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

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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Two distinct modes of metal ion binding in the nuclease active site of a viral DNA-packaging terminase: insight into the two-metal-ion catalytic mechanism

Nucleic Acids Research ◽

10.1093/nar/gkv1018 ◽

2015 ◽

Vol 43 (22) ◽

pp. 11003-11016 ◽

Cited By ~ 17

Author(s):

Haiyan Zhao ◽

Zihan Lin ◽

Anna Y. Lynn ◽

Brittany Varnado ◽

John A. Beutler ◽

...

Keyword(s):

Active Site ◽

Metal Ion ◽

Catalytic Mechanism ◽

Dna Packaging ◽

Ion Binding ◽

Metal Ion Binding ◽

Viral Dna ◽

Viral Dna Packaging ◽

Insight Into

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

Biochemical Journal ◽

10.1042/bcj20190399 ◽

2019 ◽

Vol 476 (21) ◽

pp. 3333-3353 ◽

Cited By ~ 3

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.

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Automated docking of phospholipids to the phospholipase D active site: insight into the catalytic mechanism

10.31274/rtd-20200803-109 ◽

2003 ◽

Author(s):

Christopher Lynn Aikens

Keyword(s):

Phospholipase D ◽

Active Site ◽

Catalytic Mechanism ◽

Automated Docking ◽

Insight Into

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

Acta Crystallographica Section D Biological Crystallography ◽

10.1107/s1399004714008700 ◽

2014 ◽

Vol 70 (7) ◽

pp. 1854-1872 ◽

Cited By ~ 10

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.

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Elucidating the role of metal ions in carbonic anhydrase catalysis

Nature Communications ◽

10.1038/s41467-020-18425-5 ◽

2020 ◽

Vol 11 (1) ◽

Cited By ~ 1

Author(s):

Jin Kyun Kim ◽

Cheol Lee ◽

Seon Woo Lim ◽

Aniruddha Adhikari ◽

Jacob T. Andring ◽

...

Keyword(s):

Carbonic Anhydrase ◽

Metal Ions ◽

Metal Ion ◽

Catalytic Mechanism ◽

Chemical Properties ◽

Water Network ◽

Native Metal ◽

Trigonal Bipyramidal ◽

Electrostatic Effect

Abstract Why metalloenzymes often show dramatic changes in their catalytic activity when subjected to chemically similar but non-native metal substitutions is a long-standing puzzle. Here, we report on the catalytic roles of metal ions in a model metalloenzyme system, human carbonic anhydrase II (CA II). Through a comparative study on the intermediate states of the zinc-bound native CA II and non-native metal-substituted CA IIs, we demonstrate that the characteristic metal ion coordination geometries (tetrahedral for Zn2+, tetrahedral to octahedral conversion for Co2+, octahedral for Ni2+, and trigonal bipyramidal for Cu2+) directly modulate the catalytic efficacy. In addition, we reveal that the metal ions have a long-range (~10 Å) electrostatic effect on restructuring water network in the active site. Our study provides evidence that the metal ions in metalloenzymes have a crucial impact on the catalytic mechanism beyond their primary chemical properties.

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GlcNAcstatins are nanomolar inhibitors of human O-GlcNAcase inducing cellular hyper-O-GlcNAcylation

Biochemical Journal ◽

10.1042/bj20090110 ◽

2009 ◽

Vol 420 (2) ◽

pp. 221-227 ◽

Cited By ~ 56

Author(s):

Helge C. Dorfmueller ◽

Vladimir S. Borodkin ◽

Marianne Schimpl ◽

Daan M. F. van Aalten

Keyword(s):

Active Site ◽

Cell Biology ◽

Rational Design ◽

Catalytic Mechanism ◽

Conserved Residues ◽

Cellular Processes ◽

Acetyl Group ◽

Nanomolar Range ◽

Insight Into

O-GlcNAcylation is an essential, dynamic and inducible post-translational glycosylation of cytosolic proteins in metazoa and can show interplay with protein phosphorylation. Inhibition of OGA (O-GlcNAcase), the enzyme that removes O-GlcNAc from O-GlcNAcylated proteins, is a useful strategy to probe the role of this modification in a range of cellular processes. In the present study, we report the rational design and evaluation of GlcNAcstatins, a family of potent, competitive and selective inhibitors of human OGA. Kinetic experiments with recombinant human OGA reveal that the GlcNAcstatins are the most potent human OGA inhibitors reported to date, inhibiting the enzyme in the sub-nanomolar to nanomolar range. Modification of the GlcNAcstatin N-acetyl group leads to up to 160-fold selectivity against the human lysosomal hexosaminidases which employ a similar substrate-assisted catalytic mechanism. Mutagenesis studies in a bacterial OGA, guided by the structure of a GlcNAcstatin complex, provides insight into the role of conserved residues in the human OGA active site. GlcNAcstatins are cell-permeant and, at low nanomolar concentrations, effectively modulate intracellular O-GlcNAc levels through inhibition of OGA, in a range of human cell lines. Thus these compounds are potent selective tools to study the cell biology of O-GlcNAc.

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Catalytic Mechanism of SHCHC Synthase in the Menaquinone Biosynthesis ofEscherichia coli: Identification and Mutational Analysis of the Active Site Residues

Biochemistry ◽

10.1021/bi900897h ◽

2009 ◽

Vol 48 (29) ◽

pp. 6921-6931 ◽

Cited By ~ 29

Author(s):

Ming Jiang ◽

Xiaolei Chen ◽

Xian-Hui Wu ◽

Minjiao Chen ◽

Yun-Dong Wu ◽

...

Keyword(s):

Active Site ◽

Catalytic Mechanism ◽

Mutational Analysis ◽

Ofescherichia Coli ◽

Active Site Residues

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d-Alanine–d-alanine ligase as a model for the activation of ATP-grasp enzymes by monovalent cations

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.012936 ◽

2020 ◽

Vol 295 (23) ◽

pp. 7894-7904

Author(s):

Jordan L. Pederick ◽

Andrew P. Thompson ◽

Stephen G. Bell ◽

John B. Bruning

Keyword(s):

Binding Site ◽

Active Site ◽

Drug Target ◽

Catalytic Mechanism ◽

Thermus Thermophilus ◽

Monovalent Cation ◽

Antibiotic Drug ◽

Domains Of Life ◽

Alanine Dipeptide ◽

Insight Into

The ATP-grasp superfamily of enzymes shares an atypical nucleotide-binding site known as the ATP-grasp fold. These enzymes are involved in many biological pathways in all domains of life. One ATP-grasp enzyme, d-alanine–d-alanine ligase (Ddl), catalyzes ATP-dependent formation of the d-alanyl–d-alanine dipeptide essential for bacterial cell wall biosynthesis and is therefore an important antibiotic drug target. Ddl is activated by the monovalent cation (MVC) K+, but despite its clinical relevance and decades of research, how this activation occurs has not been elucidated. We demonstrate here that activating MVCs bind adjacent to the active site of Ddl from Thermus thermophilus and used a combined biochemical and structural approach to characterize MVC activation. We found that TtDdl is a type II MVC-activated enzyme, retaining activity in the absence of MVCs. However, the efficiency of TtDdl increased ∼20-fold in the presence of activating MVCs, and it was maximally activated by K+ and Rb+ ions. A strict dependence on ionic radius of the MVC was observed, with Li+ and Na+ providing little to no TtDdl activation. To understand the mechanism of MVC activation, we solved crystal structures of TtDdl representing distinct catalytic stages in complex with K+, Rb+, or Cs+. Comparison of these structures with apo TtDdl revealed no evident conformational change on MVC binding. Of note, the identified MVC binding site is structurally conserved within the ATP-grasp superfamily. We propose that MVCs activate Ddl by altering the charge distribution of its active site. These findings provide insight into the catalytic mechanism of ATP-grasp enzymes.

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