Na+/K+exchange switches the catalytic apparatus of potassium-dependent plantL-asparaginase

2014 ◽  
Vol 70 (7) ◽  
pp. 1854-1872 ◽  
Author(s):  
Magdalena Bejger ◽  
Barbara Imiolczyk ◽  
Damien Clavel ◽  
Miroslaw Gilski ◽  
Agnieszka Pajak ◽  
...  
Keyword(s):  
Alkali Metal ◽  
Active Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Catalytic Mechanism ◽  
Structural Data ◽  
Activation Loop ◽  
Plant Type ◽  
Substrate Preferences ◽  
Autocatalytic Cleavage

Plant-type L-asparaginases, which are a subclass of the Ntn-hydrolase family, are divided into potassium-dependent and potassium-independent enzymes with different substrate preferences. While the potassium-independent enzymes have already been well characterized, there are no structural data for any of the members of the potassium-dependent group to illuminate the intriguing dependence of their catalytic mechanism on alkali-metal cations. Here, three crystal structures of a potassium-dependent plant-type L-asparaginase fromPhaseolus vulgaris(PvAspG1) differing in the type of associated alkali metal ions (K+, Na+or both) are presented and the structural consequences of the different ions are correlated with the enzyme activity. As in all plant-type L-asparaginases, immature PvAspG1 is a homodimer of two protein chains, which both undergo autocatalytic cleavage to α and β subunits, thus creating the mature heterotetramer or dimer of heterodimers (αβ)2. The αβ subunits of PvAspG1 are folded similarly to the potassium-independent enzymes, with a sandwich of two β-sheets flanked on each side by a layer of helices. In addition to the `sodium loop' (here referred to as the `stabilization loop') known from potassium-independent plant-type asparaginases, the potassium-dependent PvAspG1 enzyme contains another alkali metal-binding loop (the `activation loop') in subunit α (residues Val111–Ser118). The active site of PvAspG1 is located between these two metal-binding loops and in the immediate neighbourhood of three residues, His117, Arg224 and Glu250, acting as a catalytic switch, which is a novel feature that is identified in plant-type L-asparaginases for the first time. A comparison of the three PvAspG1 structures demonstrates how the metal ion bound in the activation loop influences its conformation, setting the catalytic switch to ON (when K+is coordinated) or OFF (when Na+is coordinated) to respectively allow or prevent anchoring of the reaction substrate/product in the active site. Moreover, it is proposed that Ser118, the last residue of the activation loop, is involved in the potassium-dependence mechanism. The PvAspG1 structures are discussed in comparison with those of potassium-independent L-asparaginases (LlA, EcAIII and hASNase3) and those of other Ntn-hydrolases (AGA and Tas1), as well as in the light of noncrystallographic studies.

scholarly journals Insight into the Function of Active Site Residues in the Catalytic Mechanism of Human Ferrochelatase

2021 ◽  
Author(s):  
Amy E. Medlock ◽  
Wided Najahi-Missaoui ◽  
Mesafint T. Shiferaw ◽  
Angela N. Albetel ◽  
William N. Lanzilotta ◽  
...  
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Catalytic Mechanism ◽  
Structural Data ◽  
Active Site Residues ◽  
Proton Abstraction ◽  
Product Release ◽  
Iron Delivery ◽  
High Level

Ferrochelatase catalyzes the insertion of ferrous iron into a porphyrin macrocycle to produce the essential cofactor, heme. In humans this enzyme not only catalyzes the terminal step, but also serves a regulatory step in the heme synthesis pathway. Over a dozen crystal structures of human ferrochelatase have been solved and many variants have been characterized kinetically. In addition, hydrogen deuterium exchange, resonance Raman, molecular dynamics, and high level quantum mechanic studies have added to our understanding of  the catalytic cycle of the enzyme. However, an understanding of how the metal ion is delivered and the specific role that active site residues play in catalysis remain open questions. Data are consistent with metal binding and insertion occurring from the side opposite from where pyrrole proton abstraction takes place. To better understand iron delivery and binding as well as the role of conserved residues in the active site, we have constructed and characterized a series of enzyme variants. Crystallographic studies as well as rescue and kinetic analysis of variants were performed. Data from these studies are consistent with the M76 residue playing a role in active site metal binding and formation of a weak iron protein ligand being necessary for product release. Additionally, structural data support a role for E343 in proton abstraction and product release in coordination with a peptide loop composed of Q302, S303 and K304 that act a metal sensor.

Structures of c-di-GMP/cGAMP degrading phosphodiesterase VcEAL: identification of a novel conformational switch and its implication

2019 ◽  
Vol 476 (21) ◽  
pp. 3333-3353 ◽  
Author(s):  
Malti Yadav ◽  
Kamalendu Pal ◽  
Udayaditya Sen
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Metal Ion ◽  
Triosephosphate Isomerase ◽  
Catalytic Mechanism ◽  
Degradation Mechanism ◽  
Structural Basis ◽  
Conserved Residues ◽  
Phosphodiester Bond ◽  
Cyclic Dinucleotides

Cyclic dinucleotides (CDNs) have emerged as the central molecules that aid bacteria to adapt and thrive in changing environmental conditions. Therefore, tight regulation of intracellular CDN concentration by counteracting the action of dinucleotide cyclases and phosphodiesterases (PDEs) is critical. Here, we demonstrate that a putative stand-alone EAL domain PDE from Vibrio cholerae (VcEAL) is capable to degrade both the second messenger c-di-GMP and hybrid 3′3′-cyclic GMP–AMP (cGAMP). To unveil their degradation mechanism, we have determined high-resolution crystal structures of VcEAL with Ca2+, c-di-GMP-Ca2+, 5′-pGpG-Ca2+ and cGAMP-Ca2+, the latter provides the first structural basis of cGAMP hydrolysis. Structural studies reveal a typical triosephosphate isomerase barrel-fold with substrate c-di-GMP/cGAMP bound in an extended conformation. Highly conserved residues specifically bind the guanine base of c-di-GMP/cGAMP in the G2 site while the semi-conserved nature of residues at the G1 site could act as a specificity determinant. Two metal ions, co-ordinated with six stubbornly conserved residues and two non-bridging scissile phosphate oxygens of c-di-GMP/cGAMP, activate a water molecule for an in-line attack on the phosphodiester bond, supporting two-metal ion-based catalytic mechanism. PDE activity and biofilm assays of several prudently designed mutants collectively demonstrate that VcEAL active site is charge and size optimized. Intriguingly, in VcEAL-5′-pGpG-Ca2+ structure, β5–α5 loop adopts a novel conformation that along with conserved E131 creates a new metal-binding site. This novel conformation along with several subtle changes in the active site designate VcEAL-5′-pGpG-Ca2+ structure quite different from other 5′-pGpG bound structures reported earlier.

Crystal structure of hexanoyl-CoA bound to β-ketoacyl reductase FabG4 of Mycobacterium tuberculosis

2013 ◽  
Vol 450 (1) ◽  
pp. 127-139 ◽  
Author(s):  
Debajyoti Dutta ◽  
Sudipta Bhattacharyya ◽  
Amlan Roychowdhury ◽  
Rupam Biswas ◽  
Amit Kumar Das
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Active Site ◽  
Ternary Complex ◽  
Catalytic Mechanism ◽  
Acyl Carrier Protein ◽  
Structural Data ◽  
Acid Synthesis ◽  
Binary Complex ◽  
C Terminus ◽  
Covalently Linked

FabGs, or β-oxoacyl reductases, are involved in fatty acid synthesis. The reaction entails NADPH/NADH-mediated conversion of β-oxoacyl-ACP (acyl-carrier protein) into β-hydroxyacyl-ACP. HMwFabGs (high-molecular-weight FabG) form a phylogenetically separate group of FabG enzymes. FabG4, an HMwFabG from Mycobacterium tuberculosis, contains two distinct domains, an N-terminal ‘flavodoxintype’ domain and a C-terminal oxoreductase domain. The catalytically active C-terminal domain utilizes NADH to reduce β-oxoacyl-CoA to β-hydroxyacyl-CoA. In the present study the crystal structures of the FabG4–NADH binary complex and the FabG4–NAD+–hexanoyl-CoA ternary complex have been determined to understand the substrate specificity and catalytic mechanism of FabG4. This is the first report to demonstrate how FabG4 interacts with its coenzyme NADH and hexanoyl-CoA that mimics an elongating fattyacyl chain covalently linked with CoA. Structural analysis shows that the binding of hexanoyl-CoA within the active site cavity of FabG significantly differs from that of the C16 fattyacyl substrate bound to mycobacterial FabI [InhA (enoyl-ACP reductase)]. The ternary complex reveals that both loop I and loop II interact with the phosphopantetheine moiety of CoA or ACP to align the covalently linked fattyacyl substrate near the active site. Structural data ACP inhibition studies indicate that FabG4 can accept both CoA- and ACP-based fattyacyl substrates. We have also shown that in the FabG4 dimer Arg146 and Arg445 of one monomer interact with the C-terminus of the second monomer to play pivotal role in substrate association and catalysis.

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

2018 ◽  
Vol 293 (21) ◽  
pp. 7993-8008 ◽  
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.

scholarly journals MeLAD: an integrated resource for metalloenzyme-ligand associations

2019 ◽  
Author(s):  
Gen Li ◽  
Yu Su ◽  
Yu-Hang Yan ◽  
Jia-Yi Peng ◽  
Qing-Qing Dai ◽  
...  
Keyword(s):  
Drug Discovery ◽  
Metal Binding ◽  
Metal Ion ◽  
Structural Data ◽  
Structural Similarity ◽  
Functional Class ◽  
Supplementary Information ◽  
Biological Processes ◽  
Ligand Interactions ◽  
Data Source

Abstract Motivation Metalloenzymes are attractive targets for therapeutic intervention owing to their central roles in various biological processes and pathological situations. The fast-growing body of structural data on metalloenzyme-ligand interactions is facilitating efficient drug discovery targeting metalloenzymes. However, there remains a shortage of specific databases that can provide centralized, interconnected information exclusive to metalloenzyme-ligand associations. Results We created a Metalloenzyme-Ligand Association Database (MeLAD), which is designed to provide curated structural data and information exclusive to metalloenzyme-ligand interactions, and more uniquely, present expanded associations that are represented by metal-binding pharmacophores (MBPs), metalloenzyme structural similarity (MeSIM) and ligand chemical similarity (LigSIM). MeLAD currently contains 6086 structurally resolved interactions of 1416 metalloenzymes with 3564 ligands, of which classical metal-binding, non-classical metal-binding, non-metal-binding and metal water-bridging interactions account for 63.0%, 2.3%, 34.4% and 0.3%, respectively. A total of 263 monodentate, 191 bidentate and 15 tridentate MBP chemotypes were included in MeLAD, which are linked to different active site metal ions and coordination modes. 3726 and 52 740 deductive metalloenzyme-ligand associations by MeSIM and LigSIM analyses, respectively, were included in MeLAD. An online server is provided for users to conduct metalloenzyme profiling prediction for small molecules of interest. MeLAD is searchable by multiple criteria, e.g. metalloenzyme name, ligand identifier, functional class, bioinorganic class, metal ion and metal-containing cofactor, which will serve as a valuable, integrative data source to foster metalloenzyme related research, particularly involved in drug discovery targeting metalloenzymes. Availability and implementation MeLAD is accessible at https://melad.ddtmlab.org. Supplementary information Supplementary data are available at Bioinformatics online.

scholarly journals An unusual metal ion configuration in a viral DNA-packaging nuclease active site

2014 ◽  
Vol 70 (a1) ◽  
pp. C489-C489
Author(s):  
Haiyan Zhao ◽  
Theodore Christensen ◽  
Zihan Lin ◽  
Annie Lynn ◽  
Liang Tang
Keyword(s):  
Nucleic Acid ◽  
Metal Ions ◽  
Active Site ◽  
Short Distance ◽  
Metal Ion ◽  
Catalytic Mechanism ◽  
Dna Packaging ◽  
Viral Dna ◽  
Viral Dna Packaging ◽  
Ion Cluster

Nucleic acid metabolism is fundamental to many biological processes. A large class of enzymes such as RNase H, reverse transcriptase, retroviral integrase, topoisomerase, DNA and RNA polymerase, transposase, Holliday-junction resolvase, RNAi slicer Argonaute, and viral DNA-packaging terminase, utilize a common two-metal-ion catalytic mechanism for cleavage or synthesis of nucleic acid chains. Here we report an unusual metal-ion cluster in the active site of the nuclease domain of a viral DNA-packaging terminase unveiled by X-ray structures up to 1.38 Angstrom resolution. Two Mg2+ ions are situated in a coupled octahedral coordination system with liganding oxygen atoms from aspartic acid residues as well as water molecules. The two Mg2+ ions are located within a strikingly short distance of ~2.5 Å, which is unusual given the 1.6 Å atomic radius of Mg2+ and is shorter than previously observed metal-metal distances in metallocluster-containing enzymes or other biological systems. This provides the structural basis for distinguishing Mg2+ from other metal ions such as Ca2+ which are well known to support binding of the nucleic acid substrate but not support catalysis. Such an ultra-short distance between two metal-ions may be essential for generation of a highly positive niche, leading to nucleophilic attack at the phosphodiester bond of DNA. These results have defined the precise chemical configuration of the active site in nucleases using two-metal-ion catalytic mechanism. Moreover, assembly of this two-metal-ion cluster in the viral DNA-packaging terminase is mediated by an adjacent Lys residue, likely serving as a regulatory mechanism for activation of the nuclease activity of the terminase during packaging of viral genome.

scholarly journals Anomalous scattering analysis of Agrobacterium radiobacter phosphotriesterase: the prominent role of iron in the heterobinuclear active site

2006 ◽  
Vol 397 (3) ◽  
pp. 501-508 ◽  
Author(s):  
Colin J. Jackson ◽  
Paul D. Carr ◽  
Hye-Kyung Kim ◽  
Jian-Wei Liu ◽  
Paul Herrald ◽  
...  
Keyword(s):  
Active Site ◽  
Metal Binding ◽  
Active Sites ◽  
Catalytic Mechanism ◽  
Catalytic Efficiency ◽  
Scattering Data ◽  
Anomalous Scattering ◽  
Agrobacterium Radiobacter

Bacterial phosphotriesterases are binuclear metalloproteins for which the catalytic mechanism has been studied with a variety of techniques, principally using active sites reconstituted in vitro from apoenzymes. Here, atomic absorption spectroscopy and anomalous X-ray scattering have been used to determine the identity of the metals incorporated into the active site in vivo. We have recombinantly expressed the phosphotriesterase from Agrobacterium radiobacter (OpdA) in Escherichia coli grown in medium supplemented with 1 mM CoCl2 and in unsupplemented medium. Anomalous scattering data, collected from a single crystal at the Fe–K, Co–K and Zn–K edges, indicate that iron and cobalt are the primary constituents of the two metal-binding sites in the catalytic centre (α and β) in the protein expressed in E. coli grown in supplemented medium. Comparison with OpdA expressed in unsupplemented medium demonstrates that the cobalt present in the supplemented medium replaced zinc at the β-position of the active site, which results in an increase in the catalytic efficiency of the enzyme. These results suggest an essential role for iron in the catalytic mechanism of bacterial phosphotriesterases, and that these phosphotriesterases are natively heterobinuclear iron–zinc enzymes.

scholarly journals Insights into Catalytic and tRNA Recognition Mechanism of the Dual-Specific tRNA Methyltransferase from Thermococcus kodakarensis

Genes ◽  
2019 ◽  
Vol 10 (2) ◽  
pp. 100 ◽  
Author(s):  
Aiswarya Krishnamohan ◽  
Samantha Dodbele ◽  
Jane Jackman
Keyword(s):  
Active Site ◽  
Metal Ion ◽  
Catalytic Mechanism ◽  
Mutational Analysis ◽  
Chemical Properties ◽  
Recognition Mechanism ◽  
Thermococcus Kodakarensis ◽  
Bifunctional Enzymes ◽  
Trna Methyltransferase ◽  
Insight Into

The tRNA methyltransferase Trm10, conserved throughout Eukarya and Archaea, catalyzes N1-methylation of purine residues at position 9 using S-adenosyl methionine as the methyl donor. The Trm10 family exhibits diverse target nucleotide specificity, with some homologs that are obligate m1G9 or m1A9-specific enzymes, while others are bifunctional enzymes catalyzing both m1G9 and m1A9. This variability is particularly intriguing given different chemical properties of the target N1 atom of guanine and adenine. Here we performed an extensive kinetic and mutational analysis of the m1G9 and m1A9-catalyzing Trm10 from Thermococcus kodakarensis to gain insight into the active site that facilitates this unique bifunctionality. These results suggest that the rate-determining step for catalysis likely involves a conformational change to correctly position the substrate tRNA in the active site. In this model, kinetic preferences for certain tRNA can be explained by variations in the overall stability of the folded substrate tRNA, consistent with tRNA-specific differences in metal ion dependence. Together, these results provide new insight into the substrate recognition, active site and catalytic mechanism of m1G/m1A catalyzing bifunctional enzymes.

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