Triosephosphate isomerase: removal of a putatively electrophilic histidine residue results in a subtle change in catalytic mechanism

Biochemistry ◽  
1988 ◽  
Vol 27 (16) ◽  
pp. 5948-5960 ◽  
Author(s):  
Elliott B. Nickbarg ◽  
Robert C. Davenport ◽  
Gregory A. Petsko ◽  
Jeremy R. Knowles
Keyword(s):  
Triosephosphate Isomerase ◽  
Catalytic Mechanism ◽  
Histidine Residue ◽  
Subtle Change

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.

Testimony of the Correlation Between the Reactive Histidine Residue and the Arginase Catalytic Mechanism Using a Biochromatographic Concept and Mutagenesis Experiments

2008 ◽  
Vol 68 (9-10) ◽  
pp. 807-810
Author(s):  
Claire André ◽  
Teddy Bagnost ◽  
Samuel Limat ◽  
Tijani Gharbi ◽  
Yves Claude Guillaume
Keyword(s):  
Catalytic Mechanism ◽  
Histidine Residue

scholarly journals Catalytic mechanism of the phospholipase D superfamily proceeds via a covalent phosphohistidine intermediate

1998 ◽  
Vol 95 (16) ◽  
pp. 9202-9207 ◽  
Author(s):  
Elizabeth B. Gottlin ◽  
Amy E. Rudolph ◽  
Yi Zhao ◽  
Harry R. Matthews ◽  
Jack E. Dixon
Keyword(s):  
Amino Acid ◽  
Phospholipase D ◽  
Inorganic Phosphate ◽  
Pathogenic Bacteria ◽  
Water Exchange ◽  
Catalytic Mechanism ◽  
Phospholipid Metabolism ◽  
Nucleophilic Attack ◽  
Sequence Motif ◽  
Histidine Residue

The phospholipase D (PLD) superfamily includes enzymes of phospholipid metabolism, nucleases, as well as ORFs of unknown function in viruses and pathogenic bacteria. These enzymes are characterized by the invariant sequence motif, H(X)K(X)4D. The endonuclease member Nuc of the PLD family was over-expressed in bacteria and purified to homogeneity. Mutation of the conserved histidine to an asparagine in the endonuclease reduced the kcat for hydrolysis by a factor of 105, suggesting that the histidine residue plays a key role in catalysis. In addition to catalyzing hydrolysis, a number of phosphohydrolases will catalyze a phosphate (oxygen)-water exchange reaction. We have taken advantage of this observation and demonstrate that a 32P-labeled protein could be trapped when the enzyme was incubated with 32P-labeled inorganic phosphate. The phosphoenzyme intermediate was stable in 1 M NaOH and labile in 1 M HCl and 1 M hydroxylamine, suggesting that the enzyme forms a phosphohistidine intermediate. The pH-stability profile of the phosphoenzyme intermediate was consistent with phosphohistidine and the only radioactive amino acid found after alkaline hydrolysis was phosphohistidine. These results suggest that the enzymes in the PLD superfamily use the conserved histidine for nucleophilic attack on the substrate phosphorus atom and most likely proceed via a common two-step catalytic mechanism.

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