Crystal Structure of Inorganic Pyrophosphatase From Schistosoma japonicum Reveals the Mechanism of Chemicals and Substrate Inhibition

Soluble inorganic pyrophosphatases (PPases) are essential for facilitating the growth and development of organisms, making them attractive functional proteins. To provide insight into the molecular basis of PPases in Schistosoma japonicum (SjPPase), we expressed the recombinant SjPPase, analyzed the hydrolysis mechanism of inorganic pyrophosphate (PPi), and measured its activity. Moreover, we solved the crystal structure of SjPPase in complex with orthophosphate (Pi) and performed PPi and methylene diphosphonic acid (MDP) docking into the active site. Our results suggest that the SjPPase possesses PPi hydrolysis activity, and the activity declines with increased MDP or NaF concentration. However, the enzyme shows unexpected substrate inhibition properties. Through PPi metabolic pathway analysis, the physiological action of substrate inhibition might be energy saving, adaptably cytoprotective, and biosynthetic rate regulating. Furthermore, the structure of apo-SjPPase and SjPPase with Pi has been solved at 2.6 and 2.3 Å, respectively. The docking of PPi into the active site of the SjPPase-Pi complex revealed that substrate inhibition might result from blocking Pi exit due to excess PPi in the SjPPase-Pi complex of the catalytic cycle. Our results revealed the structural features of apo-SjPPase and the SjPPase-Pi complex by X-ray crystallography, providing novel insights into the physiological functions of PPase in S. japonicum without the PPi transporter and the mechanism of its substrate inhibition.

Download Full-text

Mechanism of IPPase shown by high resolution Neutron and X-ray crystallography

Acta Crystallographica Section A Foundations and Advances ◽

10.1107/s2053273314087889 ◽

2014 ◽

Vol 70 (a1) ◽

pp. C1211-C1211

Author(s):

Joseph Ng ◽

Ronny Hughes ◽

Michelle Morris ◽

Leighton Coates ◽

Matthew Blakeley ◽

...

Keyword(s):

High Resolution ◽

Active Site ◽

Inorganic Pyrophosphatase ◽

Inorganic Pyrophosphate ◽

X Ray ◽

X Ray Crystallography ◽

Cellular Processes ◽

Crystallographic Structures ◽

Hydrolysis Of ◽

Low Energy Conformations

Soluble inorganic pyrophosphatase (IPPase) catalyzes the hydrolysis of inorganic pyrophosphate (PPi) to form orthophosphate (Pi). The action of this enzyme shifts the overall equilibrium in favor of synthesis during a number of ATP-dependent cellular processes such as in the polymerization of nucleic acids, production of coenzymes and proteins and sulfate assimilation pathways. Two Neutron crystallographic (2.10-2.50Å) and five high-resolution X-ray (0.99Å-1.92Å) structures of the archaeal IPPase from Thermococcus thioreducens have been determined under both cryo and room temperatures. The structures determined include the recombinant IPPase bound to Mg+2, Ca+2, Br-, SO2-2 or PO4-2 involving those with non-hydrolyzed and hydrolyzed pyrophosphate complexes. All the crystallographic structures provide snapshots of the active site corresponding to different stages of the hydrolysis of inorganic pyrophosphate. As a result, a structure-based model of IPPase catalysis is devised showing the enzyme's low-energy conformations, hydration states, movements and nucleophile generation within the active site.

Download Full-text

Structural insights into dihydroxylation of terephthalate, a product of polyethylene terephthalate degradation

Journal of Bacteriology ◽

10.1128/jb.00543-21 ◽

2022 ◽

Author(s):

Jai Krishna Mahto ◽

Neetu Neetu ◽

Monica Sharma ◽

Monika Dubey ◽

Bhanu Prakash Vellanki ◽

...

Keyword(s):

Crystal Structure ◽

Substrate Specificity ◽

Polyethylene Terephthalate ◽

Active Site ◽

Catalytic Domain ◽

Structural Features ◽

Comamonas Testosteroni ◽

Plastic Pollution ◽

Microbial Utilization ◽

Steady State Kinetics

Biodegradation of terephthalate (TPA) is a highly desired catabolic process for the bacterial utilization of this Polyethylene terephthalate (PET) depolymerization product, but to date, the structure of terephthalate dioxygenase (TPDO), a Rieske oxygenase (RO) that catalyzes the dihydroxylation of TPA to a cis -diol is unavailable. In this study, we characterized the steady-state kinetics and first crystal structure of TPDO from Comamonas testosteroni KF1 (TPDO KF1 ). The TPDO KF1 exhibited the substrate specificity for TPA ( k cat / K m = 57 ± 9 mM −1 s −1 ). The TPDO KF1 structure harbors characteristics RO features as well as a unique catalytic domain that rationalizes the enzyme’s function. The docking and mutagenesis studies reveal that its substrate specificity to TPA is mediated by Arg309 and Arg390 residues, two residues positioned on opposite faces of the active site. Additionally, residue Gln300 is also proven to be crucial for the activity, its substitution to alanine decreases the activity ( k cat ) by 80%. Together, this study delineates the structural features that dictate the substrate recognition and specificity of TPDO. Importance The global plastic pollution has become the most pressing environmental issue. Recent studies on enzymes depolymerizing polyethylene terephthalate plastic into terephthalate (TPA) show some potential in tackling this. Microbial utilization of this released product, TPA is an emerging and promising strategy for waste-to-value creation. Research from the last decade has discovered terephthalate dioxygenase (TPDO), as being responsible for initiating the enzymatic degradation of TPA in a few Gram-negative and Gram-positive bacteria. Here, we have determined the crystal structure of TPDO from Comamonas testosteroni KF1 and revealed that it possesses a unique catalytic domain featuring two basic residues in the active site to recognize TPA. Biochemical and mutagenesis studies demonstrated the crucial residues responsible for the substrate specificity of this enzyme.

Download Full-text

A trapped human PPM1A–phosphopeptide complex reveals structural features critical for regulation of PPM protein phosphatase activity

Journal of Biological Chemistry ◽

10.1074/jbc.ra117.001213 ◽

2018 ◽

Vol 293 (21) ◽

pp. 7993-8008 ◽

Cited By ~ 8

Author(s):

Subrata Debnath ◽

Dalibor Kosek ◽

Harichandra D. Tagad ◽

Stewart R. Durell ◽

Daniel H. Appella ◽

...

Keyword(s):

Crystal Structure ◽

Metal Ions ◽

Active Site ◽

Metal Binding ◽

Metal Ion ◽

Catalytic Domain ◽

Protein Phosphatases ◽

Random Order ◽

Structural Features

Metal-dependent protein phosphatases (PPM) are evolutionarily unrelated to other serine/threonine protein phosphatases and are characterized by their requirement for supplementation with millimolar concentrations of Mg2+ or Mn2+ ions for activity in vitro. The crystal structure of human PPM1A (also known as PP2Cα), the first PPM structure determined, displays two tightly bound Mn2+ ions in the active site and a small subdomain, termed the Flap, located adjacent to the active site. Some recent crystal structures of bacterial or plant PPM phosphatases have disclosed two tightly bound metal ions and an additional third metal ion in the active site. Here, the crystal structure of the catalytic domain of human PPM1A, PPM1Acat, complexed with a cyclic phosphopeptide, c(MpSIpYVA), a cyclized variant of the activation loop of p38 MAPK (a physiological substrate of PPM1A), revealed three metal ions in the active site. The PPM1Acat D146E–c(MpSIpYVA) complex confirmed the presence of the anticipated third metal ion in the active site of metazoan PPM phosphatases. Biophysical and computational methods suggested that complex formation results in a slightly more compact solution conformation through reduced conformational flexibility of the Flap subdomain. We also observed that the position of the substrate in the active site allows solvent access to the labile third metal-binding site. Enzyme kinetics of PPM1Acat toward a phosphopeptide substrate supported a random-order, bi-substrate mechanism, with substantial interaction between the bound substrate and the labile metal ion. This work illuminates the structural and thermodynamic basis of an innate mechanism regulating the activity of PPM phosphatases.

Download Full-text

The genome of a Bacteroidetes inhabitant of the human gut encodes a structurally distinct enoyl-acyl carrier protein reductase (FabI)

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.013336 ◽

2020 ◽

Vol 295 (22) ◽

pp. 7635-7652

Author(s):

Christopher D. Radka ◽

Matthew W. Frank ◽

Jiangwei Yao ◽

Jayaraman Seetharaman ◽

Darcie J. Miller ◽

...

Keyword(s):

Crystal Structure ◽

Conformational Change ◽

Conformational Changes ◽

Active Site ◽

Acyl Carrier Protein ◽

Acid Synthesis ◽

Binding Pocket ◽

Structural Features ◽

Antibacterial Drug ◽

Carrier Protein

Enoyl-acyl carrier protein reductase (FabI) catalyzes a rate-controlling step in bacterial fatty-acid synthesis and is a target for antibacterial drug development. A phylogenetic analysis shows that FabIs fall into four divergent clades. Members of clades 1–3 have been structurally and biochemically characterized, but the fourth clade, found in members of phylum Bacteroidetes, is uncharacterized. Here, we identified the unique structure and conformational changes that distinguish clade 4 FabIs. Alistipes finegoldii is a prototypical Bacteroidetes inhabitant of the gut microbiome. We found that A. finegoldii FabI (AfFabI) displays cooperative kinetics and uses NADH as a cofactor, and its crystal structure at 1.72 Å resolution showed that it adopts a Rossmann fold as do other characterized FabIs. It also disclosed a carboxyl-terminal extension that forms a helix–helix interaction that links the protomers as a unique feature of AfFabI. An AfFabI·NADH crystal structure at 1.86 Å resolution revealed that this feature undergoes a large conformational change to participate in covering the NADH-binding pocket and establishing the water channels that connect the active site to the central water well. Progressive deletion of these interactions led to catalytically compromised proteins that fail to bind NADH. This unique conformational change imparted a distinct shape to the AfFabI active site that renders it refractory to a FabI drug that targets clade 1 and 3 pathogens. We conclude that the clade 4 FabI, found in the Bacteroidetes inhabitants of the gut, have several structural features and conformational transitions that distinguish them from other bacterial FabIs.

Download Full-text

Crystal structure of human dehydroepiandrosterone sulphotransferase in complex with substrate

Biochemical Journal ◽

10.1042/bj3640165 ◽

2002 ◽

Vol 364 (1) ◽

pp. 165-171 ◽

Cited By ~ 58

Author(s):

Peter H. REHSE ◽

Ming ZHOU ◽

Sheng-Xiang LIN

Keyword(s):

Crystal Structure ◽

Hydrogen Bond ◽

Active Site ◽

Substrate Binding ◽

Substrate Inhibition ◽

Molecular Replacement ◽

Alternative Substrate ◽

Hydrophobic Residues ◽

Sequence Identity ◽

Binding Orientations

Dehydroepiandrosterone sulphotransferase (DHEA-ST) is an enzyme that converts dehydroepiandrosterone (DHEA), and some other steroids, into their sulphonated forms. The enzyme catalyses the sulphonation of DHEA on the 3α-oxygen, with 3′-phosphoadenosine-5′-phosphosulphate contributing the sulphate. The structure of human DHEA-ST in complex with its preferred substrate DHEA has been solved here to 1.99Å using molecular replacement with oestradiol sulphotransferase (37% sequence identity) as a model. Two alternative substrate-binding orientations have been identified. The primary, catalytic, orientation has the DHEA 3α-oxygen and the highly conserved catalytic histidine in nearly identical positions as are seen for the related oestradiol sulphotransferase. The substrate, however, shows rotations of up to 30°, and there is a corresponding rearrangement of the protein loops contributing to the active site. This may also reflect the low identity between the two enzymes. The second orientation penetrates further into the active site and can form a potential hydrogen bond with the desulphonated cofactor 3′,5′-phosphoadenosine (PAP). This second site contains more van der Waal interactions with hydrophobic residues than the catalytic site and may also reflect the substrate-inhibition site. The PAP position was obtained from the previously solved structure of DHEA-ST co-crystallized with PAP. This latter structure, due to the arrangement of loops within the active site and monomer interactions, cannot bind substrate. The results presented here describe details of substrate binding to DHEA-ST and the potential relationship to substrate inhibition.

Download Full-text

Preparation and Crystal Structure of a Rhenium Analogue of the Cationic Renal Agent, Tc-99m Diaminocyclohexane

Metal-Based Drugs ◽

10.1155/mbd.2000.141 ◽

2000 ◽

Vol 7 (3) ◽

pp. 141-145 ◽

Cited By ~ 2

Author(s):

Luigi G. Marzilli ◽

Lory Hansen ◽

Andrew Taylor ◽

Rene Lachicotte

Keyword(s):

Crystal Structure ◽

Plasma Flow ◽

Structural Parameters ◽

Renal Plasma Flow ◽

Chemical Study ◽

Structural Features ◽

Racemic Mixture ◽

X Ray ◽

X Ray Crystallography ◽

Renal Agent

We report here a chemical study on a Re analogue of one of the few cationic Tc-99m tracers previously investigated as an agent for effective renal plasma flow (ERPF) measurement. Cationic Tc-99m tracers have the potential for overcoming problems associated with common anionic Tc-99m tracers in patients who have developed a uremic state. The Tc-99m-DACH tracer, prepared from 1,2-diaminocyclohexane (1,2-DACH), is the only cationic renal agent tested in humans and has seven possible isomers. The complex isolated from the reaction of the racemic mixture, (±)-trans-1,2-DACH, and ReIO2(PPh3)2 after conversion to the BPh4- salt was found by X-ray crystallography to be the meso isomer, trans-[ReO2 (trans-R,R-1,2-DACH)(trans-S,S-l,2-DACH)][BPh4]·MeOH·2H2O (1). The structural parameters for 1 are normal. The complex is highly symmetrical, suggesting that the analogous meso Tc-99m-DACH agent is also symmetrical. Studies of other Tc-99m-DACH agents that were made from cis-1,2-DACH or individual trans-1,2-DACH enantiomers show that the biodistribution is not very dependent on the starting 1,2-DACH ligand stereochemistry; these agents must be less symmetrical than the meso Tc-99m-DACH agent analogue of 1. Thus, the overall charge and lipophilicity (similar for all Tc-99m-DACH isomers) exert a greater influence on biodistribution than the specific structural features of the different Tc-99m-DACH isomers.

Download Full-text

Structural insight into arginine methylation by the mouse protein arginine methyltransferase 7: a zinc finger freezes the mimic of the dimeric state into a single active site

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004714014278 ◽

2014 ◽

Vol 70 (9) ◽

pp. 2401-2412 ◽

Cited By ~ 22

Author(s):

Vincent Cura ◽

Nathalie Troffer-Charlier ◽

Jean-Marie Wurtz ◽

Luc Bonnefond ◽

Jean Cavarelli

Keyword(s):

Crystal Structure ◽

Zinc Finger ◽

Active Site ◽

Rna Splicing ◽

Structural Features ◽

Arginine Methylation ◽

Protein Arginine Methyltransferase ◽

Arginine Methyltransferase ◽

Damage Repair ◽

The Family

Protein arginine methyltransferase 7 (PRMT7) is a type III arginine methyltransferase which has been implicated in several biological processes such as transcriptional regulation, DNA damage repair, RNA splicing, cell differentiation and metastasis. PRMT7 is a unique but less characterized member of the family of PRMTs. The crystal structure of full-length PRMT7 fromMus musculusrefined at 1.7 Å resolution is described. The PRMT7 structure is composed of two catalytic modules in tandem forming a pseudo-dimer and contains only one AdoHcy molecule bound to the N-terminal module. The high-resolution crystal structure presented here revealed several structural features showing that the second active site is frozen in an inactive state by a conserved zinc finger located at the junction between the two PRMT modules and by the collapse of two degenerated AdoMet-binding loops.

Download Full-text

Crystal Structure of Complete Rhinovirus RNA Polymerase Suggests Front Loading of Protein Primer

Journal of Virology ◽

10.1128/jvi.79.1.277-288.2005 ◽

2005 ◽

Vol 79 (1) ◽

pp. 277-288 ◽

Cited By ~ 47

Author(s):

Todd C. Appleby ◽

Hartmut Luecke ◽

Jae Hoon Shim ◽

Jim Z. Wu ◽

I. Wayne Cheney ◽

...

Keyword(s):

Crystal Structure ◽

Rna Polymerase ◽

Active Site ◽

Catalytic Mechanism ◽

Structural Information ◽

Structural Features ◽

Replication Initiation ◽

Front Face ◽

Large Cleft ◽

Active Site Cavity

ABSTRACT Picornaviruses utilize virally encoded RNA polymerase and a uridylylated protein primer to ensure replication of the entire viral genome. The molecular details of this mechanism are not well understood due to the lack of structural information. We report the crystal structure of human rhinovirus 16 3D RNA-dependent RNA polymerase (HRV16 3Dpol) at a 2.4-Å resolution, representing the first complete polymerase structure from the Picornaviridae family. HRV16 3Dpol shares the canonical features of other known polymerase structures and contains an N-terminal region that tethers the fingers and thumb subdomains, forming a completely encircled active site cavity which is accessible through a small tunnel on the backside of the molecule. The small thumb subdomain contributes to the formation of a large cleft on the front face of the polymerase which also leads to the active site. The cleft appears large enough to accommodate a template:primer duplex during RNA elongation or a protein primer during the uridylylation stage of replication initiation. Based on the structural features of HRV16 3Dpo1 and the catalytic mechanism known for all polymerases, a front-loading model for uridylylation is proposed.

Download Full-text

Synthesis of the cyclopropa[c]benzofuran system of mycorrhizin A and of gilmicolin from a benzofuranol. Crystal structure of a 5,8-Methanocyclopropa-[c]naphtho[2,3-b]furan derivative

Australian Journal of Chemistry ◽

10.1071/ch9821665 ◽

1982 ◽

Vol 35 (8) ◽

pp. 1665 ◽

Cited By ~ 6

Author(s):

RFC Brown ◽

GD Fallon ◽

BM Gatehouse ◽

CM Jones ◽

ID Rae

Keyword(s):

Crystal Structure ◽

Ultraviolet Irradiation ◽

Room Temperature ◽

Cyclopropane Ring ◽

Structural Features ◽

Side Chain ◽

Furan Derivative ◽

Methoxy Derivative ◽

X Ray ◽

X Ray Crystallography

The cyclopropa[c]benzofuran derivative (17), which contains the structural features of methyl ethers of mycorrhizin A and gilmicolin save for the C3 side chain, has been synthesized from the benzofuranol (4) through the benzofuran-4,7-dione (5) and the 7a-methoxy derivative (8). The cyclopropane ring of (17) was introduced by ultraviolet irradiation of a 1-pyrazoline (13) at -78�; irradiation at room temperature gave mainly a profoundly rearranged acidic product for which the methanoindenone structure (18) is proposed. The stereochemistry of the penultimate cyclopropane (16) has been established by X-ray crystallography.

Download Full-text