Structural Enzymology of Nitrogenase Enzymes.
Ontology highlight
ABSTRACT:
SUBMITTER: Einsle O
PROVIDER: S-EPMC8606229 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Mix-and-inject serial crystallography (MISC) is a technique designed to image enzyme catalyzed reactions in which small protein crystals are mixed with a substrate just prior to being probed by an X-ray pulse. This approach offers several advantages over flow cell studies. It provides (i) room temperature structures at near atomic resolution, (ii) time resolution ranging from microseconds to seconds, and (iii) convenient reaction initiation. It outruns radiation damage by using femtosecond X-ray pulses allowing damage and chemistry to be separated. Here, we demonstrate that MISC is feasible at an X-ray free electron laser by studying the reaction of M. tuberculosis ß-lactamase microcrystals with ceftriaxone antibiotic solution. Electron density maps of the apo-ß-lactamase and of the ceftriaxone bound form were obtained at 2.8?Å and 2.4?Å resolution, respectively. These results pave the way to study cyclic and non-cyclic reactions and represent a new field of time-resolved structural dynamics for numerous substrate-triggered biological reactions.
| S-EPMC5178802 | biostudies-literature
Project description:Enzymes are remarkable catalysts that lie at the heart of biology, accelerating chemical reactions to an astounding extent with extraordinary specificity. Enormous progress in understanding the chemical basis of enzymatic transformations and the basic mechanisms underlying rate enhancements over the past decades is apparent. Nevertheless, it has been difficult to achieve a quantitative understanding of how the underlying mechanisms account for the energetics of catalysis, because of the complexity of enzyme systems and the absence of underlying energetic additivity. We review case studies from our own work that illustrate the power of precisely defined and clearly articulated questions when dealing with such complex and multifaceted systems, and we also use this approach to evaluate our current ability to design enzymes. We close by highlighting a series of questions that help frame some of what remains to be understood, and we encourage the reader to define additional questions and directions that will deepen and broaden our understanding of enzymes and their catalysis.
| S-EPMC3744632 | biostudies-literature
Project description:Three human mitochondrial diseases that directly affect lipoic acid metabolism result from heterozygous missense and nonsense mutations in the LIAS, LIPT1, and LIPT2 genes. However, the functions of the proteins encoded by these genes in lipoic acid metabolism remained uncertain due to a lack of biochemical analysis at the enzyme level. An exception was the LIPT1 protein for which a perplexing property had been reported, a ligase lacking the ability to activate its substrate. This led to several models, some contradictory, to accommodate the role of LIPT1 protein activity in explaining the phenotypes of the afflicted neonatal patients. Recent evidence indicates that this LIPT1 protein activity is a misleading evolutionary artifact and that the physiological role of LIPT1 is in transfer of lipoic acid moieties from one protein to another. This and other new biochemical data now define a straightforward pathway that fully explains each of the human disorders specific to the assembly of lipoic acid on its cognate enzyme proteins.
| S-EPMC7253636 | biostudies-literature
Project description:Nitrogenases are best known for catalyzing the reduction of dinitrogen to ammonia at a complex metallic cofactor. Recently, nitrogenases were shown to reduce carbon dioxide (CO2) and carbon monoxide to hydrocarbons, offering a pathway to recycle carbon waste into hydrocarbon products. Among the three nitrogenase isozymes, the iron nitrogenase has the highest wild-type activity for the reduction of CO2, but the molecular architecture facilitating these activities has remained unknown. Here, we report a 2.35-Å cryogenic electron microscopy structure of the ADP·AlF3-stabilized iron nitrogenase complex from Rhodobacter capsulatus, revealing an [Fe8S9C-(R)-homocitrate] cluster in the active site. The enzyme complex suggests that the iron nitrogenase G subunit is involved in cluster stabilization and substrate channeling and confers specificity between nitrogenase reductase and catalytic component proteins. Moreover, the structure highlights a different interface between the two catalytic halves of the iron and the molybdenum nitrogenase, potentially influencing the intrasubunit 'communication' and thus the nitrogenase mechanism.
| S-EPMC10803253 | biostudies-literature
Project description:The structural diversity and complexity of marine natural products have made them a rich and productive source of new bioactive molecules for drug development. The identification of these new compounds has led to extensive study of the protein constituents of the biosynthetic pathways from the producing microbes. Essential processes in the dissection of biosynthesis have been the elucidation of catalytic functions and the determination of 3D structures for enzymes of the polyketide synthases and nonribosomal peptide synthetases that carry out individual reactions. The size and complexity of these proteins present numerous difficulties in the process of going from gene to structure. Here, we review the problems that may be encountered at the various steps of this process and discuss some of the solutions devised in our and other labs for the cloning, production, purification, and structure solution of complex proteins using Escherichia coli as a heterologous host.
| S-EPMC5992914 | biostudies-literature
Project description:The roles of ATP hydrolysis in electron-transfer (ET) reactions of the nitrogenase catalytic cycle remain obscure. Here, we present a new structure of a nitrogenase complex crystallized with MgADP and MgAMPPCP, an ATP analogue. In this structure the two nucleotides are bound asymmetrically by the Fe-protein subunits connected to the two different MoFe-protein subunits. This binding mode suggests that ATP hydrolysis and phosphate release may proceed by a stepwise mechanism. Through the associated Fe-protein conformational changes, a stepwise mechanism is anticipated to prolong the lifetime of the Fe-protein-MoFe-protein complex and, in turn, could orchestrate the sequence of intracomplex ET required for substrate reduction.
| S-EPMC4304452 | biostudies-literature
Project description:Nitrogenases have the remarkable ability to catalyze the reduction of dinitrogen to ammonia under physiological conditions. How does this happen? The current view of the nitrogenase mechanism focuses on the role of hydrides, the binding of dinitrogen in a reductive elimination process coupled to loss of dihydrogen, and the binding of substrates to a binuclear site on the active site cofactor. This review focuses on recent experimental characterizations of turnover relevant forms of the enzyme determined by cryo-electron microscopy and other approaches, and comparison of these forms to the resting state enzyme and the broader family of iron sulfur clusters. Emerging themes include the following: (i) The obligatory coupling of protein and electron transfers does not occur in synthetic and small-molecule iron-sulfur clusters. The coupling of these processes in nitrogenase suggests that they may involve unique features of the cofactor, such as hydride formation on the trigonal prismatic arrangement of irons, protonation of belt sulfurs, and/or protonation of the interstitial carbon. (ii) Both the active site cofactor and protein are dynamic under turnover conditions; the changes are such that more highly reduced forms may differ in key ways from the resting-state structure. Homocitrate appears to play a key role in coupling cofactor and protein dynamics. (iii) Structural asymmetries are observed in nitrogenase under turnover-relevant conditions by cryo-electron microscopy, although the mechanistic relevance of these states (such as half-of-sites reactivity) remains to be established.
| S-EPMC10745740 | biostudies-literature
Project description:Nitrogenase-like light-independent protochlorophyllide oxidoreductase (DPOR) is involved in chlorophyll biosynthesis. Bacteriochlorophyll formation additionally requires the structurally related chlorophyllide oxidoreductase (COR). During catalysis, homodimeric subunit BchL(2) or ChlL(2) of DPOR transfers electrons to the corresponding heterotetrameric catalytic subunit, (BchNB)(2) or (ChlNB)(2). Analogously, subunit BchX(2) of the COR enzymes delivers electrons to subunit (BchYZ)(2). Various chimeric DPOR enzymes formed between recombinant subunits (BchNB)(2) and BchL(2) from Chlorobaculum tepidum or (ChlNB)(2) and ChlL(2) from Prochlorococcus marinus and Thermosynechococcus elongatus were found to be enzymatically active, indicating a conserved docking surface for the interaction of both DPOR protein subunits. Biotin label transfer experiments revealed the interaction of P. marinus ChlL(2) with both subunits, ChlN and ChlB, of the (ChlNB)(2) tetramer. Based on these findings and on structural information from the homologous nitrogenase system, a site-directed mutagenesis approach yielded 10 DPOR mutants for the characterization of amino acid residues involved in protein-protein interaction. Surface-exposed residues Tyr(127) of subunit ChlL, Leu(70) and Val(107) of subunit ChlN, and Gly(66) of subunit ChlB were found essential for P. marinus DPOR activity. Next, the BchL(2) or ChlL(2) part of DPOR was exchanged with electron-transferring BchX(2) subunits of COR and NifH(2) of nitrogenase. Active chimeric DPOR was generated via a combination of BchX(2) from C. tepidum or Roseobacter denitrificans with (BchNB)(2) from C. tepidum. No DPOR activity was observed for the chimeric enzyme consisting of NifH(2) from Azotobacter vinelandii in combination with (BchNB)(2) from C. tepidum or (ChlNB)(2) from P. marinus and T. elongatus, respectively.
| S-EPMC2708849 | biostudies-other
Project description:The iron (Fe) proteins of molybdenum (Mo)-, vanadium (V)-, and iron (Fe)-only nitrogenases are encoded by nifH, vnfH, and anfH, respectively. While the nifH-encoded Fe protein has been extensively studied over recent years, information regarding the properties of the vnfH- and anfH-encoded Fe proteins has remained scarce. Here, we present a combined biochemical, electron paramagnetic resonance (EPR) and X-ray absorption spectroscopy (XAS) analysis of the [Fe(4)S(4)] clusters of NifH, VnfH, and AnfH of Azotobacter vinelandii . Our data show that all three Fe proteins contain [Fe(4)S(4)] clusters of very similar spectroscopic and geometric structural properties, although NifH differs more from VnfH and AnfH with regard to the electronic structure. These observations have an interesting impact on the theory of the plausible sequence of evolution of nitrogenase Fe proteins. More importantly, the results presented herein provide a platform for future investigations of the differential activities of the three Fe proteins in nitrogenase biosynthesis and catalysis.
| S-EPMC3143205 | biostudies-literature
Project description:As an approach towards unraveling the nitrogenase mechanism, we have studied the binding of CO to the active-site FeMo-cofactor. CO is not only an inhibitor of nitrogenase, but it is also a substrate, undergoing reduction to hydrocarbons (Fischer-Tropsch-type chemistry). The C-C bond forming capabilities of nitrogenase suggest that multiple CO or CO-derived ligands bind to the active site. Herein, we report a crystal structure with two CO ligands coordinated to the FeMo-cofactor of the molybdenum nitrogenase at 1.33 Å resolution. In addition to the previously observed bridging CO ligand between Fe2 and Fe6 of the FeMo-cofactor, a new ligand binding mode is revealed through a second CO ligand coordinated terminally to Fe6. While the relevance of this state to nitrogenase-catalyzed reactions remains to be established, it highlights the privileged roles for Fe2 and Fe6 in ligand binding, with multiple coordination modes available depending on the ligand and reaction conditions.
| S-EPMC7920927 | biostudies-literature