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Angewandte Chemie (International Ed. in... 2009Many enzymes contain a nondiffusible organic cofactor, often termed a prosthetic group, which is located in the active site and essential for the catalytic activity of... (Review)
Review
Many enzymes contain a nondiffusible organic cofactor, often termed a prosthetic group, which is located in the active site and essential for the catalytic activity of the enzyme. These cofactors can often be extracted from the protein to yield the respective apoenzyme, which can subsequently be reconstituted with an artificial analogue of the native cofactor. Nowadays a large variety of synthetic cofactors can be used for the reconstitution of apoenzymes and, thus, generate novel semisynthetic enzymes. This approach has been refined over the past decades to become a versatile tool of structural enzymology to elucidate structure-function relationships of enzymes. Moreover, the reconstitution of apoenzymes can also be used to generate enzymes possessing enhanced or even entirely new functionality. This Review gives an overview on historical developments and the current state-of-the-art on apoenzyme reconstitution.
Topics: Apoenzymes; Biotechnology; Flavin-Adenine Dinucleotide; Heme; PQQ Cofactor; Protein Engineering; Structure-Activity Relationship
PubMed: 19165853
DOI: 10.1002/anie.200803098 -
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 -
Journal of Molecular Modeling Nov 2018Low-temperature methane oxidation is one of the greatest challenges in energy research. Although methane monooxygenase (MMO) does this catalysis naturally, how to use...
Low-temperature methane oxidation is one of the greatest challenges in energy research. Although methane monooxygenase (MMO) does this catalysis naturally, how to use this biocatalyst in a fuel cell environment where the electrons generated during the oxidation process is harvested and used for energy generation has not yet been investigated. A key requirement to use this enzyme in a fuel cell is wiring of the active site of the enzyme directly to the supporting electrode. In soluble MMO (sMMO), two cofactors, i.e., nicotinamide adenine di-nucleotide (NAD+) and flavin adenine dinucleotide (FAD) provide opportunities for direct attachment of the enzyme system to a supporting electrode. However, once modified to be compatible with a supporting metal electrode via FeS functionalization, how the two cofactors respond to complex binding phenomena is not yet understood. Using docking and molecular dynamic simulations, modified cofactors interactions with sMMO-reductase (sMMOR) were studied. Studies revealed that FAD modification with FeS did not interfere with binding phenomena. In fact, FeS introduction significantly improved the binding affinity of FAD and NAD+ on sMMOR. The simulations revealed a clear thermodynamically more favorable electron transport path for the enzyme system. This system can be used as a fuel cell and we can use FeS-modified-FAD as the anchoring molecule as opposed to using NAD+. The overall analysis suggests the strong possibility of building a fuel cell that could catalyze methane oxidation using sMMO as the anode biocatalyst.
Topics: Apoenzymes; Bacterial Proteins; Biocatalysis; Catalytic Domain; Coenzymes; Computational Biology; Electron Transport; Methane; Methylococcus capsulatus; Molecular Docking Simulation; Molecular Dynamics Simulation; Oxygenases; Protein Binding; Protein Domains; Protein Engineering; Reproducibility of Results; Substrate Specificity
PubMed: 30498917
DOI: 10.1007/s00894-018-3876-4 -
The Journal of Biological Chemistry Jan 1984The apoenzyme of Pseudomonas cepacia salicylate hydroxylase was prepared by a dialysis method. The apoprotein retains a dimeric structure and binds one FAD per monomer....
The apoenzyme of Pseudomonas cepacia salicylate hydroxylase was prepared by a dialysis method. The apoprotein retains a dimeric structure and binds one FAD per monomer. Flavin binding results in both 81 and 60% of quenching and 15- and 5-nm blue shifts of FAD and protein fluorescence, respectively. A hydrophobic environment for the flavin site and a conformational difference between apoprotein and holoenzyme are thus indicated. Prior binding of NADH markedly retards the holoenzyme activity development upon a subsequent FAD addition. Flavin 1,N6-ethenoadenine dinucleotide binds to the apoenzyme much more weakly than FAD but this reconstituted holoenzyme and the FAD X enzyme both exhibit similar activities. The adenine moiety appears to be important to binding. The formation of holoenzyme from apoprotein and FAD involves minimally a two-step reversible process, an initial flavin-binding step followed by a conformational transition. At both 6 and 23 degrees C, the rates of hydroxylase activity recovery can be correlated with the rates of FAD binding, indicating that the initial FAD X apoenzyme complex is fully active and the subsequent slow conformational change has no significant effect on the catalytic efficiency. Overall dissociation constants calculated based on kinetic data are essentially identical with those determined by equilibrium measurements.
Topics: Apoenzymes; Apoproteins; Dialysis; Flavin-Adenine Dinucleotide; Macromolecular Substances; Mixed Function Oxygenases; NAD; Pseudomonas; Spectrometry, Fluorescence
PubMed: 6693380
DOI: No ID Found -
Biochemistry May 2000The type 4 cAMP-specific phosphodiesterases (PDE4s) are Mg(2+)-dependent hydrolases that catalyze the hydrolysis of 3', 5'-cAMP to AMP. Previous studies indicate that... (Comparative Study)
Comparative Study
The type 4 cAMP-specific phosphodiesterases (PDE4s) are Mg(2+)-dependent hydrolases that catalyze the hydrolysis of 3', 5'-cAMP to AMP. Previous studies indicate that PDE4 exists in two conformations that bind the inhibitor rolipram with affinities differing by more than 100-fold. Here we report that these two conformations are the consequence of PDE4 binding to its metal cofactor such as Mg(2+). Using a fluorescence resonance energy transfer (FRET)-based equilibrium binding assay, we identified that L-791,760, a fluorescent inhibitor, binds to the apoenzyme (free enzyme) and the holoenzyme (enzyme bound to Mg(2+)) with comparable affinities (K(d) approximately 30 nM). By measuring the displacement of the bound L-791,760, we have also identified that other inhibitors bind differentially with the apoenzyme and the holoenzyme depending upon their structure. CDP-840, SB-207499, and RP-73401 bind preferentially to the holoenzyme. The conformational-sensitive inhibitor (R)-rolipram binds to the holoenzyme and apoenzyme with affinities (K(d)) of 5 and 300 nM, respectively. In contrast to its high affinity (K(d) approximately 2 microM) and active holoenzyme complex, cAMP binds to the apoenzyme nonproductively with a reduced affinity (K(d) approximately 170 microM). These results demonstrate that cofactor binding to PDE4 is responsible for eliciting its high-affinity interaction with cAMP and the activation of catalysis.
Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Animals; Apoenzymes; Binding Sites; Cell Line; Cyclic AMP; Cyclic Nucleotide Phosphodiesterases, Type 4; Energy Transfer; Humans; Kinetics; Magnesium; Phosphodiesterase Inhibitors; Protein Conformation; Recombinant Proteins; Spectrometry, Fluorescence; Spodoptera; Structure-Activity Relationship; Transfection
PubMed: 10828959
DOI: 10.1021/bi992432w -
EXS 1994The structures of horse liver alcohol dehydrogenase class I in its apoenzyme form and in different ternary complexes have been determined at high resolution. The complex... (Review)
Review
The structures of horse liver alcohol dehydrogenase class I in its apoenzyme form and in different ternary complexes have been determined at high resolution. The complex with NAD+ and the substrate analogue pentafluorobenzyl alcohol gives a detailed picture of the interactions in an enzyme-substrate complex. The alcohol is bound to the zinc and positioned so that the hydrogen atom can be directly transferred to the C4 atom of the nicotinamide ring. The structure of cod liver alcohol dehydrogenase with hybrid properties (functionally of class I but structurally overall closer to class III) has been determined by molecular replacement methods to 3 A resolution. Yeast alcohol dehydrogenase has been crystallized, and native data have been collected to 3 A resolution.
Topics: Alcohol Dehydrogenase; Amino Acid Sequence; Animals; Apoenzymes; Benzyl Alcohols; Binding Sites; Crystallography, X-Ray; Fishes; Horses; Liver; Macromolecular Substances; Models, Molecular; NAD; Protein Structure, Secondary
PubMed: 8032158
DOI: 10.1007/978-3-0348-7330-7_27 -
FEBS Letters Oct 1991A simple procedure was devised for isolating from homogenates of mitotic cells the human homolog to the fission yeast cdc2 gene product. The identity of the purified...
A simple procedure was devised for isolating from homogenates of mitotic cells the human homolog to the fission yeast cdc2 gene product. The identity of the purified protein was established with anti-p34cdc2 antibodies and p13suc 1, both specific ligands for p34cdc2. Active-site labeling with oxidized [alpha 32P]ATP showed the purified molecule to be an ATP-binding protein. Its ability to phosphorylate casein but not histone, and its phosphorylation on tyrosine, detected by anti-phosphotyrosine antibodies, indicates the form of p34cdc2 purified is the inactive or apoenzyme form. Purified quantities of human p34cdc2 should be of considerable value in establishing the mechanism of its activation at mitosis by phosphatases.
Topics: Apoenzymes; CDC2 Protein Kinase; Casein Kinases; Cell Nucleus; Cells, Cultured; Humans; Mitosis; Molecular Weight; Protein Kinases
PubMed: 1936263
DOI: 10.1016/0014-5793(91)81281-c -
Methods in Enzymology 1979
Topics: Apoenzymes; Ascorbate Oxidase; Molecular Weight; Oxidoreductases; Plants; Protein Conformation; Substrate Specificity
PubMed: 440113
DOI: 10.1016/0076-6879(79)62185-7 -
Biochemistry Jan 1994Cyclobutane pyrimidine dimers (Pyr < > Pyr) are the major DNA photoproducts induced by the UV component of solar radiation. Photoreactivating enzyme (DNA photolyase)... (Review)
Review
Cyclobutane pyrimidine dimers (Pyr < > Pyr) are the major DNA photoproducts induced by the UV component of solar radiation. Photoreactivating enzyme (DNA photolyase) repairs DNA by utilizing the energy of visible light to break the cyclobutane ring of the dimer. Photolyases are monomeric proteins of 50-60 kDa with stoichiometric amounts of two noncovalent chromophore/cofactors. One of these cofactors is FADH-, and the second chromophore is either methenyltetrahydrofolate (MTHF) or 8-hydroxy-5-deazariboflavin (8-HDF). The enzyme binds the DNA substrate in a light-independent reaction, the second chromophore of the bound enzyme absorbs a visible photon and, by dipole-dipole interaction, transfers energy to FADH- which, in turn, transfers an electron to Pyr < > Pyr in DNA; the Pyr < > Pyr- splits and back electron transfer restores the dipyrimidine and the functional form of flavin ready for a new cycle of catalysis.
Topics: Apoenzymes; Bacteria; Coenzymes; DNA; DNA Repair; Deoxyribodipyrimidine Photo-Lyase; Electron Transport; Energy Transfer; Fungi; Kinetics; Pyrimidine Dimers; Substrate Specificity; Ultraviolet Rays
PubMed: 8286340
DOI: 10.1021/bi00167a001 -
Mutation Research Jun 2000The discovery of enzymatic photoreactivation and of photolyase produced a paradigm shift in the way investigators thought about the cellular consequences of DNA damage... (Review)
Review
The discovery of enzymatic photoreactivation and of photolyase produced a paradigm shift in the way investigators thought about the cellular consequences of DNA damage and about how these consequences could be avoided. The in vitro photoreactivation system, which utilized crude extracts from Saccharomyces cerevisiae as the source of photolyase, not only provided information about the mechanism of photoreactivation, but also played an important role in the discovery of nucleotide excision repair (NER) and the identification of the pyrimidine dimer as the primary lethal lesion induced by 254 nm radiation. More recently, mechanistic studies using homogenous purified yeast photolyase have yielded insight into how DNA repair enzymes recognize specific structures in DNA, while investigations looking at the repair of lesions in chromatin have begun to elucidate how DNA repair enzymes deal with damage in the context of eukaryotic chromosomes. Additionally, genetic and molecular studies of PHR1, the S. cerevisiae gene encoding the apoenzyme of photolyase, have led to the identification of previously unknown damage-responsive transcriptional regulators.
Topics: Apoenzymes; DNA Repair; Deoxyribodipyrimidine Photo-Lyase; Enzymes; Fungal Proteins; Membrane Glycoproteins; Photochemistry; Pyrimidine Dimers; Saccharomyces cerevisiae; Thermodynamics; Transcription, Genetic
PubMed: 10915863
DOI: 10.1016/s0027-5107(00)00038-5