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Communications Biology Apr 2022Human aldehyde dehydrogenase (ALDH) participates in the oxidative stress response and retinoid metabolism, being involved in several diseases, including cancer, diabetes...
Human aldehyde dehydrogenase (ALDH) participates in the oxidative stress response and retinoid metabolism, being involved in several diseases, including cancer, diabetes and obesity. The ALDH1A3 isoform has recently elicited wide interest because of its potential use as a cancer stem cell biomarker and drug target. We report high-resolution three-dimensional ALDH1A3 structures for the apo-enzyme, the NAD complex and a binary complex with ATP. Each subunit of the ALDH1A3-ATP complex contains one ATP molecule bound to the adenosine-binding pocket of the cofactor-binding site. The ATP complex also shows a molecule, putatively identified as a polyethylene glycol aldehyde, covalently bound to the active-site cysteine. This mimics the thioacyl-enzyme catalytic intermediate, which is trapped in a dead enzyme lacking an active cofactor. At physiological concentrations, ATP inhibits the dehydrogenase activity of ALDH1A3 and other isoforms, with a K value of 0.48 mM for ALDH1A3, showing a mixed inhibition type against NAD. ATP also inhibits esterase activity in a concentration-dependent manner. The current ALDH1A3 structures at higher resolution will facilitate the rational design of potent and selective inhibitors. ATP binding to ALDH1A3 enables activity modulation by the energy status of the cell and metabolic reprogramming, which may be relevant in several disease conditions.
Topics: Adenosine Triphosphate; Aldehyde Dehydrogenase; Aldehyde Oxidoreductases; Biomarkers, Tumor; Humans; NAD; Neoplasms; Neoplastic Stem Cells
PubMed: 35418200
DOI: 10.1038/s42003-022-03311-1 -
The structure of Synechococcus elongatus enolase reveals key aspects of phosphoenolpyruvate binding.Acta Crystallographica. Section F,... Apr 2022A structure-function characterization of Synechococcus elongatus enolase (SeEN) is presented, representing the first structural report on a cyanobacterial enolase. X-ray...
A structure-function characterization of Synechococcus elongatus enolase (SeEN) is presented, representing the first structural report on a cyanobacterial enolase. X-ray crystal structures of SeEN in its apoenzyme form and in complex with phosphoenolpyruvate are reported at 2.05 and 2.30 Å resolution, respectively. SeEN displays the typical fold of enolases, with a conformationally flexible loop that closes the active site upon substrate binding, assisted by two metal ions that stabilize the negatively charged groups. The enzyme exhibits a catalytic efficiency of 1.2 × 10 M s for the dehydration of 2-phospho-D-glycerate, which is comparable to the kinetic parameters of related enzymes. These results expand the understanding of the biophysical features of these enzymes, broadening the toolbox for metabolic engineering applications.
Topics: Crystallography, X-Ray; Phosphoenolpyruvate; Phosphopyruvate Hydratase; Synechococcus
PubMed: 35400670
DOI: 10.1107/S2053230X22003612 -
Frontiers in Microbiology 2022The strict human pathogen causes infections of varying severity, ranging from self-limiting suppurative infections to life-threatening diseases like necrotizing...
The strict human pathogen causes infections of varying severity, ranging from self-limiting suppurative infections to life-threatening diseases like necrotizing fasciitis or streptococcal toxic shock syndrome. Here, we show that the non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase GapN is an essential enzyme for . GapN converts glyceraldehyde 3-phosphate into 3-phosphoglycerate coupled to the reduction of NADP to NADPH. The knock-down of by antisense peptide nucleic acids (asPNA) significantly reduces viable bacterial counts of laboratory and macrolide-resistant clinical strains . As lacks the oxidative part of the pentose phosphate pathway, GapN appears to be the major NADPH source for the bacterium. Accordingly, other streptococci that carry a complete pentose phosphate pathway are not prone to asPNA-based knock-down. Determination of the crystal structure of the GapN apo-enzyme revealed an unusual cis-peptide in proximity to the catalytic binding site. Furthermore, using a structural modeling approach, we correctly predicted competitive inhibition of GapN by erythrose 4-phosphate, indicating that our structural model can be used for screening of specific GapN inhibitors. In conclusion, the data provided here reveal that GapN is a potential target for antimicrobial substances that selectively kill and other streptococci that lack the oxidative part of the pentose phosphate pathway.
PubMed: 35242116
DOI: 10.3389/fmicb.2022.802427 -
Redox Biology May 2022Pathologies associated with tissue ischemia/reperfusion (I/R) in highly metabolizing organs such as the brain and heart are leading causes of death and disability in...
Pathologies associated with tissue ischemia/reperfusion (I/R) in highly metabolizing organs such as the brain and heart are leading causes of death and disability in humans. Molecular mechanisms underlying mitochondrial dysfunction during acute injury in I/R are tissue-specific, but their details are not completely understood. A metabolic shift and accumulation of substrates of reverse electron transfer (RET) such as succinate are observed in tissue ischemia, making mitochondrial complex I of the respiratory chain (NADH:ubiquinone oxidoreductase) the most vulnerable enzyme to the following reperfusion. It has been shown that brain complex I is predisposed to losing its flavin mononucleotide (FMN) cofactor when maintained in the reduced state in conditions of RET both in vitro and in vivo. Here we investigated the process of redox-dependent dissociation of FMN from mitochondrial complex I in brain and heart mitochondria. In contrast to the brain enzyme, cardiac complex I does not lose FMN when reduced in RET conditions. We proposed that the different kinetics of FMN loss during RET is due to the presence of brain-specific long 50 kDa isoform of the NDUFV3 subunit of complex I, which is absent in the heart where only the canonical 10 kDa short isoform is found. Our simulation studies suggest that the long NDUFV3 isoform can reach toward the FMN binding pocket and affect the nucleotide affinity to the apoenzyme. For the first time, we demonstrated a potential functional role of tissue-specific isoforms of complex I, providing the distinct molecular mechanism of I/R-induced mitochondrial impairment in cardiac and cerebral tissues. By combining functional studies of intact complex I and molecular structure simulations, we defined the critical difference between the brain and heart enzyme and suggested insights into the redox-dependent inactivation mechanisms of complex I during I/R injury in both tissues.
Topics: Brain; Dinitrocresols; Electron Transport Complex I; Flavin Mononucleotide; Heart; Humans; Ischemia; Mitochondria, Heart; Oxidation-Reduction
PubMed: 35189550
DOI: 10.1016/j.redox.2022.102258 -
International Journal of Molecular... Jan 2022Eukarya pyruvate kinases possess glutamate at position 117 (numbering of rabbit muscle enzyme), whereas bacteria have either glutamate or lysine. Those with E117 are...
Eukarya pyruvate kinases possess glutamate at position 117 (numbering of rabbit muscle enzyme), whereas bacteria have either glutamate or lysine. Those with E117 are K-dependent, whereas those with K117 are K-independent. In a phylogenetic tree, 80% of the sequences with E117 are occupied by T113/K114/T120 and 77% of those with K117 possess L113/Q114/(L,I,V)120. This work aims to understand these residues' contribution to the K-independent pyruvate kinases using the K-dependent rabbit muscle enzyme. Residues 117 and 120 are crucial in the differences between the K-dependent and -independent mutants. K-independent activity increased with L113 and Q114 to K117, but L120 induced structural differences that inactivated the enzyme. T120 appears to be key in folding the protein and closure of the lid of the active site to acquire its active conformation in the K-dependent enzymes. E117K mutant was K-independent and the enzyme acquired the active conformation by a different mechanism. In the K-independent apoenzyme of , K72 (K117) flips out of the active site; in the holoenzyme, K72 faces toward the active site bridging the substrates through water molecules. The results provide evidence that two different mechanisms have evolved for the catalysis of this reaction.
Topics: Amino Acid Sequence; Animals; Apoenzymes; Binding Sites; Catalysis; Catalytic Domain; Glutamic Acid; Lysine; Models, Molecular; Mycobacterium tuberculosis; Phylogeny; Potassium; Protein Conformation; Pyruvate Kinase; Rabbits
PubMed: 35163274
DOI: 10.3390/ijms23031347 -
Nucleic Acids Research Dec 2021G-quadruplex (G4)/hemin DNAzyme is promising horseradish peroxidase (HRP)-mimic candidate in the biological field. However, its relatively unsatisfactory catalytic...
G-quadruplex (G4)/hemin DNAzyme is promising horseradish peroxidase (HRP)-mimic candidate in the biological field. However, its relatively unsatisfactory catalytic capacity limits the potential applications. Inspired by nature protease, we conducted a proximity-enhanced cofactor assembly strategy (PECA) to form an exceptional HRP mimic, namely zippered G4/hemin DNAzyme (Z-G4/H). The hybridization of short oligonucleotides induced proximity assembly of the DNA-grafted hemin (DGH) with the complementary G4 sequences (cG4s), mimicking the tight configuration of protease cofactor and apoenzyme. The detailed investigations of catalytic efficiency and mechanism verified the higher activity, more rapid catalytic rate and high environmental tolerance of the Z-G4/H than the classical G4/hemin DNAzymes (C-G4/H). Furthermore, a proximity recognition transducer has been developed based on the PECA for sensitive detection of gene rearrangement and imaging human epidermal growth factor receptor 2 protein (HER2) dimerization on cell surfaces. Our studies demonstrate the high efficiency of Z-G4/H and its universal application potential in clinical diagnostics and biomolecule interaction research. It also may offer significant opportunities and inspiration for the engineering of the protease-free mimic enzyme.
Topics: Biocatalysis; Cell Line, Tumor; Circular Dichroism; DNA, Catalytic; Enzyme Assays; Enzyme Stability; G-Quadruplexes; Hemin; Humans; Hydrogen-Ion Concentration; Kinetics; MCF-7 Cells; Molecular Structure; Mutation; Spectrophotometry; Temperature
PubMed: 34878146
DOI: 10.1093/nar/gkab1178 -
The Journal of Biological Chemistry Nov 2021Juvenile hormone (JH) acid methyltransferase (JHAMT) is a rate-limiting enzyme that converts JH acids or inactive precursors of JHs to active JHs at the final step of JH...
Juvenile hormone (JH) acid methyltransferase (JHAMT) is a rate-limiting enzyme that converts JH acids or inactive precursors of JHs to active JHs at the final step of JH biosynthesis in insects and thus presents an excellent target for the development of insect growth regulators or insecticides. However, the three-dimensional properties and catalytic mechanism of this enzyme are not known. Herein, we report the crystal structure of the JHAMT apoenzyme, the three-dimensional holoprotein in binary complex with its cofactor S-adenosyl-l-homocysteine, and the ternary complex with S-adenosyl-l-homocysteine and its substrate methyl farnesoate. These structures reveal the ultrafine definition of the binding patterns for JHAMT with its substrate/cofactor. Comparative structural analyses led to novel findings concerning the structural specificity of the progressive conformational changes required for binding interactions that are induced in the presence of cofactor and substrate. Importantly, structural and biochemical analyses enabled identification of one strictly conserved catalytic Gln/His pair within JHAMTs required for catalysis and further provide a molecular basis for substrate recognition and the catalytic mechanism of JHAMTs. These findings lay the foundation for the mechanistic understanding of JH biosynthesis by JHAMTs and provide a rational framework for the discovery and development of specific JHAMT inhibitors as insect growth regulators or insecticides.
Topics: Animals; Bombyx; Crystallography, X-Ray; Insect Proteins; Juvenile Hormones; Methyltransferases; Protein Domains
PubMed: 34562453
DOI: 10.1016/j.jbc.2021.101234 -
Nature Aug 2021Single-particle cryogenic electron microscopy (cryo-EM) has become a standard technique for determining protein structures at atomic resolution. However, cryo-EM studies...
Single-particle cryogenic electron microscopy (cryo-EM) has become a standard technique for determining protein structures at atomic resolution. However, cryo-EM studies of protein-free RNA are in their early days. The Tetrahymena thermophila group I self-splicing intron was the first ribozyme to be discovered and has been a prominent model system for the study of RNA catalysis and structure-function relationships, but its full structure remains unknown. Here we report cryo-EM structures of the full-length Tetrahymena ribozyme in substrate-free and bound states at a resolution of 3.1 Å. Newly resolved peripheral regions form two coaxially stacked helices; these are interconnected by two kissing loop pseudoknots that wrap around the catalytic core and include two previously unforeseen (to our knowledge) tertiary interactions. The global architecture is nearly identical in both states; only the internal guide sequence and guanosine binding site undergo a large conformational change and a localized shift, respectively, upon binding of RNA substrates. These results provide a long-sought structural view of a paradigmatic RNA enzyme and signal a new era for the cryo-EM-based study of structure-function relationships in ribozymes.
Topics: Apoenzymes; Cryoelectron Microscopy; Holoenzymes; Models, Molecular; Nucleic Acid Conformation; RNA, Catalytic; Tetrahymena thermophila
PubMed: 34381213
DOI: 10.1038/s41586-021-03803-w -
Nature Communications Aug 2021There is currently a lack of effective drugs to treat people infected with SARS-CoV-2, the cause of the global COVID-19 pandemic. The SARS-CoV-2 Non-structural protein...
There is currently a lack of effective drugs to treat people infected with SARS-CoV-2, the cause of the global COVID-19 pandemic. The SARS-CoV-2 Non-structural protein 13 (NSP13) has been identified as a target for anti-virals due to its high sequence conservation and essential role in viral replication. Structural analysis reveals two "druggable" pockets on NSP13 that are among the most conserved sites in the entire SARS-CoV-2 proteome. Here we present crystal structures of SARS-CoV-2 NSP13 solved in the APO form and in the presence of both phosphate and a non-hydrolysable ATP analog. Comparisons of these structures reveal details of conformational changes that provide insights into the helicase mechanism and possible modes of inhibition. To identify starting points for drug development we have performed a crystallographic fragment screen against NSP13. The screen reveals 65 fragment hits across 52 datasets opening the way to structure guided development of novel antiviral agents.
Topics: Adenosine Triphosphate; Amino Acid Sequence; Apoenzymes; Binding Sites; Crystallography, X-Ray; Drug Design; Enzyme Inhibitors; Methyltransferases; Models, Molecular; Phosphates; Protein Conformation; RNA Helicases; RNA, Viral; SARS-CoV-2; Structure-Activity Relationship; Viral Nonstructural Proteins
PubMed: 34381037
DOI: 10.1038/s41467-021-25166-6 -
Acta Crystallographica. Section F,... Aug 2021In many prokaryotes, the first step of threonine metabolism is catalysed by the enzyme threonine dehydrogenase (TDH), which uses NAD to oxidize its substrate to...
In many prokaryotes, the first step of threonine metabolism is catalysed by the enzyme threonine dehydrogenase (TDH), which uses NAD to oxidize its substrate to 2-amino-3-ketobutyrate. The absence of a functional TDH gene in humans suggests that inhibitors of this enzyme may have therapeutic potential against pathogens which are reliant on this enzyme. Here, TDH from Clostridium difficile has been cloned and overexpressed, and the X-ray structure of the apoenzyme form has been determined at 2.6 Å resolution.
Topics: Alcohol Oxidoreductases; Amino Acid Sequence; Clostridioides difficile; Cross Infection; Crystallography, X-Ray; Humans; Protein Structure, Secondary; Protein Structure, Tertiary; X-Ray Diffraction
PubMed: 34341193
DOI: 10.1107/S2053230X21007135