Metallophosphoesterases: structural fidelity with functional promiscuity

2015 ◽  
Vol 467 (2) ◽  
pp. 201-216 ◽  
Author(s):  
Nishad Matange ◽  
Marjetka Podobnik ◽  
Sandhya S. Visweswariah
Keyword(s):  
Active Site ◽  
Metal Ion ◽  
Active Sites ◽  
Dependent Manner ◽  
The Core ◽  
Repair Enzymes ◽  
Phosphoprotein Phosphatases ◽  
Purple Acid Phosphatases ◽  
Binuclear Metal ◽  
Regulatory Effects

Calcineurin-like metallophosphoesterases (MPEs) form a large superfamily of binuclear metal-ion-centre-containing enzymes that hydrolyse phosphomono-, phosphodi- or phosphotri-esters in a metal-dependent manner. The MPE domain is found in Mre11/SbcD DNA-repair enzymes, mammalian phosphoprotein phosphatases, acid sphingomyelinases, purple acid phosphatases, nucleotidases and bacterial cyclic nucleotide phosphodiesterases. Despite this functional diversity, MPEs show a remarkably similar structural fold and active-site architecture. In the present review, we summarize the available structural, biochemical and functional information on these proteins. We also describe how diversification and specialization of the core MPE fold in various MPEs is achieved by amino acid substitution in their active sites, metal ions and regulatory effects of accessory domains. Finally, we discuss emerging roles of these proteins as non-catalytic protein-interaction scaffolds. Thus we view the MPE superfamily as a set of proteins with a highly conserved structural core that allows embellishment to result in dramatic and niche-specific diversification of function.

Photochemical mapping of the active site of myosin

1992 ◽  
Vol 336 (1276) ◽  
pp. 55-61 ◽  
Keyword(s):  
Active Site ◽  
Metal Ion ◽  
Active Sites ◽  
Cross Linking ◽  
Detailed Structure ◽  
Atp Binding Site ◽  
Adenine Ring ◽  
Skeletal Myosin ◽  
Key Residues ◽  
Specific Labelling

The active sites of myosin from skeletal, smooth and scallop muscle have been partly characterized by use of a series of photoreactive analogues of ATP. Specific labelling was attained by trapping these analogues in their diphosphate forms at the active sites by either cross-linking two reactive thiols (skeletal myosin) or by formation of stable vanadate-metal ion transition state-like complexes (smooth muscle and scallop myosin). By use of this approach combined with appropriate chemistry, several key residues in all three myosins have been identified which bind at or near the adenine ring, the ribose ring and to the γ-phosphate of ATP. This information should aid in the solution of the crystal structure of the heads of myosin and in defining a detailed structure of the ATP binding site.

scholarly journals Evolutionary Expansion of the Amidohydrolase Superfamily in Bacteria in Response to the Synthetic Compounds Molinate and Diuron

2015 ◽  
Vol 81 (7) ◽  
pp. 2612-2624 ◽  
Author(s):  
Elena Sugrue ◽  
Nicholas J. Fraser ◽  
Davis H. Hopkins ◽  
Paul D. Carr ◽  
Jeevan L. Khurana ◽  
...  
Keyword(s):  
Active Site ◽  
Metal Ion ◽  
Active Sites ◽  
Evolutionary Transition ◽  
Functional Changes ◽  
Metal Ion Analysis ◽  
Amidohydrolase Superfamily ◽  
Kinetics Of ◽  
Hydrolysis Of

ABSTRACTThe amidohydrolase superfamily has remarkable functional diversity, with considerable structural and functional annotation of known sequences. In microbes, the recent evolution of several members of this family to catalyze the breakdown of environmental xenobiotics is not well understood. An evolutionary transition from binuclear to mononuclear metal ion coordination at the active sites of these enzymes could produce large functional changes such as those observed in nature, but there are few clear examples available to support this hypothesis. To investigate the role of binuclear-mononuclear active-site transitions in the evolution of new function in this superfamily, we have characterized two recently evolved enzymes that catalyze the hydrolysis of the synthetic herbicides molinate (MolA) and phenylurea (PuhB). In this work, the crystal structures, mutagenesis, metal ion analysis, and enzyme kinetics of both MolA and PuhB establish that these enzymes utilize a mononuclear active site. However, bioinformatics and structural comparisons reveal that the closest putative ancestor of these enzymes had a binuclear active site, indicating that a binuclear-mononuclear transition has occurred. These proteins may represent examples of evolution modifying the characteristics of existing catalysts to satisfy new requirements, specifically, metal ion rearrangement leading to large leaps in activity that would not otherwise be possible.

scholarly journals Active-Site Models of Streptococcus pyogenes Cas9 in DNA Cleavage State

2021 ◽  
Vol 8 ◽  
Author(s):  
Honghai Tang ◽  
Hui Yuan ◽  
Wenhao Du ◽  
Gan Li ◽  
Dongmei Xue ◽  
...  
Keyword(s):  
Active Site ◽  
Dna Cleavage ◽  
Metal Ion ◽  
Active Sites ◽  
Structural Similarity ◽  
Atomic Model ◽  
Nucleophilic Attack ◽  
Target Dna ◽  
Coordinated Water ◽  
Dna Complex

CRISPR-Cas9 is a powerful tool for target genome editing in living cells. Significant advances have been made to understand how this system cleaves target DNA. HNH is a nuclease domain, which shares structural similarity with the HNH endonuclease characterzied by a beta-beta-alpha-metal fold. Therefore, based on one- and two-metal-ion mechanisms, homology modeling and molecular dynamics (MD) simulation are suitable tools for building an atomic model of Cas9 in the DNA cleavage state. Here, by modeling and MD, we presented an atomic model of SpCas9–sgRNA–DNA complex with the cleavage state. This model shows that the HNH and RuvC conformations resemble their DNA cleavage state where the active-sites in the complex coordinate with DNA, Mg2+ ions, and water. Among them, residues D10, E762, H983, and D986 locate at the first shell of the RuvC active-site and interact with the ions directly, residues H982 or/and H985 are general (Lewis) bases, and the coordinated water is located at the positions for nucleophilic attack of the scissile phosphate. Meanwhile, this catalytic model led us to engineer a new SpCas9 variant (SpCas9-H982A + H983D) with reduced off-target effects. Thus, our study provided new mechanistic insights into the CRISPR-Cas9 system in the DNA cleavage state and offered useful guidance for engineering new CRISPR-Cas9 editing systems with improved specificity.

scholarly journals A new family of covalent inhibitors block nucleotide binding to the active site of pyruvate kinase

2012 ◽  
Vol 448 (1) ◽  
pp. 67-72 ◽  
Author(s):  
Hugh P. Morgan ◽  
Martin J. Walsh ◽  
Elizabeth A. Blackburn ◽  
Martin A. Wear ◽  
Matthew B. Boxer ◽  
...  
Keyword(s):  
Pyruvate Kinase ◽  
Active Site ◽  
High Throughput Screening ◽  
Active Sites ◽  
Covalent Bond ◽  
Cell Types ◽  
Binding Pocket ◽  
Lysine Residue ◽  
Dependent Manner ◽  
Inhibitor Molecule

PYK (pyruvate kinase) plays a central role in the metabolism of many organisms and cell types, but the elucidation of the details of its function in a systems biology context has been hampered by the lack of specific high-affinity small-molecule inhibitors. High-throughput screening has been used to identify a family of saccharin derivatives which inhibit LmPYK (Leishmania mexicana PYK) activity in a time- (and dose-) dependent manner, a characteristic of irreversible inhibition. The crystal structure of DBS {4-[(1,1-dioxo-1,2-benzothiazol-3-yl)sulfanyl]benzoic acid} complexed with LmPYK shows that the saccharin moiety reacts with an active-site lysine residue (Lys335), forming a covalent bond and sterically hindering the binding of ADP/ATP. Mutation of the lysine residue to an arginine residue eliminated the effect of the inhibitor molecule, providing confirmation of the proposed inhibitor mechanism. This lysine residue is conserved in the active sites of the four human PYK isoenzymes, which were also found to be irreversibly inhibited by DBS. X-ray structures of PYK isoforms show structural differences at the DBS-binding pocket, and this covalent inhibitor of PYK provides a chemical scaffold for the design of new families of potentially isoform-specific irreversible inhibitors.

Active sites of β-lactamases from Bacillus cereus

1980 ◽  
Vol 289 (1036) ◽  
pp. 333-344 ◽  
Keyword(s):  
Nuclear Magnetic Resonance ◽  
Amino Acid ◽  
Bacillus Cereus ◽  
Active Site ◽  
Metal Ion ◽  
Active Sites ◽  
Extracellular Enzyme ◽  
Thiol Group ◽  
The Other ◽  
Site Group

There are two extracellular β-lactamases produced by Bacillus cereus 569. One of these enzymes, β-lactamase I, is inactivated by 6-β-bromopenicillanic acid: the site of reaction is serine-44. This is a conserved amino acid residue in the other β-lactamases whose structures have been determined, and it becomes a good candidate for an active-site group in these enzymes. The inactivation may involve a rearrangement leading to a dihydrothiazine. The other extracellular enzyme produced by B. cereus , β- lactamase II, is exceptional in requiring metal ions for activity. The Zn II and Co II enzymes (the former is more active) have been studied by nuclear magnetic resonance, and by absorption spectroscopy. The groups that bind the metal ion required for activity are three histidine residues and the enzyme’s sole thiol group.

scholarly journals A metal-dependent switch moderates activity of the hexameric M17 aminopeptidases

2018 ◽  
Author(s):  
Nyssa Drinkwater ◽  
Wei Yang ◽  
Blake T. Riley ◽  
Brooke K. Hayes ◽  
Komagal Kannan Sivaraman ◽  
...  
Keyword(s):  
Metal Ion ◽  
Active Sites ◽  
Binding Pocket ◽  
Metal Center ◽  
Processing Enzymes ◽  
Novel Structure ◽  
Wide Range ◽  
Binuclear Metal ◽  
Biochemical Analyses ◽  
Dynamics Simulations

AbstractThe metal-dependent M17 aminopeptidases are conserved throughout all kingdoms of life. The large enzyme family is characterised by a conserved binuclear metal center and a distinctive homohexameric arrangement. To understand the mechanistic role of the hexameric assembly, we undertook an investigation of the structure and dynamics of the M17 aminopeptidase from P. falciparum, PfA-M17. We describe a novel structure of PfA-M17, which shows that the active sites of each trimer are linked by a dynamic loop, and that the loop movement is coupled with a drastic rearrangement of the binuclear metal center and substrate-binding pocket. Molecular dynamics simulations, supported by biochemical analyses of PfA-M17 variants, demonstrate that this rearrangement is inherent to PfA-M17, and that the transition between the active and inactive states is part of a dynamic regulatory mechanism. Key to the mechanism is a re-modelling of the binuclear metal center, which occurs in response to a signal from the neighbouring active site, and serves to moderate the rate of proteolysis under different environmental conditions. Therefore, this work has identified the precise mechanism by which oligomerization contributes to PfA-M17 function. Further, it has described a novel role for metal cofactors in the regulation of enzymes with implications for the wide range of metalloenzymes that operate via a two-metal ion catalytic center including DNA processing enzymes and metalloproteases.

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.

The Effect of Active Site-inhibited Factor VIIa on Tissue Factor-initiated Coagulation Using Platelets before and after Aspirin Administration

1997 ◽  
Vol 78 (04) ◽  
pp. 1202-1208 ◽  
Author(s):  
Marianne Kjalke ◽  
Julie A Oliver ◽  
Dougald M Monroe ◽  
Maureane Hoffman ◽  
Mirella Ezban ◽  
...  
Keyword(s):  
Tissue Factor ◽  
Active Site ◽  
Large Scale ◽  
Thrombin Generation ◽  
Factor Viia ◽  
Dependent Manner ◽  
Antithrombotic Agent ◽  
Before And After ◽  
Ic50 Values

SummaryActive site-inactivated factor VIIa has potential as an antithrombotic agent. The effects of D-Phe-L-Phe-L-Arg-chloromethyl ketone-treated factor VIla (FFR-FVIIa) were evaluated in a cell-based system mimicking in vivo initiation of coagulation. FFR-FVIIa inhibited platelet activation (as measured by expression of P-selectin) and subsequent large-scale thrombin generation in a dose-dependent manner with IC50 values of 1.4 ± 0.8 nM (n = 8) and 0.9 ± 0.7 nM (n = 7), respectively. Kd for factor VIIa binding to monocytes ki for FFR-FVIIa competing with factor VIIa were similar (11.4 ± 0.8 pM and 10.6 ± 1.1 pM, respectively), showing that FFR-FVIIa binds to tissue factor in the tenase complex with the same affinity as factor VIIa. Using platelets from volunteers before and after ingestion of aspirin (1.3 g), there were no significant differences in the IC50 values of FFR-FVIIa [after aspirin ingestion, the IC50 values were 1.7 ± 0.9 nM (n = 8) for P-selectin expression, p = 0.37, and 1.4 ± 1.3 nM (n = 7) for thrombin generation, p = 0.38]. This shows that aspirin treatment of platelets does not influence the inhibition of tissue factor-initiated coagulation by FFR-FVIIa, probably because thrombin activation of platelets is not entirely dependent upon expression of thromboxane A2.

Catalytic Resonance Theory: Parallel Reaction Pathway Control

2019 ◽  
Author(s):  
M. Alexander Ardagh ◽  
Manish Shetty ◽  
Anatoliy Kuznetsov ◽  
Qi Zhang ◽  
Phillip Christopher ◽  
...  
Keyword(s):  
Chemical Reactions ◽  
Active Site ◽  
Active Sites ◽  
Reaction Pathway ◽  
Control Strategies ◽  
Oscillation Amplitude ◽  
Catalytic Performance ◽  
Parallel Reaction ◽  
Resonance Theory ◽  
Static Conditions

Catalytic enhancement of chemical reactions via heterogeneous materials occurs through stabilization of transition states at designed active sites, but dramatically greater rate acceleration on that same active site is achieved when the surface intermediates oscillate in binding energy. The applied oscillation amplitude and frequency can accelerate reactions orders of magnitude above the catalytic rates of static systems, provided the active site dynamics are tuned to the natural frequencies of the surface chemistry. In this work, differences in the characteristics of parallel reactions are exploited via selective application of active site dynamics (0 < ΔU < 1.0 eV amplitude, 10<sup>-6</sup> < f < 10<sup>4</sup> Hz frequency) to control the extent of competing reactions occurring on the shared catalytic surface. Simulation of multiple parallel reaction systems with broad range of variation in chemical parameters revealed that parallel chemistries are highly tunable in selectivity between either pure product, even when specific products are not selectively produced under static conditions. Two mechanisms leading to dynamic selectivity control were identified: (i) surface thermodynamic control of one product species under strong binding conditions, or (ii) catalytic resonance of the kinetics of one reaction over the other. These dynamic parallel pathway control strategies applied to a host of chemical conditions indicate significant potential for improving the catalytic performance of many important industrial chemical reactions beyond their existing static performance.

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