. Among them, the variant Y84V exhibited an 18.3% and 71.3% rise in catalytic efficiency (k /K ) for (Z)-citral and (E)-citral respectively. Meanwhile, the stereoselectivity of Y84V was improved from 89.2% to 98.0% in the reduction in (E/Z)-citral. The docking analysis and molecular dynamics simulation of OYE2p and its variants revealed that the substitution Y84V enabled (E)-citral and (Z)-citral to bind with a smaller distance to the key hydrogen donors at a modified (R)-selective binding mode. The variant Y84V was then co-expressed with glucose dehydrogenase from Bacillus megaterium in E. coli D4, in which competing prim-alcohol dehydrogenase genes were deleted to prevent the undesired reduction in the aldehyde moiety of citral and citronellal. Employing this biocatalyst, 106 g l (E/Z)-citral was completely converted into (R)-citronellal with 95.4% ee value and a high space-time yield of 121.6 g l day . The work highlights the synthetic potential of Y84V, which enabled the highest productivity of (R)-citronellal from (E/Z)-citral in high enantiopurity so far.
Topics: Acyclic Monoterpenes; Aldehydes; Escherichia coli; Glucose 1-Dehydrogenase; Molecular Docking Simulation; Oxidoreductases; Saccharomyces cerevisiae
PubMed: 34729922
DOI: 10.1111/1751-7915.13958 -
Molecular Neurobiology Jul 2023Excessive activation of aldose reductase (AR) in the brain is a risk factor for aggravating cerebral ischemia injury. Epalrestat is the only AR inhibitor with proven...
Excessive activation of aldose reductase (AR) in the brain is a risk factor for aggravating cerebral ischemia injury. Epalrestat is the only AR inhibitor with proven safety and efficacy, which is used in the clinical treatment of diabetic neuropathy. However, the molecular mechanisms underlying the neuroprotection of epalrestat remain unknown in the ischemic brain. Recent studies have found that blood-brain barrier (BBB) damage was mainly caused by increased apoptosis and autophagy of brain microvascular endothelial cells (BMVECs) and decreased expression of tight junction proteins. Thus, we hypothesized that the protective effect of epalrestat is mainly related to regulating the survival of BMVECs and tight junction protein levels after cerebral ischemia. To test this hypothesis, a mouse model of cerebral ischemia was established by permanent middle cerebral artery ligation (pMCAL), and the mice were treated with epalrestat or saline as a control. Epalrestat reduced the ischemic volume, enhanced BBB function, and improved the neurobehavior after cerebral ischemia. In vitro studies revealed that epalrestat increased the expression of tight junction proteins, and reduced the levels of cleaved-caspase3 and LC3 proteins in mouse BMVECs (bEnd.3 cells) exposed to oxygen-glucose deprivation (OGD). In addition, bicalutamide (an AKT inhibitor) and rapamycin (an mTOR inhibitor) increased the epalrestat-induced reduction in apoptosis and autophagy related protein levels in bEnd.3 cells with OGD treatment. Our findings suggest that epalrestat improves BBB function, which may be accomplished by reducing AR activation, promoting tight junction proteins expression, and upregulating AKT/mTOR signaling pathway to inhibit apoptosis and autophagy in BMVECs.
Topics: Mice; Animals; Blood-Brain Barrier; Endothelial Cells; Proto-Oncogene Proteins c-akt; Aldehyde Reductase; Brain Ischemia; Cerebral Infarction; Brain Injuries; Glucose; Tight Junction Proteins; TOR Serine-Threonine Kinases
PubMed: 36940077
DOI: 10.1007/s12035-023-03304-z -
Journal of Bacteriology Mar 2022Acetic acid bacteria catalyze the two-step oxidation of ethanol to acetic acid using the membrane-bound enzymes pyrroloquinoline quinone-dependent alcohol dehydrogenase...
Acetic acid bacteria catalyze the two-step oxidation of ethanol to acetic acid using the membrane-bound enzymes pyrroloquinoline quinone-dependent alcohol dehydrogenase and molybdopterin-dependent aldehyde dehydrogenase (ALDH). Although the reducing equivalents from the substrate are transferred to ubiquinone in the membrane, intramolecular electron transport in ALDH is not understood. Here, we purified the AldFGH complex, the membrane-bound ALDH that is physiologically relevant to acetic acid fermentation in Gluconacetobacter diazotrophicus strain PAL5. The purified AldFGH complex showed acetaldehyde:ubiquinone (Q) oxidoreductase activity. -type cytochromes of the AldFGH complex (in the AldF subunit) were reduced by acetaldehyde. Next, we genetically dissected the AldFGH complex into AldGH and AldF units and reconstituted them. The AldGH subcomplex showed acetaldehyde:ferricyanide oxidoreductase activity but not Q reductase activity. The ALDH activity of AldGH was not found in membranes but was found in the soluble fraction of the recombinant strain, suggesting that the AldF subunit is responsible for membrane binding of the AldFGH complex. The absorption spectra of the purified AldGH subcomplex suggested the presence of an [Fe-S] cluster, which can be reduced by acetaldehyde. The AldFGH complex reconstituted from the AldGH subcomplex and AldF showed Q reductase activity. We propose a model in which electrons from the substrate are abstracted by a molybdopterin in the AldH subunit and transferred to the [Fe-S] cluster(s) in the AldG subunit, followed by electron transport to -type cytochrome centers in the AldF subunit, which is the site of ubiquinone reduction in the membrane. Two membrane-bound enzymes of acetic acid bacteria, pyrroloquinoline quinone-dependent alcohol dehydrogenase and molybdopterin-dependent aldehyde dehydrogenase (ALDH), are responsible for vinegar production. Upon the oxidation of acetaldehyde, ALDH reduces ubiquinone in the cytoplasmic membrane. ALDH is an enzyme complex of three subunits. Here, we tried to understand how ALDH works by using a classical biochemical approach and genetic engineering to dissect the enzyme complex into soluble and membrane-bound parts. The soluble part had limited activity and did not reduce ubiquinone. However, the enzyme complex reconstituted from the soluble and membrane-bound parts showed ubiquinone reduction activity. The proposed working model of ALDH provides a better understanding of how the enzyme works in the vinegar fermentation process.
Topics: Acetaldehyde; Acetic Acid; Alcohol Dehydrogenase; Aldehyde Dehydrogenase; Aldehydes; Cytochromes; Electron Transport; Gluconacetobacter; PQQ Cofactor; Ubiquinone
PubMed: 35072518
DOI: 10.1128/jb.00558-21 -
Frontiers in Endocrinology 2022Aldose reductase B1 (AKR1B1) has been reported to participate in the modulation of male and female reproductive physiology in several mammalian species. In spite of...
Aldose reductase B1 (AKR1B1) has been reported to participate in the modulation of male and female reproductive physiology in several mammalian species. In spite of this, whether or not AKR1B1 could be related to sperm quality, functionality and fertilizing ability is yet to be elucidated. The present study, therefore, aimed to investigate: i) the presence of AKR1B1 in epididymal and ejaculated sperm; ii) the relationship between the AKR1B1 present in sperm and the physiology of the male gamete; iii) the liaison between the relative content of AKR1B1 in sperm and their ability to withstand preservation for 72 h; and iv) the potential link between sperm AKR1B1 and fertility outcomes. Immunoblotting revealed that AKR1B1 is present in both epididymal and ejaculated sperm with a similar relative content. Moreover, the relative levels of AKR1B1 in sperm (36 kDa band) were found to be negatively related to several kinematic parameters and intracellular calcium levels, and positively to the percentage of sperm with distal cytoplasmic droplets after storage. Finally, AKR1B1 amounts in sperm (36 kDa band) were negatively associated to fertilization rate at two days post-fertilization and embryo development at six days post-fertilization. The results of the present work suggest that AKR1B1 in sperm is probably acquired during maturation rather than at ejaculation and could play a role in that process. Moreover, AKR1B1 seems to be related to the sperm resilience to preservation and to their fertilizing capacity, as lower levels of the 36 kDa band (putative inactive form of this protein) result in better reproductive outcomes.
Topics: Aldehyde Reductase; Animals; Epididymis; Female; Fertilization; Fertilization in Vitro; Male; Mammals; Spermatozoa; Swine
PubMed: 35173684
DOI: 10.3389/fendo.2022.773249 -
Bioorganic Chemistry Jun 2022In this study, we present the concept of co-immobilization of xylose dehydrogenase and alcohol dehydrogenase from Saccharomyces cerevisiae on an XN45 nanofiltration...
In this study, we present the concept of co-immobilization of xylose dehydrogenase and alcohol dehydrogenase from Saccharomyces cerevisiae on an XN45 nanofiltration membrane for application in the process of xylose bioconversion to xylonic acid with simultaneous cofactor regeneration and membrane separation of reaction products. During the research, the effectiveness of the co-immobilization of enzymes was confirmed, and changes in the properties of the membrane after the processes were determined. Using the obtained biocatalytic system it was possible to obtain 99% xylonic acid production efficiency under optimal conditions, which were 5 mM xylose, 5 mM formaldehyde, ratio of NAD+:NADH 1:1, and 60 min of reaction. Additionally, the co-immobilization of enzymes made it possible to improve stability of the co-immobilized enzymes and to carry out xylose conversion in six consecutive cycles and after 7 days of storage at 4 °C with over 90% efficiency. The presented data confirm the effectiveness of the co-immobilization, improvement of the stability and reusability of the biocatalysts, and show that the obtained enzymatic system is promising for use in xylose bioconversion and simultaneous regeneration of nicotinamide cofactor.
Topics: Alcohol Dehydrogenase; Aldehyde Reductase; Biocatalysis; Regeneration; Xylose
PubMed: 35395447
DOI: 10.1016/j.bioorg.2022.105781 -
Protein Science : a Publication of the... Mar 2022Accumulation of formaldehyde, a highly reactive molecule, in the cell is toxic, and requires detoxification for the organism's survival. Mycothiol-dependent formaldehyde...
Accumulation of formaldehyde, a highly reactive molecule, in the cell is toxic, and requires detoxification for the organism's survival. Mycothiol-dependent formaldehyde dehydrogenase or S-nitrosomycothiol reductase (MscR) from Mycobacterium smegmatis and Mycobacterium tuberculosis was previously known for detoxifying formaldehyde and protecting the cell against nitrosative stress. We here show that M. smegmatis MscR exhibits a mycothiol-independent formaldehyde dehydrogenase (FDH) activity in vitro. Presence of zinc in the reaction enhances MscR activity, thus making it a zinc-dependent FDH. Interestingly, MscR utilizes only formaldehyde and no other primary aldehydes as its substrate in vitro, and M. smegmatis lacking mscR (ΔmscR) shows sensitivity exclusively toward formaldehyde. Bioinformatics analysis of MscRs from various bacteria reveals 10 positionally conserved cysteines, whose importance in structural stability and biological activity is not yet investigated. To explore the significance of these cysteines, we generated MscR single Cys variants by systematically replacing each cysteine with serine. All of the Cys variants except C39S and C309S are unable to show a complete rescue of ΔmscR on formaldehyde, show a significant loss of enzymatic activity in vitro, pronounced structural alterations as probed by circular dichroism, and loss of homotetramerization on size exclusion chromatography. Our data thus reveal the importance of intact cysteines in the structural stability and biological activity of MscR, which is a dedicated FDH in M. smegmatis, and shows ~84% identity with M. tuberculosis MscR. We believe that this knowledge will further help in the development of FDH as a potential drug target against M. tuberculosis infections.
Topics: Aldehyde Oxidoreductases; Bacterial Proteins; Cysteine; Mycobacterium smegmatis; Mycobacterium tuberculosis
PubMed: 34904319
DOI: 10.1002/pro.4258 -
MBio Aug 2023Malonyl-CoA reductase (MCR) is a NADPH-dependent bi-functional enzyme that performs alcohol dehydrogenase and aldehyde dehydrogenase (CoA-acylating) activities in the N-...
Malonyl-CoA reductase (MCR) is a NADPH-dependent bi-functional enzyme that performs alcohol dehydrogenase and aldehyde dehydrogenase (CoA-acylating) activities in the N- and C-terminal fragments, respectively. It catalyzes the two-step reduction of malonyl-CoA to 3-hydroxypropionate (3-HP), a key reaction in the autotrophic CO fixation cycles of green non-sulfur bacteria and the archaea . However, the structural basis underlying substrate selection, coordination, and the subsequent catalytic reactions of full-length MCR is largely unknown. For the first time, we here determined the structure of full-length MCR from the photosynthetic green non-sulfur bacterium (MCR) at 3.35 Å resolution. Furthermore, we determined the crystal structures of the N- and C-terminal fragments bound with reaction intermediates NADP and malonate semialdehyde (MSA) at 2.0 Å and 2.3 Å, respectively, and elucidated the catalytic mechanisms using a combination of molecular dynamics simulations and enzymatic analyses. Full-length MCR was a homodimer of two cross-interlocked subunits, each containing four tandemly arranged short-chain dehydrogenase/reductase (SDR) domains. Only the catalytic domains SDR1 and SDR3 incorporated additional secondary structures that changed with NADP-MSA binding. The substrate, malonyl-CoA, was immobilized in the substrate-binding pocket of SDR3 through coordination with Arg1164 and Arg799 of SDR4 and the extra domain, respectively. Malonyl-CoA was successively reduced through protonation by the Tyr743-Arg746 pair in SDR3 and the catalytic triad (Thr165-Tyr178-Lys182) in SDR1 after nucleophilic attack from NADPH hydrides. IMPORTANCE The bi-functional MCR catalyzes NADPH-dependent reduction of malonyl-CoA to 3-HP, an important metabolic intermediate and platform chemical, from biomass. The individual MCR-N and MCR-C fragments, which contain the alcohol dehydrogenase and aldehyde dehydrogenase (CoA-acylating) activities, respectively, have previously been structurally investigated and reconstructed into a malonyl-CoA pathway for the biosynthetic production of 3-HP. However, no structural information for full-length MCR has been available to illustrate the catalytic mechanism of this enzyme, which greatly limits our capacity to increase the 3-HP yield of recombinant strains. Here, we report the cryo-electron microscopy structure of full-length MCR for the first time and elucidate the mechanisms underlying substrate selection, coordination, and catalysis in the bi-functional MCR. These findings provide a structural and mechanistic basis for enzyme engineering and biosynthetic applications of the 3-HP carbon fixation pathways.
Topics: Alcohol Dehydrogenase; NADP; Cryoelectron Microscopy; Oxidoreductases; Chloroflexi; Aldehyde Dehydrogenase; Malonyl Coenzyme A
PubMed: 37278533
DOI: 10.1128/mbio.03233-22 -
Frontiers in Immunology 2023While it is considered one of the most common cancers and the leading cause of death in men worldwide, prognostic stratification and treatment modalities are still...
While it is considered one of the most common cancers and the leading cause of death in men worldwide, prognostic stratification and treatment modalities are still limited for patients with prostate cancer (PCa). Recently, the introduction of genomic profiling and the use of new techniques like next-generation sequencing (NGS) in many cancers provide novel tools for the discovery of new molecular targets that might improve our understanding of the genomic aberrations in PCa and the discovery of novel prognostic and therapeutic targets. In this study, we investigated the possible mechanisms through which (DKK3) produces its possible protective role in PCa using NGS in both the DKK3 overexpression PCa cell line (PC3) model and our patient cohort consisting of nine PCa and five benign prostatic hyperplasia. Interestingly, our results have shown that DKK3 transfection-modulated genes are involved in the regulation of cell motility, senescence-associated secretory phenotype (SASP), and cytokine signaling in the immune system, as well as in the regulation of adaptive immune response. Further analysis of our NGS using our model revealed the presence of 36 differentially expressed genes (DEGs) between DKK3 transfected cells and PC3 empty vector. In addition, both and genes were differentially expressed not only between the transfected and empty groups but also between the transfected and Mock cells. The top common DEGs between the DKK3 overexpression cell line and our patient cohort are the following: , , and . The upregulated genes including , , and showed tumor suppressor functions in various cancers including PCa. On the other hand, both and were downregulated and involved in tumor initiation, tumor progression, poor outcome, and radiotherapy resistance. Together, our results highlighted the possible role of the DKK3-related genes in protecting against PCa initiation and progression.
Topics: Humans; Male; Angiotensin-Converting Enzyme 2; Prostatic Neoplasms; Cell Line; Prostatic Hyperplasia; Aldehyde Reductase; Adaptor Proteins, Signal Transducing
PubMed: 36845147
DOI: 10.3389/fimmu.2023.978236 -
Frontiers in Plant Science 2021Green leaf volatiles (GLVs), the common constituents of herbivore-infested plant volatiles (HIPVs), play an important role in plant defense and function as chemical cues...
Green leaf volatiles (GLVs), the common constituents of herbivore-infested plant volatiles (HIPVs), play an important role in plant defense and function as chemical cues to communicate with other individuals in nature. Reportedly, in addition to endogenous GLVs, the absorbance of airborne GLVs emitted by infested neighboring plants also play a major role in plant defense. For example, the exclusive accumulation of ()-3-hexenyl vicianoside in the HIPV-exposed tomato plants occurs by the glycosylation of airborne ()-3-hexenol (Z3HOL); however, it is unclear how plants process the other absorbed GLVs. This study demonstrates that tomato plants dominantly accumulated GLV-glycosides after exposure to green leaf alcohols [Z3HOL, ()-2-hexenol, and -hexanol] using non-targeted LC-MS analysis. Three types of green leaf alcohols were independently glycosylated without isomerization or saturation/desaturation. Airborne green leaf aldehydes and esters were also glycosylated, probably through converting aldehydes and esters into alcohols. Further, we validated these findings in Arabidopsis mutants- ()-3-hexenal (Z3HAL) reductase () mutant that inhibits the conversion of Z3HAL to Z3HOL and the acetyl-CoA:()-3-hexen-1-ol acetyltransferase () mutant that impairs the conversion of Z3HOL to ()-3-hexenyl acetate. Exposure of the and mutants to Z3HAL accumulated lower and higher amounts of glycosides than their corresponding wild types (Col-0 and L), respectively. These findings suggest that plants process the exogenous GLVs by the reductase(s) and the esterase(s), and a part of the processed GLVs contribute to glycoside accumulation. Overall, the study provides insights into the understanding of the communication of the plants within their ecosystem, which could help develop strategies to protect the crops and maintain a balanced ecosystem.
PubMed: 34868107
DOI: 10.3389/fpls.2021.721572 -
International Journal of Molecular... Jan 2022Aluminum (Al) toxicity is the main factor limiting plant growth and the yield of cereal crops in acidic soils. Al-induced oxidative stress could lead to the excessive...
Aluminum (Al) toxicity is the main factor limiting plant growth and the yield of cereal crops in acidic soils. Al-induced oxidative stress could lead to the excessive accumulation of reactive oxygen species (ROS) and aldehydes in plants. Aldehyde dehydrogenase () genes, which play an important role in detoxification of aldehydes when exposed to abiotic stress, have been identified in most species. However, little is known about the function of this gene family in the response to Al stress. Here, we identified an gene in maize, , involved in protection against Al-induced oxidative stress. Al stress up-regulated expression in both the roots and leaves. The expression of only responded to Al toxicity but not to other stresses including low pH and other metals. The heterologous overexpression of in increased Al tolerance by promoting the ascorbate-glutathione cycle, increasing the transcript levels of antioxidant enzyme genes as well as the activities of their products, reducing MDA, and increasing free proline synthesis. The overexpression of also reduced Al accumulation in roots. Taken together, these findings suggest that participates in Al-induced oxidative stress and Al accumulation in roots, conferring Al tolerance in transgenic .
Topics: Adaptation, Physiological; Aldehyde Dehydrogenase; Aluminum; Amino Acid Sequence; Antioxidants; Arabidopsis; Ascorbate Peroxidases; Ascorbic Acid; Cloning, Molecular; Gene Expression Regulation, Plant; Genes, Plant; Glutathione; Glutathione Reductase; Hydrogen Peroxide; Lipid Peroxidation; Oxidative Stress; Phylogeny; Plant Leaves; Plant Roots; Plants, Genetically Modified; Proline; RNA, Messenger; Subcellular Fractions; Superoxides; Nicotiana; Zea mays
PubMed: 35008903
DOI: 10.3390/ijms23010477