From the Eukaryotic Molybdenum Cofactor Biosynthesis to the Moonlighting Enzyme mARC

All eukaryotic molybdenum (Mo) enzymes contain in their active site a Mo Cofactor (Moco), which is formed by a tricyclic pyranopterin with a dithiolene chelating the Mo atom. Here, the eukaryotic Moco biosynthetic pathway and the eukaryotic Moco enzymes are overviewed, including nitrate reductase (NR), sulfite oxidase, xanthine oxidoreductase, aldehyde oxidase, and the last one discovered, the moonlighting enzyme mitochondrial Amidoxime Reducing Component (mARC). The mARC enzymes catalyze the reduction of hydroxylated compounds, mostly N-hydroxylated (NHC), but as well of nitrite to nitric oxide, a second messenger. mARC shows a broad spectrum of NHC as substrates, some are prodrugs containing an amidoxime structure, some are mutagens, such as 6-hydroxylaminepurine and some others, which most probably will be discovered soon. Interestingly, all known mARC need the reducing power supplied by different partners. For the NHC reduction, mARC uses cytochrome b5 and cytochrome b5 reductase, however for the nitrite reduction, plant mARC uses NR. Despite the functional importance of mARC enzymatic reactions, the structural mechanism of its Moco-mediated catalysis is starting to be revealed. We propose and compare the mARC catalytic mechanism of nitrite versus NHC reduction. By using the recently resolved structure of a prokaryotic MOSC enzyme, from the mARC protein family, we have modeled an in silico three-dimensional structure of a eukaryotic homologue.

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The first crystal structure of the peptidase domain of the U32 peptidase family

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004715019549 ◽

2015 ◽

Vol 71 (12) ◽

pp. 2505-2512 ◽

Cited By ~ 4

Author(s):

Magdalena Schacherl ◽

Angelika A. M. Montada ◽

Elena Brunstein ◽

Ulrich Baumann

Keyword(s):

Crystal Structure ◽

Catalytic Mechanism ◽

Quaternary Structure ◽

Catalytic Domain ◽

Three Dimensional ◽

Zinc Ion ◽

Crystal Structure Analysis ◽

Dimensional Structure ◽

Sequence Motifs ◽

Conserved Sequence

The U32 family is a collection of over 2500 annotated peptidases in the MEROPS database with unknown catalytic mechanism. They mainly occur in bacteria and archaea, but a few representatives have also been identified in eukarya. Many of the U32 members have been linked to pathogenicity, such as proteins fromHelicobacterandSalmonella. The first crystal structure analysis of a U32 catalytic domain fromMethanopyrus kandleri(genemk0906) reveals a modified (βα)8TIM-barrel fold with some unique features. The connecting segment between strands β7 and β8 is extended and helix α7 is located on top of the C-terminal end of the barrel body. The protein exhibits a dimeric quaternary structure in which a zinc ion is symmetrically bound by histidine and cysteine side chains from both monomers. These residues reside in conserved sequence motifs. No typical proteolytic motifs are discernible in the three-dimensional structure, and biochemical assays failed to demonstrate proteolytic activity. A tunnel in which an acetate ion is bound is located in the C-terminal part of the β-barrel. Two hydrophobic grooves lead to a tunnel at the C-terminal end of the barrel in which an acetate ion is bound. One of the grooves binds to aStrep-Tag II of another dimer in the crystal lattice. Thus, these grooves may be binding sites for hydrophobic peptides or other ligands.

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Substitution of murine ferrochelatase glutamate-287 with glutamine or alanine leads to porphyrin substrate-bound variants

Biochemical Journal ◽

10.1042/bj3560217 ◽

2001 ◽

Vol 356 (1) ◽

pp. 217-222 ◽

Cited By ~ 5

Author(s):

Ricardo FRANCO ◽

Alice S. PEREIRA ◽

Pedro TAVARES ◽

Arianna MANGRAVITA ◽

Michael J. BARBER ◽

...

Keyword(s):

Absorption Spectra ◽

Catalytic Mechanism ◽

Protoporphyrin Ix ◽

Three Dimensional ◽

Iron Chelation ◽

Enzymic Activity ◽

Dimensional Structure ◽

Wild Type ◽

Wild Type Enzyme ◽

Flow Experiments

Ferrochelatase (EC 4.99.1.1) is the terminal enzyme of the haem biosynthetic pathway and catalyses iron chelation into the protoporphyrin IX ring. Glutamate-287 (E287) of murine mature ferrochelatase is a conserved residue in all known sequences of ferrochelatase, is present at the active site of the enzyme, as inferred from the Bacillus subtilis ferrochelatase three-dimensional structure, and is critical for enzyme activity. Substitution of E287 with either glutamine (Q) or alanine (A) yielded variants with lower enzymic activity than that of the wild-type ferrochelatase and with different absorption spectra from the wild-type enzyme. In contrast to the wild-type enzyme, the absorption spectra of the variants indicate that these enzymes, as purified, contain protoporphyrin IX. Identification and quantification of the porphyrin bound to the E287-directed variants indicate that approx. 80% of the total porphyrin corresponds to protoporphyrin IX. Significantly, rapid stopped-flow experiments of the E287A and E287Q variants demonstrate that reaction with Zn2+ results in the formation of bound Zn-protoporphyrin IX, indicating that the endogenously bound protoporphyrin IX can be used as a substrate. Taken together, these findings suggest that the structural strain imposed by ferrochelatase on the porphyrin substrate as a critical step in the enzyme catalytic mechanism is also accomplished by the E287A and E287Q variants, but without the release of the product. Thus E287 in murine ferrochelatase appears to be critical for the catalytic process by controlling the release of the product.

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On the three-dimensional structure and catalytic mechanism of triose phosphate isomerase

Philosophical Transactions of the Royal Society of London Series B Biological Sciences ◽

10.1098/rstb.1981.0069 ◽

1981 ◽

Vol 293 (1063) ◽

pp. 159-171 ◽

Cited By ~ 170

Keyword(s):

Catalytic Mechanism ◽

Polypeptide Chain ◽

Three Dimensional ◽

Triose Phosphate Isomerase ◽

Dimensional Structure ◽

Dihydroxyacetone Phosphate ◽

Triose Phosphate ◽

Independent Determination ◽

Chicken Muscle

Triose phosphate isomerase is a dimeric enzyme of molecular mass 56000 which catalyses the interconversion of dihydroxyacetone phosphate (DHAP) and D-glyceraldehyde-3-phosphate. The crystal structure of the enzyme from chicken muscle has been determined at a resolution of 2.5 A, and an independent determination of the structure of the yeast enzyme has just been completed at 3 A resolution. The conformation of the polypeptide chain is essentially identical in the two structures, and consists of an inner cylinder of eight strands of parallel |3-pleated sheet, with mostly helical segments connecting each strand. The active site is a pocket containing glutamic acid 165, which is believed to act as a base in the reaction. Crystallographic studies of the binding of DHAP to both the chicken and the yeast enzymes reveal a common mode of binding and suggest a mechanism for catalysis involving polarization of the substrate carbonyl group.

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A forty-year journey in plant research: original contributions to flavonoid biochemistry

Canadian Journal of Botany ◽

10.1139/b05-030 ◽

2005 ◽

Vol 83 (5) ◽

pp. 433-450 ◽

Cited By ~ 18

Author(s):

Ragai K Ibrahim

Keyword(s):

Three Dimensional ◽

Biological Significance ◽

Flavonoid Biosynthesis ◽

Dimensional Structure ◽

Enzymatic Reactions ◽

Research Career ◽

Substrate Preferences ◽

Genes Encoding ◽

The Common ◽

New Compounds

This review highlights original contributions by the author to the field of flavonoid biochemistry during his research career of more than four decades. These include elucidation of novel aspects of some of the common enzymatic reactions involved in the later steps of flavonoid biosynthesis, with emphasis on methyltransferases, glucosyltransferases, sulfotransferases, and an oxoglutarate-dependent dioxygenase, as well as cloning, and inferences about phylogenetic relationships, of the genes encoding some of these enzymes. The three-dimensional structure of a flavonol O-methyltransferase was studied through homology-based modeling, using a caffeic acid O-methyltransferase as a template, to explain their strict substrate preferences. In addition, the biological significance of enzymatic prenylation of isoflavones, as well as their role as phytoanticipins and inducers of nodulation genes, are emphasized. Finally, the potential application of knowledge about the genes encoding these enzyme reactions is discussed in terms of improving plant productivity and survival, modification of flavonoid profiles, and the search for new compounds with pharmaceutical and (or) nutraceutical value.Key words: flavonoid enzymology, metabolite localization, gene cloning, 3-D structure, phylogeny.

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Redox-Regulation of α-Globin in Vascular Physiology

Antioxidants ◽

10.3390/antiox11010159 ◽

2022 ◽

Vol 11 (1) ◽

pp. 159

Author(s):

Laurent Kiger ◽

Julia Keith ◽

Abdullah Freiwan ◽

Alfonso G. Fernandez ◽

Heather Tillman ◽

...

Keyword(s):

Nitric Oxide ◽

Cellular Localization ◽

Redox Regulation ◽

Cytochrome B5 ◽

Three Dimensional ◽

Redox Status ◽

Dimensional Structure ◽

Redox Systems ◽

Circulatory Control ◽

Endothelial Nitric Oxide

Interest in the structure, function, and evolutionary relations of circulating and intracellular globins dates back more than 60 years to the first determination of the three-dimensional structure of these proteins. Non-erythrocytic globins have been implicated in circulatory control through reactions that couple nitric oxide (NO) signaling with cellular oxygen availability and redox status. Small artery endothelial cells (ECs) express free α-globin, which causes vasoconstriction by degrading NO. This reaction converts reduced (Fe2+) α-globin to the oxidized (Fe3+) form, which is unstable, cytotoxic, and unable to degrade NO. Therefore, (Fe3+) α-globin must be stabilized and recycled to (Fe2+) α-globin to reinitiate the catalytic cycle. The molecular chaperone α-hemoglobin-stabilizing protein (AHSP) binds (Fe3+) α-globin to inhibit its degradation and facilitate its reduction. The mechanisms that reduce (Fe3+) α-globin in ECs are unknown, although endothelial nitric oxide synthase (eNOS) and cytochrome b5 reductase (CyB5R3) with cytochrome b5 type A (CyB5a) can reduce (Fe3+) α-globin in solution. Here, we examine the expression and cellular localization of eNOS, CyB5a, and CyB5R3 in mouse arterial ECs and show that α-globin can be reduced by either of two independent redox systems, CyB5R3/CyB5a and eNOS. Together, our findings provide new insights into the regulation of blood vessel contractility.

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Role of Pro-297 in the catalytic mechanism of sheep liver serine hydroxymethyltransferase

Biochemical Journal ◽

10.1042/bj3500849 ◽

2000 ◽

Vol 350 (3) ◽

pp. 849-853 ◽

Cited By ~ 1

Author(s):

Rashmi TALWAR ◽

Vijayapandian LEELAVATHY ◽

Jala V. KRISHNA RAO ◽

Naropantul APPAJI RAO ◽

Handanahal S. SAVITHRI

Keyword(s):

Aspartate Aminotransferase ◽

Catalytic Mechanism ◽

Three Dimensional ◽

Proton Exchange ◽

Serine Hydroxymethyltransferase ◽

Dimensional Structure ◽

Transamination Reaction ◽

Reaction Specificity ◽

High Degree

Serine hydroxymethyltransferase belongs to the α class of pyridoxal-5´-phosphate enzymes along with aspartate aminotransferase. Recent reports on the three-dimensional structure of human liver cytosolic serine hydroxymethyltransferase had suggested a high degree of similarity between the active-site geometries of the two enzymes. A comparison of the sequences of serine hydroxymethyltransferases revealed the presence of several highly conserved residues, including Pro-297. This residue is equivalent to residue Arg-292 of aspartate aminotransferase, which binds the γ-carboxy group of aspartate. In an attempt to change the reaction specificity of the hydroxymethyltransferase to that of an aminotransferase and to assign a possible reason for the conserved nature of Pro-297, it was mutated to Arg. The mutation decreased the hydroxymethyltransferase activity significantly (by 85–90%) and abolished the ability to catalyse alternative reactions, without alteration in the oligomeric structure, pyridoxal 5´-phosphate content or substrate binding. However, the concentration of the quinonoid intermediate and the extent of proton exchange was decreased considerably (by approx. 85%) corresponding to the decrease in catalytic activity. Interestingly, mutant Pro-297 Arg was unable to perform the transamination reaction with l-aspartate. All these results suggest that although Pro-297 is indirectly involved in catalysis, it might not have any role in imparting substrate specificity, unlike the similarly positioned Arg-292 in aspartate aminotransferase.

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Complex structure of type VI peptidoglycan muramidase effector and a cognate immunity protein

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s090744491301576x ◽

2013 ◽

Vol 69 (10) ◽

pp. 1889-1900 ◽

Cited By ~ 15

Author(s):

Tianyu Wang ◽

Jinjing Ding ◽

Ying Zhang ◽

Da-Cheng Wang ◽

Wei Liu

Keyword(s):

Calcium Binding ◽

Catalytic Mechanism ◽

Catalytic Domain ◽

Complex Structure ◽

Three Dimensional ◽

Dimensional Structure ◽

Structural Basis ◽

Type Vi Secretion System ◽

Lytic Enzymes ◽

Lytic Transglycosylase

The type VI secretion system (T6SS) is a bacterial protein-export machine that is capable of delivering virulence effectors between Gram-negative bacteria. The T6SS ofPseudomonas aeruginosatransports two lytic enzymes, Tse1 and Tse3, to degrade cell-wall peptidoglycan in the periplasm of rival bacteria that are competing for nichesviaamidase and muramidase activities, respectively. Two cognate immunity proteins, Tsi1 and Tsi3, are produced by the bacterium to inactivate the two antibacterial effectors, thereby protecting its siblings from self-intoxication. Recently, Tse1–Tsi1 has been structurally characterized. Here, the structure of the Tse3–Tsi3 complex is reported at 1.9 Å resolution. The results reveal that Tse3 contains a C-terminal catalytic domain that adopts a soluble lytic transglycosylase (SLT) fold in which three calcium-binding sites were surprisingly observed close to the catalytic Glu residue. The electrostatic properties of the substrate-binding groove are also distinctive from those of known structures with a similar fold. All of these features imply that a unique catalytic mechanism is utilized by Tse3 in cleaving glycosidic bonds. Tsi3 comprises a single domain showing a β-sandwich architecture that is reminiscent of the immunoglobulin fold. Three loops of Tsi3 insert deeply into the groove of Tse3 and completely occlude its active site, which forms the structural basis of Tse3 inactivation. This work is the first crystallographic report describing the three-dimensional structure of the Tse3–Tsi3 effector–immunity pair.

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