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Accounts of Chemical Research Nov 2017Nitrogenase is known for its remarkable ability to catalyze the reduction of N to NH, and C substrates to short-chain hydrocarbon products, under ambient conditions. The...
Nitrogenase is known for its remarkable ability to catalyze the reduction of N to NH, and C substrates to short-chain hydrocarbon products, under ambient conditions. The best-studied Mo-nitrogenase utilizes a complex metallocofactor as the site of substrate binding and reduction. Designated the M-cluster, this [MoFeSC(R-homocitrate)] cluster can be viewed as [MoFeS] and [FeS] subclusters bridged by three μ-sulfides and one μ-interstitial carbide, with its Mo end further coordinated by an R-homocitrate moiety. The unique cofactor has attracted considerable attention ever since its discovery; however, the complexity of its structure has hindered mechanistic understanding and chemical synthesis of this cofactor. Motivated by the pressing questions related to the structure and function of the nitrogenase cofactor, one major thrust of our research has been to unravel the key biosynthetic steps of this metallocluster to cultivate a deeper understanding of these reactions and their effects on functionalizing the cofactor. In this Account, we will discuss our recent work that provides insights into how simple Fe and S atoms, along with a single C atom, a heterometallic Mo atom and an organic homocitrate entity, are assembled into one of the most complex metalloclusters known in Nature. Combined biochemical, spectroscopic and structural studies have led us to a working model of M-cluster assembly, which starts with the sequential synthesis of small [FeS] and [FeS] units by NifS/U, followed by the coupling and rearrangement of two [FeS] clusters on NifB concomitant with the insertion of an interstitial carbide and a "9th sulfur" that give rise to a [FeSC] core that is nearly indistinguishable in structure to the M-cluster except for the absence of Mo and homocitrate. This 8Fe core is then matured into an M-cluster on NifEN upon substitution of a Mo-homocitrate conjugate for one terminal Fe atom of the cluster prior to transfer of the M-cluster to its target binding site in the catalytic component of Mo-nitrogenase. Taking stock of the elemental inventory during the cofactor assembly process, the core Fe and S atoms are derived from modular fusion of FeS building blocks, going through 2Fe, 4Fe and 8Fe stages to generate an 8Fe core of the cofactor. However, such a flow of Fe/S along the biosynthetic pathway of the M-cluster is "intervened" by the insertion of C and Mo, which renders the cofactor unique in structure and reactivity. Insertion of C occurs through a novel, radical SAM-dependent mechanism, which involves SN2-type methyl transfer from SAM to a [FeS] cluster pair, hydrogen abstraction of the transferred methyl group by a SAM-derived 5'-dA· radical, and further deprotonation of the resultant methylene radical concomitant with radical chemistry-based coupling and rearrangement of the [FeS] cluster pair into an [FeSC] core. Insertion of Mo, on the other hand, employs an ATPase-dependent mechanism that parallels metal trafficking in the biosynthesis of molybdopterin and CO dehydrogenase cofactors. These findings provide a nice framework for further exploration of the "black box" of nitrogenase cofactor assembly and function.
Topics: Coenzymes; Metalloproteins; Molybdenum Cofactors; Nitrogenase; Pteridines
PubMed: 29064664
DOI: 10.1021/acs.accounts.7b00417 -
Journal of the American Chemical Society Dec 2022Phosphine ligands are the most important class of ligands for cross-coupling reactions due to their unique electronic and steric properties. However, metalloproteins...
Phosphine ligands are the most important class of ligands for cross-coupling reactions due to their unique electronic and steric properties. However, metalloproteins generally rely on nitrogen, sulfur, or oxygen ligands. Here, we report the genetic incorporation of P3BF, which contains a biocompatible borane-protected phosphine, into proteins. This step is followed by a straightforward one-pot strategy to perform deboronation and palladium coordination in aqueous and aerobic conditions. The genetically encoded phosphine ligand P3BF should significantly expand our ability to design functional metalloproteins.
Topics: Metalloproteins; Ligands; Phosphines; Palladium
PubMed: 36417425
DOI: 10.1021/jacs.2c09683 -
Nucleic Acids Research Jan 2023Metalloenzymes are attractive research targets in fields of chemistry, biology, and medicine. Given that metalloenzymes can manifest conservation of metal-coordination...
Metalloenzymes are attractive research targets in fields of chemistry, biology, and medicine. Given that metalloenzymes can manifest conservation of metal-coordination and ligand binding modes, the excavation and expansion of metalloenzyme-specific knowledge is of interest in bridging metalloenzyme-related fields. Building on our previous metalloenzyme-ligand association database, MeLAD, we have expanded the scope of metalloenzyme-specific knowledge and services, by forming a versatile platform, termed the Metalloenzyme Data Bank and Analysis (MeDBA). The MeDBA provides: (i) manual curation of metalloenzymes into different categories, that this M-I, M-II and M-III; (ii) comprehensive information on metalloenzyme activities, expression profiles, family and disease links; (iii) structural information on metalloenzymes, in particular metal binding modes; (iv) metalloenzyme substrates and bioactive molecules acting on metalloenzymes; (v) excavated metal-binding pharmacophores and (vi) analysis tools for structure/metal active site comparison and metalloenzyme profiling. The MeDBA is freely available at https://medba.ddtmlab.org.
Topics: Catalytic Domain; Ligands; Metalloproteins; Metals; Enzymes; Databases, Protein
PubMed: 36243971
DOI: 10.1093/nar/gkac860 -
Bioscience Reports Apr 2017Metal ions play pivotal roles in protein structure, function and stability. The functional and structural diversity of proteins in nature expanded with the incorporation... (Review)
Review
Metal ions play pivotal roles in protein structure, function and stability. The functional and structural diversity of proteins in nature expanded with the incorporation of metal ions or clusters in proteins. Approximately one-third of these proteins in the databases contain metal ions. Many biological and chemical processes in nature involve metal ion-binding proteins, aka metalloproteins. Many cellular reactions that underpin life require metalloproteins. Most of the remarkable, complex chemical transformations are catalysed by metalloenzymes. Realization of the importance of metal-binding sites in a variety of cellular events led to the advancement of various computational methods for their prediction and characterization. Furthermore, as structural and functional knowledgebase about metalloproteins is expanding with advances in computational and experimental fields, the focus of the research is now shifting towards design and redesign of metalloproteins to extend nature's own diversity beyond its limits. In this review, we will focus on the computational toolbox for prediction of metal ion-binding sites, metalloprotein design and redesign. We will also give examples of tailor-made artificial metalloproteins designed with the computational toolbox.
Topics: Amino Acid Motifs; Binding Sites; Computational Biology; Databases, Protein; Humans; Metalloproteins; Metals; Models, Molecular; Protein Engineering
PubMed: 28167677
DOI: 10.1042/BSR20160179 -
Journal Francais D'ophtalmologie Mar 2022Central serous chorioretinopathy (CSCR) is an eye disease of unknown etiology that presents with reduced visual acuity, choroidal thickening (distance between Bruch's...
PURPOSE
Central serous chorioretinopathy (CSCR) is an eye disease of unknown etiology that presents with reduced visual acuity, choroidal thickening (distance between Bruch's membrane and the chorioscleral border), and subretinal fluid leakage. In the present study, the goal was to investigate the role of the interrelated tenascin C, metalloprotein-1, BAX, BCL2, subfatin and asprosin molecules in the pathogenesis of CSCR.
METHOD
Thirty CSCR patients and 30 controls were included. CSCR was diagnosed by optical coherence tomography imaging. A 5mL blood sample was collected from all participants after overnight fasting. Compounds in the blood samples were studied with the Enzyme-Linked Immunosorbent Assay (ELISA) method.
RESULTS
Patients with CSCR were found to have macular thickening (P: 0.08) and statistically significantly reduced visual acuity (P: 0.034) compared to controls. With regard to serum parameters, there were statistically significant increases in tenascin C, metalloprotein-1, BAX, BCL2, subfatin and asprosin levels compared to controls. We found a positive correlation between macular thickness and tenascin C (r+0.670, P<0.001), metaloprotein-1 (r+0.714, P<0.001), BAX, BCL2 (r+0.771, P<0.001), subfatin and asprosin levels and a negative correlation between visual acuity and tenascin C (r+0.605 P<0.001), metaloprotein-1 (r+0.704, P<0.001), BAX, BCL2 (r+0.738, P<0.001), subfatin and asprosin levels.
CONCLUSION
The molecules studied herein were negatively correlated with visual acuity and positively correlated with macular thickness, suggesting that these molecules might have a role in the pathogenesis of CSCR. Thus, we predict that these molecules could be new candidates for the diagnosis and follow-up of CSCR in the future.
Topics: Central Serous Chorioretinopathy; Fluorescein Angiography; Humans; Laboratories; Metalloproteins; Proto-Oncogene Proteins c-bcl-2; Retrospective Studies; Tenascin; Tomography, Optical Coherence; bcl-2-Associated X Protein
PubMed: 35123814
DOI: 10.1016/j.jfo.2021.09.011 -
Archives of Biochemistry and Biophysics Sep 2015In this contribution, recent developments in the design of biocatalysts are reviewed with particular emphasis in the de novo strategy. Studies based on three different... (Review)
Review
In this contribution, recent developments in the design of biocatalysts are reviewed with particular emphasis in the de novo strategy. Studies based on three different reactions, Kemp elimination, Diels-Alder and Retro-Aldolase, are used to illustrate different success achieved during the last years. Finally, a section is devoted to the particular case of designed metalloenzymes. As a general conclusion, the interplay between new and more sophisticated engineering protocols and computational methods, based on molecular dynamics simulations with Quantum Mechanics/Molecular Mechanics potentials and fully flexible models, seems to constitute the bed rock for present and future successful design strategies.
Topics: Computer-Aided Design; Drug Design; Enzymes; Metalloproteins; Molecular Dynamics Simulation
PubMed: 25797438
DOI: 10.1016/j.abb.2015.03.013 -
Journal of Inorganic Biochemistry Jun 2021Artificial metalloenzymes (ArMs) consist of an unnatural metal or cofactor embedded in a protein scaffold, and are an excellent platform for applying the concepts of... (Review)
Review
Artificial metalloenzymes (ArMs) consist of an unnatural metal or cofactor embedded in a protein scaffold, and are an excellent platform for applying the concepts of protein engineering to catalysis. In this Focused Review, we describe the application of ArMs as simple, tunable artificial models of the active sites of complex natural metalloenzymes for small-molecule activation. In this sense, ArMs expand the strategies of synthetic model chemistry to protein-based supporting ligands with potential for participation from the second coordination sphere. We focus specifically on ArMs that are structural, spectroscopic, and functional models of enzymes for activation of small molecules like CO, CO, O, N, and NO, as well as production/consumption of H. These ArMs give insight into the identities and roles of metalloenzyme structural features within and near the cofactor. We give examples of ArM work relevant to hydrogenases, acetyl-coenzyme A synthase, superoxide dismutase, heme oxygenases, nitric oxide reductase, methyl-coenzyme M reductase, copper-O enzymes, and nitrogenases.
Topics: Catalysis; Catalytic Domain; Coordination Complexes; Hydrogenase; Ligands; Metalloproteins; Metals; Models, Theoretical; Nitrogenase; Oxidoreductases; Protein Engineering
PubMed: 33873051
DOI: 10.1016/j.jinorgbio.2021.111430 -
Accounts of Chemical Research Feb 2019Nature puts to use only a small fraction of metal ions in the periodic table. Yet, when incorporated into protein scaffolds, this limited set of metal ions carry out...
Nature puts to use only a small fraction of metal ions in the periodic table. Yet, when incorporated into protein scaffolds, this limited set of metal ions carry out innumerable cellular functions and execute essential biochemical transformations such as photochemical HO oxidation, O or CO reduction, and N fixation, highlighting the outsized importance of metalloproteins in biology. Not surprisingly, elucidating the intricate interplay between metal ions and protein structures has been the focus of extensive structural and mechanistic scrutiny over the last several decades. As a result of such top-down efforts, we have gained a reasonably detailed understanding of how metal ions shape protein structures and how protein structures in turn influence metal reactivity. It is fair to say that we now have some idea-and in some cases, a good idea-about how most known metalloproteins function and we possess enough insight to quickly assess the modus operandi of newly discovered ones. However, translating this knowledge into an ability to construct functional metalloproteins from scratch represents a challenge at a whole different level: it is one thing to know how an automobile works; it is another to build one. In our quest to build new metalloproteins, we have taken an original approach in which folded, monomeric proteins are used as ligands or synthons for building supramolecular complexes through metal-mediated self-assembly (MDPSA, Metal-Directed Protein Self-Assembly). The interfaces in the resulting protein superstructures are subsequently tailored with covalent, noncovalent, or additional metal-coordination interactions for stabilization and incorporation of new functionalities (MeTIR, Metal Templated Interface Redesign). In an earlier Account, we had described the proof-of-principle studies for MDPSA and MeTIR, using a four-helix bundle, heme protein cytochrome cb (cyt cb), as a model building block. By the end of those studies, we were able to demonstrate that a tetrameric, Zn-directed cyt cb complex (Zn:M1) could be stabilized through computationally prescribed noncovalent interactions inserted into the nascent protein-protein interfaces. In this Account, we first describe the rationale and motivation for our particular metalloprotein engineering strategy and a brief summary of our earlier work. We then describe the next steps in the "evolution" of bioinorganic complexity on the Zn:M1 scaffold, namely, (a) the generation of a self-standing protein assembly that can stably and selectively bind metal ions, (b) the creation of reactive metal centers within the protein assembly, and (c) the coupling of metal coordination and reactivity to external stimuli through allosteric effects.
Topics: Catalytic Domain; Cytochrome b Group; Esterases; Metalloproteins; Point Mutation; Protein Conformation; Protein Engineering; Zinc; beta-Lactamases
PubMed: 30698941
DOI: 10.1021/acs.accounts.8b00617 -
Nature Communications Nov 2022Metallohydrolases are ubiquitous in nearly all subclasses of hydrolases, utilizing metal elements to activate a water molecule and facilitate its subsequent dissociation...
Metallohydrolases are ubiquitous in nearly all subclasses of hydrolases, utilizing metal elements to activate a water molecule and facilitate its subsequent dissociation of diverse chemical bonds. However, such a catalytic role of metal ions is rarely found with glycosidases that hydrolyze the glycosidic bonds in sugars. Herein, we design metalloglycosidases by constructing a hydrolytically active Zn-binding site within a barrel-shaped outer membrane protein OmpF. Structure- and mechanism-based redesign and directed evolution have led to the emergence of Zn-dependent glycosidases with catalytic proficiency of 2.8 × 10 and high β-stereoselectivity. Biochemical characterizations suggest that the Zn-binding site constitutes a key catalytic motif along with at least one adjacent acidic residue. This work demonstrates that unprecedented metalloenzymes can be tailor-made, expanding the scope of inorganic reactivities in proteinaceous environments, resetting the structural and functional diversity of metalloenzymes, and providing the potential molecular basis of unidentified metallohydrolases and novel whole-cell biocatalysts.
Topics: Metalloproteins; Binding Sites; Catalytic Domain; Catalysis; Glycoside Hydrolases; Metals
PubMed: 36369431
DOI: 10.1038/s41467-022-34713-8 -
Current Opinion in Structural Biology Oct 2015Although the structure of enzymes and the chemistry at the catalytic sites have been studied intensively, an understanding of the atomic-scale chemistry requires a new... (Review)
Review
Although the structure of enzymes and the chemistry at the catalytic sites have been studied intensively, an understanding of the atomic-scale chemistry requires a new approach beyond steady state X-ray crystallography and X-ray spectroscopy at cryogenic temperatures. Following the dynamic changes in the geometric and electronic structure of metallo-enzymes at ambient conditions, while overcoming the severe X-ray-induced changes to the redox active catalytic center, is key for deriving reaction mechanisms. Such studies become possible by the intense and ultra-short femtosecond (fs) X-ray pulses from an X-ray free electron laser (XFEL) by acquiring a signal before the sample is destroyed. This review describes the recent and pioneering uses of XFELs to study the protein structure and dynamics of metallo-enzymes using crystallography and scattering, as well as the chemical structure and dynamics of the catalytic complexes (charge, spin, and covalency) using spectroscopy during the reaction to understand the electron-transfer processes and elucidate the mechanism.
Topics: Binding Sites; Crystallography, X-Ray; Lasers; Metalloproteins; Metals; Models, Molecular; Protein Binding; Protein Conformation; Spectrum Analysis; Temperature
PubMed: 26342144
DOI: 10.1016/j.sbi.2015.07.014