scholarly journals The structure of the Moco carrier protein from Rippkaea orientalis

2020 ◽  
Vol 76 (9) ◽  
pp. 453-463 ◽  
Author(s):  
Joern Krausze ◽  
Thomas W. Hercher ◽  
Archna Archna ◽  
Tobias Kruse
Keyword(s):  
Quaternary Structure ◽  
Chloride Ions ◽  
Free Form ◽  
Carrier Protein ◽  
Prosthetic Group ◽  
Computational Docking ◽  
Recombinant Production ◽  
Enzymatic Function ◽  
Nucleotide Binding Proteins ◽  
Positively Charged

The molybdenum cofactor (Moco) is the prosthetic group of all molybdenum-dependent enzymes except for nitrogenase. The multistep biosynthesis pathway of Moco and its function in molybdenum-dependent enzymes are already well understood. The mechanisms of Moco transfer, storage and insertion, on the other hand, are not. In the cell, Moco is usually not found in its free form and remains bound to proteins because of its sensitivity to oxidation. The green alga Chlamydomonas reinhardtii harbors a Moco carrier protein (MCP) that binds and protects Moco but is devoid of enzymatic function. It has been speculated that this MCP acts as a means of Moco storage and transport. Here, the search for potential MCPs has been extended to the prokaryotes, and many MCPs were found in cyanobacteria. A putative MCP from Rippkaea orientalis (RoMCP) was selected for recombinant production, crystallization and structure determination. RoMCP has a Rossmann-fold topology that is characteristic of nucleotide-binding proteins and a homotetrameric quaternary structure similar to that of the MCP from C. reinhardtii. In each protomer, a positively charged crevice was identified that accommodates up to three chloride ions, hinting at a potential Moco-binding site. Computational docking experiments supported this notion and gave an impression of the RoMCP–Moco complex.

scholarly journals Blood-borne, albumin-bound prostaglandin E2 may be involved in fever

1999 ◽  
Vol 276 (6) ◽  
pp. R1840-R1844 ◽  
Author(s):  
Andrej A. Romanovsky ◽  
Andrei I. Ivanov ◽  
Elena K. Karman
Keyword(s):  
Human Serum Albumin ◽  
Rectal Temperature ◽  
Enzymatic Degradation ◽  
False Negative ◽  
Free Ligand ◽  
Free Form ◽  
Carrier Protein ◽  
Negative Results ◽  
False Negative Results ◽  
Pyrogenic Activity

Although the involvement of blood-borne PGE2 in fever has been hypothesized by several authors and has substantial experimental support, the current literature often rejects this hypothesis because several attempts to induce fever by a peripheral PGE2 failed. However, it is usually ignored that the amphipathic molecules of PGE2 are readily self-associating and that such an aggregation could have prevented the peripherally administered PGE2 (free form) from expressing its pyrogenic activity, thus leading to false negative results. To ensure disaggregation of PGE2, we prepared its complex within a carrier protein, human serum albumin (HSA). HSA was purified with activated charcoal and polymixin B-polyacrylamide gel and incubated with PGE2 for 1 h at 37°C. Adult Chinchilla rabbits were injected intravenously with PGE2-HSA complex in either the higher (75 μg/kg PGE2:30 mg/kg HSA) or the lower (15 μg/kg:6 mg/kg) dose, and the rectal temperature (Tr) was measured. In the controls, either the ligand alone or the carrier alone was administered. At the higher dose, neither free PGE2 nor albumin alone was pyrogenic, whereas the PGE2-HSA complex produced a fever characterized by a short latency (<10 min) and a maximal Tr rise of 0.7 ± 0.2°C. At the lower dose, none of the substances affected the Tr. This study demonstrates a marked pyrogenic activity of the intravenous PGE2-HSA, but not of the free PGE2. Administration of a preformed complex may be more physiologically relevant than administration of the free ligand because of the ligand’s disaggregation, protection from enzymatic degradation, and facilitated delivery to targets. Our study supports the hypothesis that peripheral PGE2 is involved in fever genesis.

scholarly journals Sticky swinging arm dynamics: studies of an acyl carrier protein domain from the mycolactone polyketide synthase

2016 ◽  
Vol 473 (8) ◽  
pp. 1097-1110 ◽  
Author(s):  
Steven Vance ◽  
Olga Tkachenko ◽  
Ben Thomas ◽  
Mona Bassuni ◽  
Hui Hong ◽  
...  
Keyword(s):  
Polyketide Synthase ◽  
Acyl Carrier Protein ◽  
Protein Domain ◽  
Carrier Protein ◽  
Protein Surface ◽  
Prosthetic Group ◽  
Polyketide Biosynthesis ◽  
Covalently Linked

When covalently linked to an acyl carrier protein (ACP) and loaded with acyl substrate-mimics, some 4′-phosphopantetheine prosthetic group arms swing freely, whereas others stick to the protein surface, suggesting a possible mode of interaction with enzyme domains during polyketide biosynthesis.

scholarly journals Identification of an Oligosaccharide Dehydrogenase from Pycnoporus Cinnabarinus Provides Insights into Fungal Breakdown of Lignocellulose

2021 ◽  
Author(s):  
Gabriele Cerutti ◽  
Elena Gugole ◽  
Linda Celeste Montemiglio ◽  
Annick Turbé-Doan ◽  
Dehbia Chena ◽  
...  
Keyword(s):  
Function Analysis ◽  
Sequence Similarity ◽  
Functional Characterization ◽  
Physiological Role ◽  
Free Form ◽  
Lignocellulose Degradation ◽  
Carbohydrate Active Enzymes ◽  
Recognition Mechanism ◽  
Pycnoporus Cinnabarinus ◽  
Enzymatic Function

Abstract Background: Fungal glucose dehydrogenases (GDHs) are FAD-dependent enzymes belonging to the glucose-methanol-choline oxidoreductase superfamily. These enzymes are classified in the “Auxiliary Activity” family 3 (AA3) of the Carbohydrate-Active enZymes database, and more specifically in subfamily AA3_2, that also includes the closely related flavoenzymes aryl-alcohol oxidase and glucose 1-oxidase. Based on sequence similarity to known fungal GDHs, an AA3_2 enzyme active on glucose was identified in the genome of Pycnoporus cinnabarinus, a model Basidiomycete able to completely degrade lignin.Results: In our work, substrate screening and functional characterization showed an unexpected preferential activity of this enzyme toward oligosaccharides containing a b(1à3) glycosidic bond, with the highest efficiency observed for the disaccharide laminaribiose. Despite its sequence similarity to GDHs, we defined a novel enzymatic activity, namely oligosaccharide dehydrogenase (ODH), for this enzyme. The crystallographic structures of ODH in the sugar-free form and in complex with glucose and laminaribiose unveiled a peculiar saccharide recognition mechanism which is not shared with previously characterized AA3 oxidoreductases and accounts for ODH preferential activity toward oligosaccharides. The sugar molecules in the active site of ODH are mainly stabilized through CH-p interactions with aromatic residues rather than through hydrogen bonds with highly conserved residues, as observed instead for the fungal glucose dehydrogenases and oxidases characterized to date. Finally, three sugar-binding sites were identified on ODH external surface, which were not previously observed and might be of importance in the physiological scenario.Conclusions: Structure-function analysis of ODH is consistent with its role as an auxiliary enzyme in lignocellulose degradation and unveils yet another enzymatic function within the AA3 family of the Carbohydrate-Active enZymes database. Our findings allow deciphering the molecular determinants of substrate binding and provide insight into the physiological role of ODH, opening new perspectives to exploit biodiversity for lignocellulose transformation into fuels and chemicals.

scholarly journals Plate-like Alginate Microparticles with Disulfiram–SPIO–Coencapsulation: An In Vivo Study for Combined Therapy on Ovarian Cancer

Pharmaceutics ◽  
2021 ◽  
Vol 13 (9) ◽  
pp. 1348
Author(s):  
Meng-Yi Bai ◽  
Mu-Hsien Yu ◽  
Ting-Teng Wang ◽  
Shiu-Hsin Chen ◽  
Yu-Chi Wang
Keyword(s):  
Ovarian Cancer ◽  
Combined Therapy ◽  
Superparamagnetic Iron Oxide ◽  
Drug Carriers ◽  
Treated Group ◽  
Chloride Ions ◽  
Free Form ◽  
Ovarian Cancer Cells ◽  
Cross Linking

Disulfiram is a drug used to support the treatment of chronic alcoholism. Recently, it has been found to have an off-label ability to inhibit the growth of ovarian cancer cells. However, the original formulation was designed for use via oral administration, which is not suitable to be given by a direct spray on the affected area. Therefore, in this study, we designed and prepared alginate (ALG) microparticles loaded with disulfiram and superparamagnetic iron oxide (cross-linking disulfiram/SPIO/ALG MPs), which have great potential application for inhibiting the growth of ovarian cancer simultaneously via two treatments, i.e., chemotherapy and hyperthermia. The drug-encapsulating alginate microparticles were prepared using an electrospray system and then cross-linked with calcium chloride ions. The particles were observed by optical microscopy and scanning electron microscopy, and found to be approximately 200 μm in diameter. The disc-shape morphology of the microparticles could be controlled by up to 95%. The drug-encapsulation efficiency of the microparticles reached 98%, and the suppression of tumor growth for the free-form disulfiram-treated group and disulfiram/SPIO/ALG MPs-treated group were 48.2% and 55.9% of tumor volume reduction, respectively, compared with a cisplatin-treated group. A hyperthermic effect can be achieved by applying a magnetic field to oscillate SPIO. The results of this study showed that these cross-linking disulfiram/SPIO/ALG MPs are potential drug carriers for the treatment of ovarian cancer.

scholarly journals T-to-R switch of muscle fructose-1,6-bisphosphatase involves fundamental changes of secondary and quaternary structure

2016 ◽  
Vol 72 (4) ◽  
pp. 536-550 ◽  
Author(s):  
Jakub Barciszewski ◽  
Janusz Wisniewski ◽  
Robert Kolodziejczyk ◽  
Mariusz Jaskolski ◽  
Dariusz Rakus ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Catalytic Mechanism ◽  
Quaternary Structure ◽  
Homo Sapiens ◽  
Helical Structure ◽  
Smooth Transition ◽  
Muscle Enzyme ◽  
Enzymatic Function ◽  
Fructose 1,6 Bisphosphatase ◽  
Key Enzyme

Fructose-1,6-bisphosphatase (FBPase) catalyzes the hydrolysis of fructose 1,6-bisphosphate to fructose 6-phosphate and is a key enzyme of gluconeogenesis and glyconeogenesis and, more generally, of the control of energy metabolism and glucose homeostasis. Vertebrates, and notablyHomo sapiens, express two FBPase isoforms. The liver isozyme is expressed mainly in gluconeogenic organs, where it functions as a regulator of glucose synthesis. The muscle isoform is expressed in all cells, and recent studies have demonstrated that its role goes far beyond the enzymatic function, as it can interact with various nuclear and mitochondrial proteins. Even in its enzymatic function, the muscle enzyme is different from the liver isoform, as it is 100-fold more susceptible to allosteric inhibition by AMP and this effect can be abrogated by complex formation with aldolase. All FBPases are homotetramers composed of two intimate dimers: the upper dimer and the lower dimer. They oscillate between two conformational states: the inactive T form when in complex with AMP, and the active R form. Parenthetically, it is noted that bacterial FBPases behave somewhat differently, and in the absence of allosteric activators exist in a tetramer–dimer equilibrium even at relatively high concentrations. [Hineset al.(2007),J. Biol. Chem.282, 11696–11704]. The T-to-R transition is correlated with the conformation of the key loop L2, which in the T form becomes `disengaged' and unable to participate in the catalytic mechanism. The T states of both isoforms are very similar, with a small twist of the upper dimer relative to the lower dimer. It is shown that at variance with the well studied R form of the liver enzyme, which is flat, the R form of the muscle enzyme is diametrically different, with a perpendicular orientation of the upper and lower dimers. The crystal structure of the muscle-isozyme R form shows that in this arrangement of the tetramer completely new protein surfaces are exposed that are most likely targets for the interactions with various cellular and enzymatic partners. The cruciform R structure is stabilized by a novel `leucine lock', which prevents the key residue, Asp187, from locking loop L2 in the disengaged conformation. In addition, the crystal structures of muscle FBPase in the T conformation with and without AMP strongly suggest that the T-to-R transition is a discrete jump rather than a shift of an equilibrium smooth transition through multiple intermediate states. Finally, using snapshots from three crystal structures of human muscle FBPase, it is conclusively demonstrated that the AMP-binding event is correlated with a β→α transition at the N-terminus of the protein and with the formation of a new helical structure.

Acyl carrier protein: structure–function relationships in a conserved multifunctional protein family

2007 ◽  
Vol 85 (6) ◽  
pp. 649-662 ◽  
Author(s):  
David M. Byers ◽  
Huansheng Gong
Keyword(s):  
Fatty Acid ◽  
Active Sites ◽  
Fatty Acid Synthase ◽  
Acyl Carrier Protein ◽  
Divalent Cations ◽  
Structural Features ◽  
Carrier Protein ◽  
Type I ◽  
Prosthetic Group

Acyl carrier protein (ACP) is a universal and highly conserved carrier of acyl intermediates during fatty acid synthesis. In yeast and mammals, ACP exists as a separate domain within a large multifunctional fatty acid synthase polyprotein (type I FAS), whereas it is a small monomeric protein in bacteria and plastids (type II FAS). Bacterial ACPs are also acyl donors for synthesis of a variety of products, including endotoxin and acylated homoserine lactones involved in quorum sensing; the distinct and essential nature of these processes in growth and pathogenesis make ACP-dependent enzymes attractive antimicrobial drug targets. Additionally, ACP homologues are key components in the production of secondary metabolites such as polyketides and nonribosomal peptides. Many ACPs exhibit characteristic structural features of natively unfolded proteins in vitro, with a dynamic and flexible conformation dominated by 3 parallel α helices that enclose the thioester-linked acyl group attached to a phosphopantetheine prosthetic group. ACP conformation may also be influenced by divalent cations and interaction with partner enzymes through its “recognition” helix II, properties that are key to its ability to alternately sequester acyl groups and deliver them to the active sites of ACP-dependent enzymes. This review highlights recent progress in defining how the structural features of ACP are related to its multiple carrier roles in fatty acid metabolism.

Structures of haemoglobin from woolly mammoth in liganded and unliganded states

2012 ◽  
Vol 68 (11) ◽  
pp. 1441-1449 ◽  
Author(s):  
Hiroki Noguchi ◽  
Kevin L. Campbell ◽  
Chien Ho ◽  
Satoru Unzai ◽  
Sam-Yong Park ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Crystal Structures ◽  
High Latitude ◽  
Quaternary Structure ◽  
Globin Gene ◽  
Oxygen Affinity ◽  
Chloride Ions ◽  
Structural Basis ◽  
Structure Changes ◽  
Woolly Mammoth

The haemoglobin (Hb) of the extinct woolly mammoth has been recreated using recombinant genes expressed inEscherichia coli. The globin gene sequences were previously determined using DNA recovered from frozen cadavers. Although highly similar to the Hb of existing elephants, the woolly mammoth protein shows rather different responses to chloride ions and temperature. In particular, the heat of oxygenation is found to be much lower in mammoth Hb, which appears to be an adaptation to the harsh high-latitude climates of the Pleistocene Ice Ages and has been linked to heightened sensitivity of the mammoth protein to protons, chloride ions and organic phosphates relative to that of Asian elephants. To elucidate the structural basis for the altered homotropic and heterotropic effects, the crystal structures of mammoth Hb have been determined in the deoxy, carbonmonoxy and aquo-met forms. These models, which are the first structures of Hb from an extinct species, show many features reminiscent of human Hb, but underline how the delicate control of oxygen affinity relies on much more than simple overall quaternary-structure changes.

scholarly journals Structural analysis of a novel substrate-free form of the aminoglycoside 6′-N-acetyltransferase from Enterococcus faecium

2020 ◽  
Vol 76 (8) ◽  
pp. 364-371
Author(s):  
Hyunseok Jang ◽  
Sunghark Kwon ◽  
Chang-Sook Jeong ◽  
Chang Woo Lee ◽  
Jisub Hwang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Structural Analysis ◽  
Mechanism Of Action ◽  
Enterococcus Faecium ◽  
Free Form ◽  
Amino Group ◽  
Detailed Mechanism ◽  
Conformational Selection ◽  
Enzymatic Function ◽  
Acetyl Group

Aminoglycoside acetyltransferases (AACs) catalyze the transfer of an acetyl group between acetyl-CoA and an aminoglycoside, producing CoA and an acetylated aminoglycoside. AAC(6′)-Ii enzymes target the amino group linked to the 6′ C atom in an aminoglycoside. Several structures of the AAC(6′)-Ii from Enterococcus faecium [Ef-AAC(6′)-Ii] have been reported to date. However, the detailed mechanism of its enzymatic function remains elusive. In this study, the crystal structure of Ef-AAC(6′)-Ii was determined in a novel substrate-free form. Based on structural analysis, it is proposed that Ef-AAC(6′)-Ii sequentially undergoes conformational selection and induced fit for substrate binding. These results therefore provide a novel viewpoint on the mechanism of action of Ef-AAC(6′)-Ii.

scholarly journals Flavin-dependent alcohol oxidase from the yeast Pichia pinus. Spatial localization of the coenzyme FAD in the protein structure: hot-tritium bombardment and ESR experiments

1995 ◽  
Vol 310 (2) ◽  
pp. 601-604 ◽  
Author(s):  
A Z Averbakh ◽  
N D Pekel ◽  
V I Seredenko ◽  
A V Kulikov ◽  
R I Gvozdev ◽  
...  
Keyword(s):  
Protein Structure ◽  
Quaternary Structure ◽  
Specific Radioactivity ◽  
Alcohol Oxidase ◽  
Spatial Localization ◽  
Prosthetic Group ◽  
Tritium Planigraphy ◽  
Pichia Pinus

The spatial localization of the coenzyme FAD in the quaternary structure of the alcohol oxidase from the yeast Pichia pinus was studied by tritium planigraphy and ESR methods. In the present paper we measured the specific radioactivity of FAD labelled as a part of the alcohol oxidase complex. The specific-radioactivity ratio for two FAD portions (FMN and AMP) was calculated. ESR experiments show 4 A (0.4 nm) to be the depth of immersion of paramagnetic isoalloxazines into alcohol oxidase octamer molecules. It is suggested that FAD molecules are bound to the surface of the octamer, rather than to the subunit interfaces. The orientation of the prosthetic group FAD in the alcohol oxidase protein is discussed.

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