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Current Opinion in Biotechnology Dec 2013Cofactors provide redox carriers for biosynthetic reactions, catabolic reactions and act as important agents in transfer of energy for the cell. Recent advances in... (Review)
Review
Cofactors provide redox carriers for biosynthetic reactions, catabolic reactions and act as important agents in transfer of energy for the cell. Recent advances in manipulating cofactors include culture conditions or additive alterations, genetic modification of host pathways for increased availability of desired cofactor, changes in enzyme cofactor specificity, and introduction of novel redox partners to form effective circuits for biochemical processes and biocatalysts. Genetic strategies to employ ferredoxin, NADH and NADPH most effectively in natural or novel pathways have improved yield and efficiency of large-scale processes for fuels and chemicals and have been demonstrated with a variety of microbial organisms.
Topics: Biotechnology; Coenzymes; Ferredoxins; Metabolic Engineering; NAD; NADP; Oxidation-Reduction; Substrate Specificity
PubMed: 23611567
DOI: 10.1016/j.copbio.2013.03.022 -
Current Pharmaceutical Design 2013Xanthine oxidoreductase (XOR), a complex flavoprotein, catalyzes the metabolic reactions leading from hypoxanthine to xanthine and from xanthine to urate, and both... (Review)
Review
Xanthine oxidoreductase (XOR), a complex flavoprotein, catalyzes the metabolic reactions leading from hypoxanthine to xanthine and from xanthine to urate, and both reactions take place at the molybdenum cofactor. The enzyme is a target of drugs for therapy of gout or hyperuricemia. We review the chemical nature and reaction mechanisms of the molybdenum cofactor of XOR, focusing on molybdenum-dependent reactions of actual or potential medical importance, including nitric oxide (NO) synthesis. It is now generally accepted that XOR transfers the water-exchangeable -OH ligand of the molybdenum atom to the substrate. The hydroxyl group at OH-Mo(IV) can be replaced by urate, oxipurinol and FYX-051 derivatives and the structures of these complexes have been determined by xray crystallography under anaerobic conditions. Although formation of NO from nitrite or formation of xanthine from urate by XOR ischemically feasible, it is not yet clear whether these reactions have any physiological significance since the reactions are catalyzed at a slow rate even under anaerobic conditions.
Topics: Animals; Coenzymes; Drug Discovery; Electron Transport; Humans; Hydrogen Bonding; Hydroxylation; Metalloproteins; Models, Molecular; Molybdenum Cofactors; Nitric Oxide; Oxidation-Reduction; Protein Binding; Pteridines; Substrate Specificity; Uric Acid; Xanthine; Xanthine Dehydrogenase
PubMed: 23116398
DOI: 10.2174/1381612811319140010 -
Journal of the American Chemical Society Mar 2023Semi-artificial approaches to solar-to-chemical conversion can achieve chemical transformations that are beyond the capability of natural enzymes, but face marked...
Semi-artificial approaches to solar-to-chemical conversion can achieve chemical transformations that are beyond the capability of natural enzymes, but face marked challenges to facilitate cascades, due to their inevitable need for cofactor shuttling and regeneration. Here, we report on an enzyme grafting strategy to build a metal-organic capsule-docking artificial enzyme (metal-organic-enzyme, MOE) that comprised the self-assembly of a cofactor-decorated capsule and the supramolecular enzyme-recognition features between the enzyme scaffold and the capsule to bypass cofactor shuttling and regeneration. The incorporated NADH mimics within the metal-organic capsule interacted with the imine intermediate that formed from the condensation of the amines and the dehydrogenation of alcohol substrates in the microenvironment to form complexes within the capsule and subsequently served as an -generated photoresponsive cofactor. Upon illumination, the photoresponsive cofactor facilitates efficient proton/electron transport between the inner space (supramolecular hydrogenation) and outer space (enzymatic dehydrogenation) of the capsule to dehydrogenize the alcohols and hydrogenize the imine intermediates, respectively, circumventing the conventionally complex multistep cofactor shuttling and regeneration. The semi-artificial enzyme endows the conversion of diverse types of alcohol to amine products in both aqueous/organic solutions and with high efficiency, offering a wide range of opportunities for sustainable and environmentally friendly biomanufacturing of commodity and fine chemicals.
Topics: Electron Transport; Metals; Alcohols; Escherichia coli; Amines; Imines
PubMed: 36916689
DOI: 10.1021/jacs.2c12636 -
Angewandte Chemie (International Ed. in... Jul 2022Cofactors are required for almost half of all enzyme reactions, but their functions and binding partners are not fully understood even after decades of research.... (Review)
Review
Cofactors are required for almost half of all enzyme reactions, but their functions and binding partners are not fully understood even after decades of research. Functionalised cofactor mimics that bind in place of the unmodified cofactor can provide answers, as well as expand the scope of cofactor activity. Through chemical proteomics approaches such as activity-based protein profiling, the interactome and localisation of the native cofactor in its physiological environment can be deciphered and previously uncharacterised proteins annotated. Furthermore, cofactors that supply functional groups to substrate biomolecules can be hijacked by mimics to site-specifically label targets and unravel the complex biology of post-translational protein modification. The diverse activity of cofactors has inspired the design of mimics for use as inhibitors, antibiotic therapeutics, and chemo- and biosensors, and cofactor conjugates have enabled the generation of novel enzymes and artificial DNAzymes.
Topics: DNA, Catalytic; Proteomics
PubMed: 35286003
DOI: 10.1002/anie.202201136 -
Methods in Molecular Biology (Clifton,... 2009Cofactors are organic molecules, most of them originating from vitamins, that bind to enzymes making them able to catalyze defined reactions. A cofactor-based... (Review)
Review
Cofactors are organic molecules, most of them originating from vitamins, that bind to enzymes making them able to catalyze defined reactions. A cofactor-based chemogenomics approach exploits the presence of a cofactor-binding domain to develop compound scaffolds tailored to mimic the cofactor and to replace it within target enzyme classes. As a result, a loss of function is observed. An expansion of the cofactor scaffold to include structural/chemical features derived from the substrate, that usually binds at cofactor adjacent sites, increases the specificity of the enzyme fishing. This approach has been so far applied only to NAD(P)(+)-dependent enzymes. However, it is suitable for all other cofactors, with difficulties, for some of them, originated by very tight binding. In the case of cofactors covalently bound to the enzyme, the competition between the natural cofactor and the cofactor scaffold mimic can only occur during enzyme folding.
Topics: Binding Sites; Coenzymes; Drug Design; Drug Discovery; Enzymes; Genomics; Models, Molecular; Molecular Biology; Protein Binding; Protein Conformation
PubMed: 19727612
DOI: 10.1007/978-1-60761-274-2_4 -
Applied Microbiology and Biotechnology Apr 2010Whereas the majority of O(2)-metabolizing enzymes depend on transition metal ions or organic cofactors for catalysis, a significant number of oxygenases and oxidases... (Review)
Review
Whereas the majority of O(2)-metabolizing enzymes depend on transition metal ions or organic cofactors for catalysis, a significant number of oxygenases and oxidases neither contain nor require any cofactor. Among the cofactor-independent oxidases, urate oxidase, coproporphyrinogen oxidase, and formylglycine-generating enzyme are of mechanistic as well as medical interest. Formylglycine-generating enzyme is also a promising tool for protein engineering as it can be used to equip proteins with a reactive aldehyde function. PqqC, an oxidase in the biosynthesis of the bacterial cofactor pyrroloquinoline quinone, catalyzes an eight-electron ring-closure oxidation reaction. Among bacterial oxygenases, quinone-forming monooxygenases involved in the tailoring of polyketides, the dioxygenase DpgC found in the biosynthesis of a building block of vancomycin and teicoplanin antibiotics, luciferase monooxygenase from Renilla sp., and bacterial ring-cleaving 2,4-dioxygenases active towards 3-hydroxy-4(1H)-quinolones have been identified as cofactor-independent enzymes. Interestingly, the 3-hydroxy-4(1H)-quinolone 2,4-dioxygenases as well as Renilla luciferase use an alpha/beta-hydrolase architecture for oxygenation reactions. Cofactor-independent oxygenases and oxidases catalyze very different reactions and belong to several different protein families, reflecting their diverse origin. Nevertheless, they all may share the common mechanistic concept of initial base-catalyzed activation of their organic substrate and "substrate-assisted catalysis".
Topics: Coenzymes; Metabolic Networks and Pathways; Models, Chemical; Models, Molecular; Oxidoreductases; Oxygen
PubMed: 20157809
DOI: 10.1007/s00253-010-2455-0 -
Biotechnology Advances Dec 2017Cofactors are involved in numerous intracellular reactions and critically influence redox balance and cellular metabolism. Cofactor engineering can support and promote... (Review)
Review
Cofactors are involved in numerous intracellular reactions and critically influence redox balance and cellular metabolism. Cofactor engineering can support and promote the biocatalysis process, even help driving thermodynamically unfavorable reactions forwards. To achieve efficient production of chemicals and biofuels, cofactor engineering strategies such as altering cofactor supply or modifying reactants' cofactor preference have been developed to maintain redox balance. This review focuses primarily on the effects of cofactor engineering on carbon and energy metabolism. Coupling carbon metabolism with cofactor engineering can promote large-scale production, and even offer possibilities for producing new products or converting new materials.
Topics: Biofuels; Carbon; Coenzymes; Cyanobacteria; Energy Metabolism; Metabolic Engineering; Synthetic Biology
PubMed: 28939499
DOI: 10.1016/j.biotechadv.2017.09.008 -
Methods in Molecular Biology (Clifton,... 2022Noncanonical redox cofactor systems utilize nicotinamide adenine dinucleotide (phosphate), NAD(P)H, mimics to perform biotransformation reactions. Compared to systems...
Noncanonical redox cofactor systems utilize nicotinamide adenine dinucleotide (phosphate), NAD(P)H, mimics to perform biotransformation reactions. Compared to systems utilizing native NAD(P)H, these noncanonical redox cofactors can offer decreased cost of cofactor supply, improved system activities, and can even supply reducing power directly to targeted reactions in complex biological environments. When these systems are operated in cell-free settings, the high level of user control afforded by direct access to the reaction system enables specific tuning of cofactor parameters, enzyme activity, and reaction progression to maximize system productivity. In this chapter, we will describe methods for constructing these cell-free noncanonical redox cofactor systems. Specifically, methods, design concepts, and system adaptation will be discussed for applying noncanonical redox cofactors to both purified protein-based and crude lysate-based biotransformation systems.
Topics: NAD; NADP; Oxidation-Reduction
PubMed: 34985745
DOI: 10.1007/978-1-0716-1998-8_11 -
The FEBS Journal Sep 2023Formate dehydrogenases (Fdhs) mediate the oxidation of formate to carbon dioxide and concomitant reduction of nicotinamide adenine dinucleotide (NAD ). The low cost of...
Formate dehydrogenases (Fdhs) mediate the oxidation of formate to carbon dioxide and concomitant reduction of nicotinamide adenine dinucleotide (NAD ). The low cost of the substrate formate and importance of the product NADH as a cellular source of reducing power make this reaction attractive for biotechnological applications. However, the majority of Fdhs are sensitive to inactivation by thiol-modifying reagents. In this study, we report a chemically resistant Fdh (Fdh ) from the soil bacterium Starkeya novella strictly specific for NAD . We present its recombinant overproduction, purification and biochemical characterization. The mechanistic basis of chemical resistance was found to be a valine in position 255 (rather than a cysteine as in other Fdhs) preventing the inactivation by thiol-modifying compounds. To further improve the usefulness of Fdh as for generating reducing power, we rationally engineered the protein to reduce the coenzyme nicotinamide adenine dinucleotide phosphate (NADP ) with better catalytic efficiency than NAD . The single mutation D221Q enabled the reduction of NADP with a catalytic efficiency k /K of 0.4 s ·mm at 200 mm formate, while a quadruple mutant (A198G/D221Q/H379K/S380V) resulted in a fivefold increase in catalytic efficiency for NADP compared with the single mutant. We determined the cofactor-bound structure of the quadruple mutant to gain mechanistic evidence behind the improved specificity for NADP . Our efforts to unravel the key residues for the chemical resistance and cofactor specificity of Fdh may lead to wider use of this enzymatic group in a more sustainable (bio)manufacture of value-added chemicals, as for instance the biosynthesis of chiral compounds.
Topics: NAD; Formate Dehydrogenases; NADP; Formates; Sulfhydryl Compounds
PubMed: 37213112
DOI: 10.1111/febs.16871 -
Current Opinion in Biotechnology Feb 2022Nicotinamide adenine dinucleotide(NAD(P)H) is a metabolically interconnected redox cofactor serving as a hydride source for the majority of oxidoreductases, and... (Review)
Review
Nicotinamide adenine dinucleotide(NAD(P)H) is a metabolically interconnected redox cofactor serving as a hydride source for the majority of oxidoreductases, and consequently constituting a significant cost factor for bioprocessing. Much research has been devoted to the development of efficient, affordable, and sustainable methods for the regeneration of these cofactors through chemical, electrochemical, and photochemical approaches. However, the enzymatic approach using formate dehydrogenase is still the most abundantly employed in industrial applications, even though it suffers from system complexity and product purity issues. In this review, we summarize non-enzymatic and enzymatic electrochemical approaches for cofactor regeneration, then discuss recent developments to solve major issues. Issues discussed include Rh-catalyst mediated enzyme mutual inactivation, electron-transfer rates, catalyst sustainability, product selectivity and simplifying product purification. Recently reported remedies are discussed, such as heterogeneous metal catalysts generating H as the sole byproduct or high activity and stability redox-polymer immobilized enzymatic systems for sustainable organic synthesis.
Topics: Catalysis; Electron Transport; NAD; Oxidation-Reduction
PubMed: 34246871
DOI: 10.1016/j.copbio.2021.06.013