Mutational and computational analysis of the role of conserved residues in the active site of a family 18 chitinase

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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Ribokinase family evolution and the role of conserved residues at the active site of the PfkB subfamily representative, Pfk-2 from Escherichia coli

Archives of Biochemistry and Biophysics ◽

10.1016/j.abb.2010.06.024 ◽

2010 ◽

Vol 502 (1) ◽

pp. 23-30 ◽

Cited By ~ 20

Author(s):

Ricardo Cabrera ◽

Jorge Babul ◽

Victoria Guixé

Keyword(s):

Escherichia Coli ◽

Active Site ◽

Conserved Residues

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Kinetic and crystallographic analyses of the catalytic domain of chitinase from Pyrococcus furiosus- the role of conserved residues in the active site

FEBS Journal ◽

10.1111/j.1742-4658.2010.07685.x ◽

2010 ◽

Vol 277 (12) ◽

pp. 2683-2695 ◽

Cited By ~ 23

Author(s):

Hiroaki Tsuji ◽

Shigenori Nishimura ◽

Takashi Inui ◽

Yuji Kado ◽

Kazuhiko Ishikawa ◽

...

Keyword(s):

Active Site ◽

Catalytic Domain ◽

Pyrococcus Furiosus ◽

Conserved Residues

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Molecular dynamics studies unravel role of conserved residues responsible for movement of ions into active site of DHBPS

Scientific Reports ◽

10.1038/srep40452 ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 1

Author(s):

Ranajit Nivrutti Shinde ◽

Subramanian Karthikeyan ◽

Balvinder Singh

Keyword(s):

Molecular Dynamics ◽

Active Site ◽

Conserved Residues

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Crystal Structure of the Extended-Spectrum β-Lactamase PER-2 and Insights into the Role of Specific Residues in the Interaction with β-Lactams and β-Lactamase Inhibitors

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00089-14 ◽

2014 ◽

Vol 58 (10) ◽

pp. 5994-6002 ◽

Cited By ~ 10

Author(s):

Melina Ruggiero ◽

Frédéric Kerff ◽

Raphaël Herman ◽

Frédéric Sapunaric ◽

Moreno Galleni ◽

...

Keyword(s):

Active Site ◽

Amino Acid Identity ◽

Conserved Residues ◽

Class A ◽

Extended Spectrum ◽

Active Site Cavity ◽

Bond Network ◽

Hydrolysis Of ◽

Structure And Activity

ABSTRACTPER-2 belongs to a small (7 members to date) group of extended-spectrum β-lactamases. It has 88% amino acid identity with PER-1 and both display high catalytic efficiencies toward most β-lactams. In this study, we determined the X-ray structure of PER-2 at 2.20 Å and evaluated the possible role of several residues in the structure and activity toward β-lactams and mechanism-based inhibitors. PER-2 is defined by the presence of a singulartransbond between residues 166 to 167, which generates an inverted Ω loop, an expanded fold of this domain that results in a wide active site cavity that allows for efficient hydrolysis of antibiotics like the oxyimino-cephalosporins, and a series of exclusive interactions between residues not frequently involved in the stabilization of the active site in other class A β-lactamases. PER β-lactamases might be included within a cluster of evolutionarily related enzymes harboring the conserved residues Asp136 and Asn179. Other signature residues that define these enzymes seem to be Gln69, Arg220, Thr237, and probably Arg/Lys240A (“A” indicates an insertion according to Ambler's scheme for residue numbering in PER β-lactamases), with structurally important roles in the stabilization of the active site and proper orientation of catalytic water molecules, among others. We propose, supported by simulated models of PER-2 in combination with different β-lactams, the presence of a hydrogen-bond network connecting Ser70-Gln69-water-Thr237-Arg220 that might be important for the proper activity and inhibition of the enzyme. Therefore, we expect that mutations occurring in these positions will have impacts on the overall hydrolytic behavior.

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Clarification of the role of key active site residues of glutathione transferase Zeta/maleylacetoacetate isomerase by a new spectrophotometric technique

Biochemical Journal ◽

10.1042/bj20030625 ◽

2003 ◽

Vol 374 (3) ◽

pp. 731-737 ◽

Cited By ~ 32

Author(s):

Philip G. BOARD ◽

Matthew C. TAYLOR ◽

Marjorie COGGAN ◽

Michael W. PARKER ◽

Hoffman B. LANTUM ◽

...

Keyword(s):

Crystal Structure ◽

Active Site ◽

Glutathione Transferase ◽

Salt Bridge ◽

Side Chain ◽

Active Site Residues ◽

Conserved Residues ◽

Isomerase Activity ◽

Catalytic Role

hGSTZ1-1 (human glutathione transferase Zeta 1-1) catalyses a range of glutathione-dependent reactions and plays an important role in the metabolism of tyrosine via its maleylacetoacetate isomerase activity. The crystal structure and sequence alignment of hGSTZ1 with other GSTs (glutathione transferases) focused attention on three highly conserved residues (Ser-14, Ser-15, Cys-16) as candidates for an important role in catalysis. Progress in the investigation of these residues has been limited by the absence of a convenient assay for kinetic analysis. In this study we have developed a new spectrophotometric assay with a novel substrate [(±)-2-bromo-3-(4-nitrophenyl)propionic acid]. The assay has been used to rapidly assess the potential catalytic role of several residues in the active site. Despite its less favourable orientation in the crystal structure, Ser-14 was the only residue found to be essential for catalysis. It is proposed that a conformational change may favourably reposition the hydroxyl of Ser-14 during the catalytic cycle. The Cys16→Ala (Cys-16 mutated to Ala) mutation caused a dramatic increase in the Km for glutathione, indicating that Cys-16 plays an important role in the binding and orientation of glutathione in the active site. Previous structural studies implicated Arg-175 in the orientation of α-halo acid substrates in the active site of hGSTZ1-1. Mutation of Arg-175 to Lys or Ala resulted in a significant lowering of the kcat in the Ala-175 variant. This result is consistent with the proposal that the charged side chain of Arg-175 forms a salt bridge with the carboxylate of the α-halo acid substrates.

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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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