Structure and allosteric regulation of eukaryotic 6-phosphofructokinases

2013 ◽  
Vol 394 (8) ◽  
pp. 977-993 ◽  
Author(s):  
Torsten Schöneberg ◽  
Marco Kloos ◽  
Antje Brüser ◽  
Jürgen Kirchberger ◽  
Norbert Sträter
Keyword(s):  
Crystal Structures ◽  
Structural Information ◽  
Current Knowledge ◽  
Allosteric Regulation ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Directed Mutagenesis ◽  
Special Focus ◽  
Molecular Architecture ◽  
Key Enzyme

Abstract Although the crystal structures of prokaryotic 6-phosphofructokinase, a key enzyme of glycolysis, have been available for almost 25 years now, structural information about the more complex and highly regulated eukaryotic enzymes is still lacking until now. This review provides an overview of the current knowledge of eukaryotic 6-phosphofructokinase based on recent crystal structures, kinetic analyses and site-directed mutagenesis data with special focus on the molecular architecture and the structural basis of allosteric regulation.

Crystal structures of Magnaporthe oryzae trehalose-6-phosphate synthase (MoTps1) suggest a model for catalytic process of Tps1

2019 ◽  
Vol 476 (21) ◽  
pp. 3227-3240 ◽  
Author(s):  
Shanshan Wang ◽  
Yanxiang Zhao ◽  
Long Yi ◽  
Minghe Shen ◽  
Chao Wang ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Conformational Changes ◽  
Magnaporthe Oryzae ◽  
Structural Information ◽  
Complex Structure ◽  
Critical Role ◽  
Catalytic Process ◽  
Structural Basis ◽  
Salt Bridges ◽  
Terminal Domain

Trehalose-6-phosphate (T6P) synthase (Tps1) catalyzes the formation of T6P from UDP-glucose (UDPG) (or GDPG, etc.) and glucose-6-phosphate (G6P), and structural basis of this process has not been well studied. MoTps1 (Magnaporthe oryzae Tps1) plays a critical role in carbon and nitrogen metabolism, but its structural information is unknown. Here we present the crystal structures of MoTps1 apo, binary (with UDPG) and ternary (with UDPG/G6P or UDP/T6P) complexes. MoTps1 consists of two modified Rossmann-fold domains and a catalytic center in-between. Unlike Escherichia coli OtsA (EcOtsA, the Tps1 of E. coli), MoTps1 exists as a mixture of monomer, dimer, and oligomer in solution. Inter-chain salt bridges, which are not fully conserved in EcOtsA, play primary roles in MoTps1 oligomerization. Binding of UDPG by MoTps1 C-terminal domain modifies the substrate pocket of MoTps1. In the MoTps1 ternary complex structure, UDP and T6P, the products of UDPG and G6P, are detected, and substantial conformational rearrangements of N-terminal domain, including structural reshuffling (β3–β4 loop to α0 helix) and movement of a ‘shift region' towards the catalytic centre, are observed. These conformational changes render MoTps1 to a ‘closed' state compared with its ‘open' state in apo or UDPG complex structures. By solving the EcOtsA apo structure, we confirmed that similar ligand binding induced conformational changes also exist in EcOtsA, although no structural reshuffling involved. Based on our research and previous studies, we present a model for the catalytic process of Tps1. Our research provides novel information on MoTps1, Tps1 family, and structure-based antifungal drug design.

scholarly journals Structural basis of the stereoselective formation of the spirooxindole ring in the biosynthesis of citrinadins

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Zhiwen Liu ◽  
Fanglong Zhao ◽  
Boyang Zhao ◽  
Jie Yang ◽  
Joseph Ferrara ◽  
...  
Keyword(s):  
High Resolution ◽  
Organic Synthesis ◽  
Indole Alkaloids ◽  
Site Directed Mutagenesis ◽  
Structural Basis ◽  
Directed Mutagenesis ◽  
Computational Studies ◽  
X Ray ◽  
Evolutionary Branch ◽  
Catalytic Mechanisms

AbstractPrenylated indole alkaloids featuring spirooxindole rings possess a 3R or 3S carbon stereocenter, which determines the bioactivities of these compounds. Despite the stereoselective advantages of spirooxindole biosynthesis compared with those of organic synthesis, the biocatalytic mechanism for controlling the 3R or 3S-spirooxindole formation has been elusive. Here, we report an oxygenase/semipinacolase CtdE that specifies the 3S-spirooxindole construction in the biosynthesis of 21R-citrinadin A. High-resolution X-ray crystal structures of CtdE with the substrate and cofactor, together with site-directed mutagenesis and computational studies, illustrate the catalytic mechanisms for the possible β-face epoxidation followed by a regioselective collapse of the epoxide intermediate, which triggers semipinacol rearrangement to form the 3S-spirooxindole. Comparing CtdE with PhqK, which catalyzes the formation of the 3R-spirooxindole, we reveal an evolutionary branch of CtdE in specific 3S spirocyclization. Our study provides deeper insights into the stereoselective catalytic machinery, which is important for the biocatalysis design to synthesize spirooxindole pharmaceuticals.

scholarly journals Structural basis for the functional properties of the P2X7 receptor for extracellular ATP

2021 ◽  
Author(s):  
Lin-Hua Jiang ◽  
Emily A. Caseley ◽  
Steve P. Muench ◽  
Sébastien Roger
Keyword(s):  
Functional Properties ◽  
P2x7 Receptor ◽  
Site Directed Mutagenesis ◽  
P2x Receptor ◽  
Structural Basis ◽  
Structure Characteristic ◽  
Purinergic Signalling ◽  
Directed Mutagenesis ◽  
Important Mediator ◽  
Receptor Family

AbstractThe P2X7 receptor, originally known as the P2Z receptor due to its distinctive functional properties, has a structure characteristic of the ATP-gated ion channel P2X receptor family. The P2X7 receptor is an important mediator of ATP-induced purinergic signalling and is involved the pathogenesis of numerous conditions as well as in the regulation of diverse physiological functions. Functional characterisations, in conjunction with site-directed mutagenesis, molecular modelling, and, recently, structural determination, have provided significant insights into the structure–function relationships of the P2X7 receptor. This review discusses the current understanding of the structural basis for the functional properties of the P2X7 receptor.

scholarly journals Domain sliding of two Staphylococcus aureus N-acetylglucosaminidases enables their substrate-binding prior to its catalysis

2020 ◽  
Vol 3 (1) ◽  
Author(s):  
Sara Pintar ◽  
Jure Borišek ◽  
Aleksandra Usenik ◽  
Andrej Perdih ◽  
Dušan Turk
Keyword(s):  
Crystal Structures ◽  
Active Site ◽  
Drug Targets ◽  
Substrate Binding ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Human Pathogen ◽  
Active Site Cleft ◽  
Novel Drug ◽  
Novel Drug Targets

AbstractTo achieve productive binding, enzymes and substrates must align their geometries to complement each other along an entire substrate binding site, which may require enzyme flexibility. In pursuit of novel drug targets for the human pathogen S. aureus, we studied peptidoglycan N-acetylglucosaminidases, whose structures are composed of two domains forming a V-shaped active site cleft. Combined insights from crystal structures supported by site-directed mutagenesis, modeling, and molecular dynamics enabled us to elucidate the substrate binding mechanism of SagB and AtlA-gl. This mechanism requires domain sliding from the open form observed in their crystal structures, leading to polysaccharide substrate binding in the closed form, which can enzymatically process the bound substrate. We suggest that these two hydrolases must exhibit unusual extents of flexibility to cleave the rigid structure of a bacterial cell wall.

scholarly journals Structural Insights into 6-Hydroxypseudooxynicotine Amine Oxidase from Pseudomonas geniculata N1, the Key Enzyme Involved in Nicotine Degradation

2020 ◽  
Vol 86 (19) ◽  
Author(s):  
Gongquan Liu ◽  
Weiwei Wang ◽  
Fangyuan He ◽  
Peng Zhang ◽  
Ping Xu ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Structural Information ◽  
Amine Oxidase ◽  
Substrate Binding ◽  
Docking Study ◽  
Site Directed Mutagenesis ◽  
Flavin Mononucleotide ◽  
Structure Comparison ◽  
Content Type ◽  
Key Enzyme

ABSTRACT Bacteria degrade nicotine mainly using pyridine and pyrrolidine pathways. Previously, we discovered a hybrid of the pyridine and pyrrolidine pathways (the VPP pathway) in Pseudomonas geniculata N1 and characterized its key enzyme, 6-hydroxypseudooxynicotine amine oxidase (HisD). It catalyzes oxidative deamination of 6-hydroxypseudooxynicotine to 6-hydroxy-3-succinoylsemialdehyde-pyridine, which is the crucial step connecting upstream and downstream portions of the VPP pathway. We determined the crystal structure of wild-type HisD to 2.6 Å. HisD is a monomer that contains a flavin mononucleotide, an iron-sulfur cluster, and ADP. On the basis of sequence alignment and structure comparison, a difference has been found among HisD, closely related trimethylamine dehydrogenase (TMADH), and histamine dehydrogenase (HADH). The flavin mononucleotide (FMN) cofactor is not covalently bound to any residue, and the FMN isoalloxazine ring is planar in HisD compared to TMADH or HADH, which forms a 6-S-cysteinyl flavin mononucleotide cofactor and has an FMN isoalloxazine ring in a “butterfly bend” conformation. Based on the structure, docking study, and site-directed mutagenesis, the residues Glu60, Tyr170, Asp262, and Trp263 may be involved in substrate binding. The expanded understanding of the substrate binding mode from this study may guide rational engineering of such enzymes for biodegradation of potential pollutants or for bioconversion to generate desired products. IMPORTANCE Nicotine is a major tobacco alkaloid in tobacco waste. Pyridine and pyrrolidine pathways are the two best-elucidated nicotine metabolic pathways; Pseudomonas geniculata N1 catabolizes nicotine via a hybrid between the pyridine and pyrrolidine pathways. The crucial enzyme, 6-hydroxypseudooxynicotine amine oxidase (HisD), links the upstream and downstream portions of the VPP pathway; however, there is little structural information about this important enzyme. In this study, we determined the crystal structure of HisD from Pseudomonas geniculata N1. Its basic insights about the structure may help us to guide the engineering of such enzymes for bioremediation and bioconversion applications.

A special halide-stabilizing pair and enantioselectivity mechanism of DatA

2014 ◽  
Vol 70 (a1) ◽  
pp. C450-C450
Author(s):  
Lijun Guan ◽  
Hideya Yabuki ◽  
Masahiko Okai ◽  
Jun Ohtsuka ◽  
Masaru Tanokura
Keyword(s):  
Reaction Mechanism ◽  
Crystal Structures ◽  
Structural Information ◽  
Docking Simulation ◽  
Competitive Inhibitor ◽  
Residue Pair ◽  
Structural Basis ◽  
Chiral Compounds ◽  
In The Wild ◽  
Agrobacterium Tumefaciens C58

A novel haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 belongs to the HLD-II subfamily and hydrolyzes brominated and iodinated compounds, leading to the generation of the corresponding alcohol, a halide ion, and a proton. DatA possesses a unique Asn-Tyr residue pair instead of the Asn-Trp residue pair conserved among the subfamily members, thus the structural basis for its reaction mechanism merits elucidation. In addition, DatA is potentially useful for pharmaceutical and environmental applications, though several crystal structures of HLD-II dehalogenases have been reported so far, the determination of the DatA structure will provide an important contribution to those fields. This work provided insight into the reaction mechanism of DatA via a combination of X-ray crystallographic and computational analysis. The crystal structures of DatA and the Y109W mutant were determined at 1.70 Å [1] and 1.95 Å, respectively. The location of the active site was confirmed by using its novel competitive inhibitor, CHES. The structural information from these two crystal structures and the docking simulation with 1,3-dibromopropane revealed that the replacement of the Asn-Tyr pair with the Asn-Trp pair increases the binding affinity for 1,3-dibromopropane, due to the extra hydrogen bond between Trp109 and halogenated compounds; and that the key residue to bind halogenated substrate is only Asn43 in the wild-type DatA, while those in the Y109W mutant are the Asn-Trp pair. Furthermore, docking simulation using the crystal structures of DatA and some chiral compounds indicated that enantioselectivity of DatA toward brominated alkanes is determined by the large and small spaces around the halogen binding site.

scholarly journals Conserved Residues Control the T1R3-Specific Allosteric Signaling Pathway of the Mammalian Sweet-Taste Receptor

2019 ◽  
Vol 44 (5) ◽  
pp. 303-310 ◽  
Author(s):  
Jean-Baptiste Chéron ◽  
Amanda Soohoo ◽  
Yi Wang ◽  
Jérôme Golebiowski ◽  
Serge Antonczak ◽  
...  
Keyword(s):  
Transmembrane Domain ◽  
Taste Receptor ◽  
Receptor Activation ◽  
Sweet Taste ◽  
Site Directed Mutagenesis ◽  
Activation Mechanism ◽  
Structural Basis ◽  
Directed Mutagenesis ◽  
Conserved Residues ◽  
Sweet Taste Receptor

Abstract Mammalian sensory systems detect sweet taste through the activation of a single heteromeric T1R2/T1R3 receptor belonging to class C G-protein-coupled receptors. Allosteric ligands are known to interact within the transmembrane domain, yet a complete view of receptor activation remains elusive. By combining site-directed mutagenesis with computational modeling, we investigate the structure and dynamics of the allosteric binding pocket of the T1R3 sweet-taste receptor in its apo form, and in the presence of an allosteric ligand, cyclamate. A novel positively charged residue at the extracellular loop 2 is shown to interact with the ligand. Molecular dynamics simulations capture significant differences in the behavior of a network of conserved residues with and without cyclamate, although they do not directly interact with the allosteric ligand. Structural models show that they adopt alternate conformations, associated with a conformational change in the transmembrane region. Site-directed mutagenesis confirms that these residues are unequivocally involved in the receptor function and the allosteric signaling mechanism of the sweet-taste receptor. Similar to a large portion of the transmembrane domain, they are highly conserved among mammals, suggesting an activation mechanism that is evolutionarily conserved. This work provides a structural basis for describing the dynamics of the receptor, and for the rational design of new sweet-taste modulators.

scholarly journals Structure of lipoprotein lipase in complex with GPIHBP1

2019 ◽  
Vol 116 (21) ◽  
pp. 10360-10365 ◽  
Author(s):  
Rishi Arora ◽  
Amitabh V. Nimonkar ◽  
Daniel Baird ◽  
Chunhua Wang ◽  
Chun-Hao Chiu ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Lipoprotein Lipase ◽  
Structural Information ◽  
Density Lipoprotein ◽  
Lipid Binding ◽  
Structural Basis ◽  
Maturation Factor ◽  
Lipase Maturation Factor 1 ◽  
Hydrolysis Of

Lipoprotein lipase (LPL) plays a central role in triglyceride (TG) metabolism. By catalyzing the hydrolysis of TGs present in TG-rich lipoproteins (TRLs), LPL facilitates TG utilization and regulates circulating TG and TRL concentrations. Until very recently, structural information for LPL was limited to homology models, presumably due to the propensity of LPL to unfold and aggregate. By coexpressing LPL with a soluble variant of its accessory protein glycosylphosphatidylinositol-anchored high-density lipoprotein binding protein 1 (GPIHBP1) and with its chaperone protein lipase maturation factor 1 (LMF1), we obtained a stable and homogenous LPL/GPIHBP1 complex that was suitable for structure determination. We report here X-ray crystal structures of human LPL in complex with human GPIHBP1 at 2.5–3.0 Å resolution, including a structure with a novel inhibitor bound to LPL. Binding of the inhibitor resulted in ordering of the LPL lid and lipid-binding regions and thus enabled determination of the first crystal structure of LPL that includes these important regions of the protein. It was assumed for many years that LPL was only active as a homodimer. The structures and additional biochemical data reported here are consistent with a new report that LPL, in complex with GPIHBP1, can be active as a monomeric 1:1 complex. The crystal structures illuminate the structural basis for LPL-mediated TRL lipolysis as well as LPL stabilization and transport by GPIHBP1.

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