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Nature Communications Mar 2020Long-chain alk(a/e)nes represent the major constituents of conventional transportation fuels. Biosynthesis of alkanes is ubiquitous in many kinds of organisms....
Long-chain alk(a/e)nes represent the major constituents of conventional transportation fuels. Biosynthesis of alkanes is ubiquitous in many kinds of organisms. Cyanobacteria possess two enzymes, acyl-acyl carrier protein (acyl-ACP) reductase (AAR) and aldehyde-deformylating oxygenase (ADO), which function in a two-step alkane biosynthesis pathway. These two enzymes act in series and possibly form a complex that efficiently converts long chain fatty acyl-ACP/fatty acyl-CoA into hydrocarbon. While the structure of ADO has been previously described, structures of both AAR and AAR-ADO complex have not been solved, preventing deeper understanding of this pathway. Here, we report a ligand-free AAR structure, and three AAR-ADO complex structures in which AARs bind various ligands. Our results reveal the binding pattern of AAR with its substrate/cofactor, and suggest a potential aldehyde-transferring channel from AAR to ADO. Based on our structural and biochemical data, we proposed a model for the complete catalytic cycle of AAR.
Topics: Acyl Carrier Protein; Aldehyde Oxidoreductases; Aldehyde-Lyases; Alkanes; Bacterial Proteins; Biocatalysis; Crystallography, X-Ray; Synechococcus
PubMed: 32251275
DOI: 10.1038/s41467-020-15268-y -
Applied Microbiology and Biotechnology Aug 2021Deoxyribose-5-phosphate aldolases (DERAs, EC 4.1.2.4) are acetaldehyde-dependent, Class I aldolases catalyzing in nature a reversible aldol reaction between an... (Review)
Review
Deoxyribose-5-phosphate aldolases (DERAs, EC 4.1.2.4) are acetaldehyde-dependent, Class I aldolases catalyzing in nature a reversible aldol reaction between an acetaldehyde donor (C2 compound) and glyceraldehyde-3-phosphate acceptor (C3 compound, C3P) to generate deoxyribose-5-phosphate (C5 compound, DR5P). DERA enzymes have been found to accept also other types of aldehydes as their donor, and in particular as acceptor molecules. Consequently, DERA enzymes can be applied in C-C bond formation reactions to produce novel compounds, thus offering a versatile biocatalytic alternative for synthesis. DERA enzymes, found in all kingdoms of life, share a common TIM barrel fold despite the low overall sequence identity. The catalytic mechanism is well-studied and involves formation of a covalent enzyme-substrate intermediate. A number of protein engineering studies to optimize substrate specificity, enzyme efficiency, and stability of DERA aldolases have been published. These have employed various engineering strategies including structure-based design, directed evolution, and recently also machine learning-guided protein engineering. For application purposes, enzyme immobilization and usage of whole cell catalysis are preferred methods as they improve the overall performance of the biocatalytic processes, including often also the stability of the enzyme. Besides single-step enzymatic reactions, DERA aldolases have also been applied in multi-enzyme cascade reactions both in vitro and in vivo. The DERA-based applications range from synthesis of commodity chemicals and flavours to more complicated and high-value pharmaceutical compounds. KEY POINTS: • DERA aldolases are versatile biocatalysts able to make new C-C bonds. • Synthetic utility of DERAs has been improved by protein engineering approaches. • Computational methods are expected to speed up the future DERA engineering efforts.
Topics: Aldehyde-Lyases; Fructose-Bisphosphate Aldolase; Ribosemonophosphates; Substrate Specificity
PubMed: 34410440
DOI: 10.1007/s00253-021-11462-0 -
Biochimica Et Biophysica Acta Jan 2013Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid whose actions are essential for many physiological processes including angiogenesis, lymphocyte trafficking and... (Review)
Review
Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid whose actions are essential for many physiological processes including angiogenesis, lymphocyte trafficking and development. In addition, S1P serves as a muscle trophic factor that enables efficient muscle regeneration. This is due in part to S1P's ability to activate quiescent muscle stem cells called satellite cells (SCs) that are needed for muscle repair. However, the molecular mechanism by which S1P activates SCs has not been well understood. Further, strategies for harnessing S1P signaling to recruit SCs for therapeutic benefit have been lacking. S1P is irreversibly catabolized by S1P lyase (SPL), a highly conserved enzyme that catalyzes the cleavage of S1P at carbon bond C(2-3), resulting in formation of hexadecenal and ethanolamine-phosphate. SPL enhances apoptosis through substrate- and product-dependent events, thereby regulating cellular responses to chemotherapy, radiation and ischemia. SPL is undetectable in resting murine skeletal muscle. However, we recently found that SPL is dynamically upregulated in skeletal muscle after injury. SPL upregulation occurred in the context of a tightly orchestrated genetic program that resulted in a transient S1P signal in response to muscle injury. S1P activated quiescent SCs via a sphingosine-1-phosphate receptor 2 (S1P2)/signal transducer and activator of transcription 3 (STAT3)-dependent pathway, thereby facilitating skeletal muscle regeneration. Mdx mice, which serve as a model for muscular dystrophy (MD), exhibited skeletal muscle SPL upregulation and S1P deficiency. Pharmacological SPL inhibition raised skeletal muscle S1P levels, enhanced SC recruitment and improved mdx skeletal muscle regeneration. These findings reveal how S1P can activate SCs and indicate that SPL suppression may provide a therapeutic strategy for myopathies. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.
Topics: Aldehyde-Lyases; Animals; Disease Models, Animal; Humans; Lysophospholipids; Muscle, Skeletal; Regeneration; Satellite Cells, Skeletal Muscle; Sphingosine
PubMed: 22750505
DOI: 10.1016/j.bbalip.2012.06.009 -
Nature May 2016Two-dimensional (2D) crystalline materials possess unique structural, mechanical and electronic properties that make them highly attractive in many applications....
Two-dimensional (2D) crystalline materials possess unique structural, mechanical and electronic properties that make them highly attractive in many applications. Although there have been advances in preparing 2D materials that consist of one or a few atomic or molecular layers, bottom-up assembly of 2D crystalline materials remains a challenge and an active area of development. More challenging is the design of dynamic 2D lattices that can undergo large-scale motions without loss of crystallinity. Dynamic behaviour in porous three-dimensional (3D) crystalline solids has been exploited for stimuli-responsive functions and adaptive behaviour. As in such 3D materials, integrating flexibility and adaptiveness into crystalline 2D lattices would greatly broaden the functional scope of 2D materials. Here we report the self-assembly of unsupported, 2D protein lattices with precise spatial arrangements and patterns using a readily accessible design strategy. Three single- or double-point mutants of the C4-symmetric protein RhuA were designed to assemble via different modes of intermolecular interactions (single-disulfide, double-disulfide and metal-coordination) into crystalline 2D arrays. Owing to the flexibility of the single-disulfide interactions, the lattices of one of the variants ((C98)RhuA) are essentially defect-free and undergo substantial, but fully correlated, changes in molecular arrangement, yielding coherently dynamic 2D molecular lattices. (C98)RhuA lattices display a Poisson's ratio of -1-the lowest thermodynamically possible value for an isotropic material-making them auxetic.
Topics: Aldehyde-Lyases; Crystallization; Disulfides; Escherichia coli; Metals; Microscopy, Electron, Transmission; Models, Molecular; Mutant Proteins; Pliability; Protein Conformation; Rotation; Stress, Mechanical; Thermodynamics
PubMed: 27135928
DOI: 10.1038/nature17633 -
The Journal of Biological Chemistry Jan 2020Carbon-carbon bond formation is one of the most important reactions in biocatalysis and organic chemistry. In nature, aldolases catalyze the reversible stereoselective...
Carbon-carbon bond formation is one of the most important reactions in biocatalysis and organic chemistry. In nature, aldolases catalyze the reversible stereoselective aldol addition between two carbonyl compounds, making them attractive catalysts for the synthesis of various chemicals. In this work, we identified several 2-deoxyribose-5-phosphate aldolases (DERAs) having acetaldehyde condensation activity, which can be used for the biosynthesis of ()-1,3-butanediol (1,3BDO) in combination with aldo-keto reductases (AKRs). Enzymatic screening of 20 purified DERAs revealed the presence of significant acetaldehyde condensation activity in 12 of the enzymes, with the highest activities in BH1352 from , TM1559 from , and DeoC from The crystal structures of BH1352 and TM1559 at 1.40-2.50 Å resolution are the first full-length DERA structures revealing the presence of the C-terminal Tyr (Tyr in BH1352). The results from structure-based site-directed mutagenesis of BH1352 indicated a key role for the catalytic Lys and other active-site residues in the 2-deoxyribose-5-phosphate cleavage and acetaldehyde condensation reactions. These experiments also revealed a 2.5-fold increase in acetaldehyde transformation to 1,3BDO (in combination with AKR) in the BH1352 F160Y and F160Y/M173I variants. The replacement of the WT BH1352 by the F160Y or F160Y/M173I variants in cells expressing the DERA + AKR pathway increased the production of 1,3BDO from glucose five and six times, respectively. Thus, our work provides detailed insights into the molecular mechanisms of substrate selectivity and activity of DERAs and identifies two DERA variants with enhanced activity for and 1,3BDO biosynthesis.
Topics: Aldehyde-Lyases; Bacillus; Biosynthetic Pathways; Butylene Glycols; Catalytic Domain; Crystallography, X-Ray; Escherichia coli; Industrial Microbiology; Models, Molecular; Mutagenesis, Site-Directed; Phylogeny; Protein Engineering; Thermotoga maritima
PubMed: 31806708
DOI: 10.1074/jbc.RA119.011363 -
Scientific Reports Oct 2015Autophagy is an important homeostatic mechanism that eliminates long-lived proteins, protein aggregates and damaged organelles. Its dysregulation is involved in many...
Autophagy is an important homeostatic mechanism that eliminates long-lived proteins, protein aggregates and damaged organelles. Its dysregulation is involved in many neurodegenerative disorders. Autophagy is therefore a promising target for blunting neurodegeneration. We searched for novel autophagic pathways in primary neurons and identified the cytosolic sphingosine-1-phosphate (S1P) pathway as a regulator of neuronal autophagy. S1P, a bioactive lipid generated by sphingosine kinase 1 (SK1) in the cytoplasm, is implicated in cell survival. We found that SK1 enhances flux through autophagy and that S1P-metabolizing enzymes decrease this flux. When autophagy is stimulated, SK1 relocalizes to endosomes/autophagosomes in neurons. Expression of a dominant-negative form of SK1 inhibits autophagosome synthesis. In a neuron model of Huntington's disease, pharmacologically inhibiting S1P-lyase protected neurons from mutant huntingtin-induced neurotoxicity. These results identify the S1P pathway as a novel regulator of neuronal autophagy and provide a new target for developing therapies for neurodegenerative disorders.
Topics: Aldehyde-Lyases; Animals; Autophagy; Biomarkers; Cell Survival; Cytoplasm; Endoplasmic Reticulum; Endosomes; Enzyme Inhibitors; Gene Expression; Lysophospholipids; Neurons; Phagosomes; Phosphotransferases (Alcohol Group Acceptor); Protein Binding; Protein Transport; Rats; Signal Transduction; Sphingosine
PubMed: 26477494
DOI: 10.1038/srep15213 -
The Journal of Steroid Biochemistry and... Sep 2020Deficiency in Sphingosine-1-phosphate lyase (S1P lyase) is associated with a multi-systemic disorder incorporating primary adrenal insufficiency (PAI), steroid resistant...
Deficiency in Sphingosine-1-phosphate lyase (S1P lyase) is associated with a multi-systemic disorder incorporating primary adrenal insufficiency (PAI), steroid resistant nephrotic syndrome and neurological dysfunction. Accumulation of sphingolipid intermediates, as seen with loss of function mutations in SGPL1, has been implicated in mitochondrial dysregulation, including alterations in mitochondrial membrane potentials and initiation of mitochondrial apoptosis. For the first time, we investigate the impact of S1P lyase deficiency on mitochondrial morphology and function using patient-derived human dermal fibroblasts and CRISPR engineered SGPL1-knockout HeLa cells. Reduced cortisol output in response to progesterone stimulation was observed in two patient dermal fibroblast cell lines. Mass spectrometric analysis of patient dermal fibroblasts revealed significantly elevated levels of sphingosine-1-phosphate, sphingosine, ceramide species and sphingomyelin when compared to control. Total mitochondrial volume was reduced in both S1P lyase deficient patient and HeLa cell lines. Mitochondrial dynamics and parameters of oxidative phosphorylation were altered when compared to matched controls, though differentially across the cell lines. Mitochondrial dysfunction may represent a major event in the pathogenesis of this disease, associated with severity of phenotype.
Topics: Adrenal Insufficiency; Aldehyde-Lyases; Cell Respiration; Cells, Cultured; Fibroblasts; Humans; Hydrocortisone; Mitochondria; Mitochondrial Diseases; Phosphoproteins; Progesterone; Skin
PubMed: 32682944
DOI: 10.1016/j.jsbmb.2020.105730 -
Nature Communications Mar 2019Acetyl-CoA is a fundamental metabolite for all life on Earth, and is also a key starting point for the biosynthesis of a variety of industrial chemicals and natural...
Acetyl-CoA is a fundamental metabolite for all life on Earth, and is also a key starting point for the biosynthesis of a variety of industrial chemicals and natural products. Here we design and construct a Synthetic Acetyl-CoA (SACA) pathway by repurposing glycolaldehyde synthase and acetyl-phosphate synthase. First, we design and engineer glycolaldehyde synthase to improve catalytic activity more than 70-fold, to condense two molecules of formaldehyde into one glycolaldehyde. Second, we repurpose a phosphoketolase to convert glycolaldehyde into acetyl-phosphate. We demonstrated the feasibility of the SACA pathway in vitro, achieving a carbon yield ~50%, and confirmed the SACA pathway by C-labeled metabolites. Finally, the SACA pathway was verified by cell growth using glycolaldehyde, formaldehyde and methanol as supplemental carbon source. The SACA pathway is proved to be the shortest, ATP-independent, carbon-conserving and oxygen-insensitive pathway for acetyl-CoA biosynthesis, opening possibilities for producing acetyl-CoA-derived chemicals from one-carbon resources in the future.
Topics: Acetaldehyde; Acetyl Coenzyme A; Aldehyde-Lyases; Carbon; Escherichia coli; Formaldehyde; Metabolic Engineering; Organophosphates; Plasmids
PubMed: 30914637
DOI: 10.1038/s41467-019-09095-z -
Biochimica Et Biophysica Acta Sep 2008Sphingosine-1-phosphate lyase (SPL) is a highly conserved enzyme that catalyses the final step of sphingolipid degradation, namely the irreversible cleavage of the... (Review)
Review
Sphingosine-1-phosphate lyase (SPL) is a highly conserved enzyme that catalyses the final step of sphingolipid degradation, namely the irreversible cleavage of the carbon chain at positions 2-3 of a long-chain base phosphate (LCBP), thereby yielding a long-chain aldehyde and phosphoethanolamine. LCBPs are potent signaling molecules involved in cell proliferation, survival, migration, cell-cell interactions and cell stress responses. Therefore, tight regulation of LCBP signaling is required for proper cell function, and perturbations of this system can lead to alterations in biological processes including development, reproduction and physiology. SPL is a key enzyme in regulating the intracellular and circulating levels of LCBPs and is, therefore, gaining attention as a putative target for pharmacological intervention. This review provides an overview of our current understanding of SPL structure and function, mechanisms involved in SPL regulation and the role of SPL in development and disease.
Topics: Aldehyde-Lyases; Animals; Apoptosis; Disease; Gene Expression Regulation, Developmental; Humans; Sphingosine
PubMed: 18558101
DOI: 10.1016/j.bbalip.2008.05.005 -
Expert Opinion on Therapeutic Targets Aug 2009Sphingosine 1-phosphate (S1P) is a bioactive lipid that regulates cell proliferation, survival and migration and plays an essential role in angiogenesis and lymphocyte... (Review)
Review
BACKGROUND
Sphingosine 1-phosphate (S1P) is a bioactive lipid that regulates cell proliferation, survival and migration and plays an essential role in angiogenesis and lymphocyte trafficking. S1P levels in the circulation and tissues are tightly regulated for proper cell functioning, and dysregulation of this system may contribute to the pathophysiology of certain human diseases. Sphingosine phosphate lyase (SPL) irreversibly degrades S1P and thereby acts as a gatekeeper that regulates S1P signaling by modulating intracellular S1P levels and the chemical S1P gradient that exists between lymphoid organs and circulating blood and lymph. However, SPL also generates biochemical products that may be relevant in human disease. SPL has been directly implicated in various physiological and pathological processes, including cell stress responses, cancer, immunity, hematopoietic function, muscle homeostasis, inflammation and development.
OBJECTIVE/METHODS
This review summarizes the current know-ledge of SPL structure, function and regulation, its involvement in various disease states and currently available small molecules known to modulate SPL activity.
RESULTS/CONCLUSION
This review provides evidence that SPL is a potential target for pharmacological manipulation for the treatment of malignant, autoimmune, inflammatory and other diseases.
Topics: Aldehyde-Lyases; Animals; Autoimmune Diseases; Drug Delivery Systems; Enzyme Inhibitors; Gene Targeting; Humans; Inflammation Mediators; Signal Transduction
PubMed: 19534571
DOI: 10.1517/14728220903039722