Crystal Structure and Catalytic Mechanism of the MJ0109 Gene Product:  A Bifunctional Enzyme with Inositol Monophosphatase and Fructose 1,6-Bisphosphatase Activities†,‡

Biochemistry ◽  
2001 ◽  
Vol 40 (3) ◽  
pp. 618-630 ◽  
Author(s):  
Kenneth A. Johnson ◽  
Liangjing Chen ◽  
Hongying Yang ◽  
Mary F. Roberts ◽  
Boguslaw Stec
Keyword(s):  
Crystal Structure ◽  
Gene Product ◽  
Catalytic Mechanism ◽  
Bifunctional Enzyme ◽  
Inositol Monophosphatase ◽  
Fructose 1,6 Bisphosphatase

scholarly journals Structural and Functional Characterization of NadR from Lactococcus lactis

Molecules ◽  
2020 ◽  
Vol 25 (8) ◽  
pp. 1940
Author(s):  
Artem Stetsenko ◽  
Rajkumar Singh ◽  
Michael Jaehme ◽  
Albert Guskov ◽  
Dirk Jan Slotboom
Keyword(s):  
Crystal Structure ◽  
Lactococcus Lactis ◽  
Catalytic Mechanism ◽  
Adenine Nucleotides ◽  
Functional Characterization ◽  
Bifunctional Enzyme ◽  
Structural Determinants ◽  
Nicotinamide Riboside ◽  
Atp Concentration ◽  
Nicotinamide Mononucleotide

NadR is a bifunctional enzyme that converts nicotinamide riboside (NR) into nicotinamide mononucleotide (NMN), which is then converted into nicotinamide adenine dinucleotide (NAD). Although a crystal structure of the enzyme from the Gram-negative bacterium Haemophilus influenzae is known, structural understanding of its catalytic mechanism remains unclear. Here, we purified the NadR enzyme from Lactococcus lactis and established an assay to determine the combined activity of this bifunctional enzyme. The conversion of NR into NAD showed hyperbolic dependence on the NR concentration, but sigmoidal dependence on the ATP concentration. The apparent cooperativity for ATP may be explained because both reactions catalyzed by the bifunctional enzyme (phosphorylation of NR and adenylation of NMN) require ATP. The conversion of NMN into NAD followed simple Michaelis-Menten kinetics for NMN, but again with the sigmoidal dependence on the ATP concentration. In this case, the apparent cooperativity is unexpected since only a single ATP is used in the NMN adenylyltransferase catalyzed reaction. To determine the possible structural determinants of such cooperativity, we solved the crystal structure of NadR from L. lactis (NadRLl). Co-crystallization with NAD, NR, NMN, ATP, and AMP-PNP revealed a ‘sink’ for adenine nucleotides in a location between two domains. This sink could be a regulatory site, or it may facilitate the channeling of substrates between the two domains.

Rv2131c gene product: An unconventional enzyme that is both inositol monophosphatase and fructose-1,6-bisphosphatase

2006 ◽  
Vol 339 (3) ◽  
pp. 897-904 ◽  
Author(s):  
Xiaoling Gu ◽  
Mao Chen ◽  
Hongbo Shen ◽  
Xin Jiang ◽  
Yishu Huang ◽  
...  
Keyword(s):  
Gene Product ◽  
Inositol Monophosphatase ◽  
Fructose 1,6 Bisphosphatase

scholarly journals Structural basis of Naa20 activity towards a canonical NatB substrate

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Dominik Layer ◽  
Jürgen Kopp ◽  
Miriam Fontanillo ◽  
Maja Köhn ◽  
Karine Lapouge ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Drug Development ◽  
Catalytic Mechanism ◽  
Peptide Binding ◽  
Competitive Inhibitor ◽  
Structural Basis ◽  
Substrate Affinity ◽  
Protein Homeostasis ◽  
Auxiliary Subunit

AbstractN-terminal acetylation is one of the most common protein modifications in eukaryotes and is carried out by N-terminal acetyltransferases (NATs). It plays important roles in protein homeostasis, localization, and interactions and is linked to various human diseases. NatB, one of the major co-translationally active NATs, is composed of the catalytic subunit Naa20 and the auxiliary subunit Naa25, and acetylates about 20% of the proteome. Here we show that NatB substrate specificity and catalytic mechanism are conserved among eukaryotes, and that Naa20 alone is able to acetylate NatB substrates in vitro. We show that Naa25 increases the Naa20 substrate affinity, and identify residues important for peptide binding and acetylation activity. We present the first Naa20 crystal structure in complex with the competitive inhibitor CoA-Ac-MDEL. Our findings demonstrate how Naa20 binds its substrates in the absence of Naa25 and support prospective endeavors to derive specific NAT inhibitors for drug development.

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

2015 ◽  
Vol 71 (12) ◽  
pp. 2505-2512 ◽  
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.

Crystal Structure of the Mycobacterium tuberculosis dUTPase: Insights into the Catalytic Mechanism

2004 ◽  
Vol 341 (2) ◽  
pp. 503-517 ◽  
Author(s):  
Sum Chan ◽  
Brent Segelke ◽  
Timothy Lekin ◽  
Heike Krupka ◽  
Uhn Soo Cho ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Mycobacterium Tuberculosis ◽  
Catalytic Mechanism

Crystal structure of the wild-type and D30A mutant thioredoxin h of Chlamydomonas reinhardtii and implications for the catalytic mechanism

2001 ◽  
Vol 359 (1) ◽  
pp. 65-75 ◽  
Author(s):  
Valeria MENCHISE ◽  
Catherine CORBIER ◽  
Claude DIDIERJEAN ◽  
Michele SAVIANO ◽  
Ettore BENEDETTI ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Proton Transfer ◽  
Chlamydomonas Reinhardtii ◽  
Catalytic Mechanism ◽  
Structural Data ◽  
Careful Analysis ◽  
Wild Type ◽  
Thioredoxin H ◽  
Dynamics Simulations

Thioredoxins are ubiquitous proteins which catalyse the reduction of disulphide bridges on target proteins. The catalytic mechanism proceeds via a mixed disulphide intermediate whose breakdown should be enhanced by the involvement of a conserved buried residue, Asp-30, as a base catalyst towards residue Cys-39. We report here the crystal structure of wild-type and D30A mutant thioredoxin h from Chlamydomonas reinhardtii, which constitutes the first crystal structure of a cytosolic thioredoxin isolated from a eukaryotic plant organism. The role of residue Asp-30 in catalysis has been revisited since the distance between the carboxylate OD1 of Asp-30 and the sulphur SG of Cys-39 is too great to support the hypothesis of direct proton transfer. A careful analysis of all available crystal structures reveals that the relative positioning of residues Asp-30 and Cys-39 as well as hydrophobic contacts in the vicinity of residue Asp-30 do not allow a conformational change sufficient to bring the two residues close enough for a direct proton transfer. This suggests that protonation/deprotonation of Cys-39 should be mediated by a water molecule. Molecular-dynamics simulations, carried out either in vacuo or in water, as well as proton-inventory experiments, support this hypothesis. The results are discussed with respect to biochemical and structural data.

scholarly journals Crystal structure of human mARC1 reveals its exceptional position among eukaryotic molybdenum enzymes

2018 ◽  
Vol 115 (47) ◽  
pp. 11958-11963 ◽  
Author(s):  
Christian Kubitza ◽  
Florian Bittner ◽  
Carsten Ginsel ◽  
Antje Havemeyer ◽  
Bernd Clement ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Catalytic Mechanism ◽  
Full Range ◽  
3D Structure ◽  
Cytochrome P450 Enzymes ◽  
Oxygenated Compounds ◽  
Online Databases ◽  
Starting Point ◽  
Wide Range ◽  
Nitrogen Containing Compounds

Biotransformation enzymes ensure a viable homeostasis by regulating reversible cycles of oxidative and reductive reactions. The metabolism of nitrogen-containing compounds is of high pharmaceutical and toxicological relevance because N-oxygenated metabolites derived from reactions mediated by cytochrome P450 enzymes or flavin-dependent monooxygenases are in some cases highly toxic or mutagenic. The molybdenum-dependent mitochondrial amidoxime-reducing component (mARC) was found to be an extremely efficient counterpart, which is able to reduce the full range of N-oxygenated compounds and thereby mediates detoxification reactions. However, the 3D structure of this enzyme was unknown. Here we present the high-resolution crystal structure of human mARC. We give detailed insight into the coordination of its molybdenum cofactor (Moco), the catalytic mechanism, and its ability to reduce a wide range of N-oxygenated compounds. The identification of two key residues will allow future discrimination between mARC paralogs and ensure correct annotation. Since our structural findings contradict in silico predictions that are currently made by online databases, we propose domain definitions for members of the superfamily of Moco sulfurase C-terminal (MOSC) domain-containing proteins. Furthermore, we present evidence for an evolutionary role of mARC for the emergence of the xanthine oxidase protein superfamily. We anticipate the hereby presented crystal structure to be a starting point for future descriptions of MOSC proteins, which are currently poorly structurally characterized.

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