metal cofactor
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2021 ◽  
Vol 23 (1) ◽  
pp. 230
Author(s):  
Eva Balint ◽  
Ildiko Unk

DNA polymerase η (Polη) is a translesion synthesis polymerase that can bypass different DNA lesions with varying efficiency and fidelity. Its most well-known function is the error-free bypass of ultraviolet light-induced cyclobutane pyrimidine dimers. The lack of this unique ability in humans leads to the development of a cancer-predisposing disease, the variant form of xeroderma pigmentosum. Human Polη can insert rNTPs during DNA synthesis, though with much lower efficiency than dNTPs, and it can even extend an RNA chain with ribonucleotides. We have previously shown that Mn2+ is a specific activator of the RNA synthetic activity of yeast Polη that increases the efficiency of the reaction by several thousand-fold over Mg2+. In this study, our goal was to investigate the metal cofactor dependence of RNA synthesis by human Polη. We found that out of the investigated metal cations, only Mn2+ supported robust RNA synthesis. Steady state kinetic analysis showed that Mn2+ activated the reaction a thousand-fold compared to Mg2+, even during DNA damage bypass opposite 8-oxoG and TT dimer. Our results revealed a two order of magnitude higher affinity of human Polη towards ribonucleotides in the presence of Mn2+ compared to Mg2+. It is noteworthy that activation occurred without lowering the base selectivity of the enzyme on undamaged templates, whereas the fidelity decreased across a TT dimer. In summary, our data strongly suggest that, like with its yeast homolog, Mn2+ is the proper metal cofactor of hPolη during RNA chain extension, and selective metal cofactor utilization contributes to switching between its DNA and RNA synthetic activities.


2021 ◽  
Vol 12 ◽  
Author(s):  
Minyoung So ◽  
Johnny Stiban ◽  
Grzegorz L. Ciesielski ◽  
Stacy L. Hovde ◽  
Laurie S. Kaguni

Recent evidence suggests that iron-sulfur clusters (ISCs) in DNA replicative proteins sense DNA-mediated charge transfer to modulate nuclear DNA replication. In the mitochondrial DNA replisome, only the replicative DNA helicase (mtDNA helicase) from Drosophila melanogaster (Dm) has been shown to contain an ISC in its N-terminal, primase-like domain (NTD). In this report, we confirm the presence of the ISC and demonstrate the importance of a metal cofactor in the structural stability of the Dm mtDNA helicase. Further, we show that the NTD also serves a role in membrane binding. We demonstrate that the NTD binds to asolectin liposomes, which mimic phospholipid membranes, through electrostatic interactions. Notably, membrane binding is more specific with increasing cardiolipin content, which is characteristically high in the mitochondrial inner membrane (MIM). We suggest that the N-terminal domain of the mtDNA helicase interacts with the MIM to recruit mtDNA and initiate mtDNA replication. Furthermore, Dm NUBPL, the known ISC donor for respiratory complex I and a putative donor for Dm mtDNA helicase, was identified as a peripheral membrane protein that is likely to execute membrane-mediated ISC delivery to its target proteins.


Author(s):  
Gabriela C. Schröder ◽  
Flora Meilleur

Metalloproteins catalyze a range of reactions, with enhanced chemical functionality due to their metal cofactor. The reaction mechanisms of metalloproteins have been experimentally characterized by spectroscopy, macromolecular crystallography and cryo-electron microscopy. An important caveat in structural studies of metalloproteins remains the artefacts that can be introduced by radiation damage. Photoreduction, radiolysis and ionization deriving from the electromagnetic beam used to probe the structure complicate structural and mechanistic interpretation. Neutron protein diffraction remains the only structural probe that leaves protein samples devoid of radiation damage, even when data are collected at room temperature. Additionally, neutron protein crystallography provides information on the positions of light atoms such as hydrogen and deuterium, allowing the characterization of protonation states and hydrogen-bonding networks. Neutron protein crystallography has further been used in conjunction with experimental and computational techniques to gain insight into the structures and reaction mechanisms of several transition-state metal oxidoreductases with iron, copper and manganese cofactors. Here, the contribution of neutron protein crystallography towards elucidating the reaction mechanism of metalloproteins is reviewed.


mBio ◽  
2021 ◽  
Author(s):  
Rob A. Schmitz ◽  
Nunzia Picone ◽  
Helena Singer ◽  
Andreas Dietl ◽  
Kerstin-Anikó Seifert ◽  
...  

Lanthanides comprise a group of 15 elements with atomic numbers 57 to 71 that are essential in a variety of high-tech devices, such as mobile phones, but were considered biologically inert for a long time. The biological relevance of lanthanides became evident when the acidophilic methanotroph Methylacidiphilum fumariolicum SolV, isolated from a volcanic mud pot, could only grow when lanthanides were supplied to the growth medium.


2021 ◽  
Vol 22 (18) ◽  
pp. 9820
Author(s):  
Viktor A. Anashkin ◽  
Anssi M. Malinen ◽  
Alexander V. Bogachev ◽  
Alexander A. Baykov

Membrane-bound inorganic pyrophosphatase (mPPase) resembles the F-ATPase in catalyzing polyphosphate-energized H+ and Na+ transport across lipid membranes, but differs structurally and mechanistically. Homodimeric mPPase likely uses a “direct coupling” mechanism, in which the proton generated from the water nucleophile at the entrance to the ion conductance channel is transported across the membrane or triggers Na+ transport. The structural aspects of this mechanism, including subunit cooperation, are still poorly understood. Using a refined enzyme assay, we examined the inhibition of K+-dependent H+-transporting mPPase from Desulfitobacterium hafniensee by three non-hydrolyzable PPi analogs (imidodiphosphate and C-substituted bisphosphonates). The kinetic data demonstrated negative cooperativity in inhibitor binding to two active sites, and reduced active site performance when the inhibitor or substrate occupied the other active site. The nonequivalence of active sites in PPi hydrolysis in terms of the Michaelis constant vanished at a low (0.1 mM) concentration of Mg2+ (essential cofactor). The replacement of K+, the second metal cofactor, by Na+ increased the substrate and inhibitor binding cooperativity. The detergent-solubilized form of mPPase exhibited similar active site nonequivalence in PPi hydrolysis. Our findings support the notion that the mPPase mechanism combines Mitchell’s direct coupling with conformational coupling to catalyze cation transport across the membrane.


2021 ◽  
Author(s):  
Jaya Joshi ◽  
Qiang Li ◽  
Jorge D. García-García ◽  
Bryan J. Leong ◽  
You Hu ◽  
...  

Plant and fungal THI4 thiazole synthases produce the thiamin thiazole moiety in aerobic conditions via a single-turnover suicide reaction that uses an active-site Cys residue as sulfur donor. Multiple-turnover (i.e. catalytic) THI4s lacking an active-site Cys (non-Cys THI4s) that use sulfide as sulfur donor have been biochemically characterized – but only from archaeal methanogens that are anaer­obic, O2-sensitive hyperthermophiles from sulfide-rich habitats. These THI4s prefer iron as cofactor. A survey of prokaryote genomes uncovered non-Cys THI4s in aerobic mesophiles from sulfide-poor habitats, suggesting that multiple-turnover THI4 operation is possible in aerobic, mild, low-sulfide conditions. This was confirmed by testing 23 representative non-Cys THI4s for complementation of an Escherichia coli ΔthiG thiazole auxotroph in aerobic conditions. Sixteen were clearly active, and more so when intracellular sulfide level was raised by supplying Cys, demonstrating catalytic function in the presence of O2 at mild temperatures and indicating use of sulfide or a sulfide metabolite as sulfur donor. Comparative genomic evidence linked non-Cys THI4s with proteins from families that bind, transport, or metabolize cobalt or other heavy metals. The crystal structure of the aerotolerant bacterial Thermovibrio ammonificans THI4 was determined to probe the molecular basis of aerotolerance. The structure suggested no large deviations compared to the structures of THI4s from O2-sensitive methanogens, but is consistent with an alternative catalytic metal. Together with complementation data, use of cobalt rather than iron was supported. We conclude that catalytic THI4s can indeed operate aerobically and that the metal cofactor inserted is a likely natural determinant of aerotolerance.


Molecules ◽  
2021 ◽  
Vol 26 (16) ◽  
pp. 5009
Author(s):  
Alexander K. H. Weiss ◽  
Richard Wurzer ◽  
Patrycia Klapec ◽  
Manuel Philip Eder ◽  
Johannes R. Loeffler ◽  
...  

FAH domain containing protein 1 (FAHD1) acts as oxaloacetate decarboxylase in mitochondria, contributing to the regulation of the tricarboxylic acid cycle. Guided by a high-resolution X-ray structure of FAHD1 liganded by oxalate, the enzymatic mechanism of substrate processing is analyzed in detail. Taking the chemical features of the FAHD1 substrate oxaloacetate into account, the potential inhibitor structures are deduced. The synthesis of drug-like scaffolds afforded first-generation FAHD1-inhibitors with activities in the low micromolar IC50 range. The investigations disclosed structures competing with the substrate for binding to the metal cofactor, as well as scaffolds, which may have a novel binding mode to FAHD1.


2021 ◽  
Author(s):  
Jaya Joshi ◽  
Qiang Li ◽  
Jorge D. Garcia-Garcia ◽  
Bryan J. Leong ◽  
You Hu ◽  
...  

Plant and fungal THI4 thiazole synthases produce the thiamin thiazole moiety in aerobic conditions via a single–turnover suicide reaction that uses an active–site Cys residue as sulfur donor. Multiple turnover (i.e. catalytic) THI4s lacking an active–site Cys (non–Cys THI4s) that use sulfide as sulfur donor have been characterized—but only from archaeal methanogens that are anaerobic, O2–sensitivehyperthermophiles from sulfide–rich habitats. These THI4s prefer iron as cofactor. A survey of prokaryote genomes uncovered non–Cys THI4s in aerobic mesophiles from sulfide–poor habitats, suggesting that multiple–turnover THI4 operation is possible in aerobic, mild, low–sulfide conditions. This was confirmed by testing 23 representative non–Cys THI4s for complementation of an Escherichia coli ΔthiG thiazole auxotroph in aerobic conditions. Sixteen were active, and more so when intracellular sulfidelevel was raised by supplying Cys, demonstrating that they function in the presence of O2 at mild temperatures and indicating they use sulfide or a sulfide metabolite as sulfur donor. Comparative genomic evidence linked non–Cys THI4s with proteins from families that bind, transport, or metabolize cobalt or other heavy metals. The crystal structure of the aerotolerant bacterial Thermovibrio ammonificans THI4 was determined to probe the molecular basis of aerotolerance. The structure suggested no large deviations compared to the structures of THI4s from O2–sensitive methanogens but is consistent with an alternative catalytic metal. Together with complementation data, the use of cobalt rather than iron was supported. We conclude that catalytic THI4s can indeed operate aerobically and that the metal cofactor inserted is a likely natural determinant of aerotolerance.


2021 ◽  
Author(s):  
Juan Pinheiro Oliveira Martinez ◽  
Leonardo Vazquez ◽  
Matheus Mikio Takeyama ◽  
Tarcizio José Santos Filho ◽  
Fernanda Sampaio Cavalcante ◽  
...  

Abstract Background Staphylococcus lugdunensis is a commensal skin microorganism that, unlike other coagulase-negative staphylococci, presents increasing clinical importance. This species yields a metalloprotease called lugdulysin that may contribute to its higher degree of virulence. This study aimed to determine the biochemical characterization of the lugdulysin produced by S. lugdunensis clinical isolates and investigate its effect on the formation and disruption of biofilm of Staphylococcus aureus isolates. The protease was isolated and characterized for its optimal pH and temperature, activity in the presence of inhibitors and enzymatic kinetics. The influence of metal cofactor supplementation on proteolysis was also evaluated, with and without inhibitors. Finally, the protease capacity to inhibit and disrupt biofilms of different S. aureus lineages and biofilm matrix was analyzed. Results The protease optimal pH and temperature were 7.0 and 37° C, respectively. EDTA inhibited the protease, and the activity was not recovered by divalent ion supplementation. In addition, divalent ions did not change enzymatic activity without inhibitors, which was stable for up to 3 hours. Its structure was determined via homology modelling. The protease significantly inhibited the formation and disrupted established biofilms of S. aureus isolates with protein biofilm. Conclusions This study confirmed features of the lugdulysin metalloprotease and showed that this S. lugdunensis virulence factor may be a new competition mechanism and/or modulation of the staphylococcal biofilm.


Molecules ◽  
2021 ◽  
Vol 26 (11) ◽  
pp. 3220
Author(s):  
Dóra Lakk-Bogáth ◽  
Natalija Pantalon Juraj ◽  
Bashdar I. Meena ◽  
Berislav Perić ◽  
Srećko I. Kirin ◽  
...  

Heme and nonheme-type flavone synthase enzymes, FS I and FS II are responsible for the synthesis of flavones, which play an important role in various biological processes, and have a wide range of biomedicinal properties including antitumor, antimalarial, and antioxidant activities. To get more insight into the mechanism of this curious enzyme reaction, nonheme structural and functional models were carried out by the use of mononuclear iron, [FeII(CDA-BPA*)]2+ (6) [CDA-BPA = N,N,N’,N’-tetrakis-(2-pyridylmethyl)-cyclohexanediamine], [FeII(CDA-BQA*)]2+ (5) [CDA-BQA = N,N,N’,N’-tetrakis-(2-quinolilmethyl)-cyclohexanediamine], [FeII(Bn-TPEN)(CH3CN)]2+ (3) [Bn-TPEN = N-benzyl-N,N’,N’-tris(2-pyridylmethyl)-1,2- diaminoethane], [FeIV(O)(Bn-TPEN)]2+ (9), and manganese, [MnII(N4Py*)(CH3CN)]2+ (2) [N4Py* = N,N-bis(2-pyridylmethyl)-1,2-di(2-pyridyl)ethylamine)], [MnII(Bn-TPEN)(CH3CN)]2+ (4) complexes as catalysts, where the possible reactive intermediates, high-valent FeIV(O) and MnIV(O) are known and well characterised. The results of the catalytic and stoichiometric reactions showed that the ligand framework and the nature of the metal cofactor significantly influenced the reactivity of the catalyst and its intermediate. Comparing the reactions of [FeIV(O)(Bn-TPEN)]2+ (9) and [MnIV(O)(Bn-TPEN)]2+ (10) towards flavanone under the same conditions, a 3.5-fold difference in reaction rate was observed in favor of iron, and this value is three orders of magnitude higher than was observed for the previously published [FeIV(O)(N2Py2Q*)]2+ [N,N-bis(2-quinolylmethyl)-1,2-di(2-pyridyl)ethylamine] species.


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