Mutational Analysis of the Interaction between Active Site Residues and the Loop Region in Mammalian Purple Acid Phosphatases†

Biochemistry ◽  
2001 ◽  
Vol 40 (38) ◽  
pp. 11614-11622 ◽  
Author(s):  
Enrico G. Funhoff ◽  
Jenny Ljusberg ◽  
Yunling Wang ◽  
Goran Andersson ◽  
Bruce A. Averill
Keyword(s):  
Active Site ◽  
Mutational Analysis ◽  
Acid Phosphatases ◽  
Active Site Residues ◽  
Loop Region ◽  
Purple Acid Phosphatases

scholarly journals Important Role for Phylogenetically Invariant PP2Acα Active Site and C-Terminal Residues Revealed by Mutational Analysis in Saccharomyces cerevisiae

Genetics ◽  
2000 ◽  
Vol 156 (1) ◽  
pp. 21-29 ◽  
Author(s):  
David R H Evans ◽  
Brian A Hemmings
Keyword(s):  
Protein Interactions ◽  
Active Site ◽  
Protein Function ◽  
Mutational Analysis ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Active Site Residues ◽  
Catalytic Function

Abstract PP2A is a central regulator of eukaryotic signal transduction. The human catalytic subunit PP2Acα functionally replaces the endogenous yeast enzyme, Pph22p, indicating a conservation of function in vivo. Therefore, yeast cells were employed to explore the role of invariant PP2Ac residues. The PP2Acα Y127N substitution abolished essential PP2Ac function in vivo and impaired catalysis severely in vitro, consistent with the prediction from structural studies that Tyr-127 mediates substrate binding and its side chain interacts with the key active site residues His-118 and Asp-88. The V159E substitution similarly impaired PP2Acα catalysis profoundly and may cause global disruption of the active site. Two conditional mutations in the yeast Pph22p protein, F232S and P240H, were found to cause temperature-sensitive impairment of PP2Ac catalytic function in vitro. Thus, the mitotic and cell lysis defects conferred by these mutations result from a loss of PP2Ac enzyme activity. Substitution of the PP2Acα C-terminal Tyr-307 residue by phenylalanine impaired protein function, whereas the Y307D and T304D substitutions abolished essential function in vivo. Nevertheless, Y307D did not reduce PP2Acα catalytic activity significantly in vitro, consistent with an important role for the C terminus in mediating essential protein-protein interactions. Our results identify key residues important for PP2Ac function and characterize new reagents for the study of PP2A in vivo.

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

Biochemistry ◽  
2009 ◽  
Vol 48 (29) ◽  
pp. 6921-6931 ◽  
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

The Roles of Active-Site Residues in the Catalytic Mechanism oftrans-3-Chloroacrylic Acid Dehalogenase:  A Kinetic, NMR, and Mutational Analysis†

Biochemistry ◽  
2004 ◽  
Vol 43 (14) ◽  
pp. 4082-4091 ◽  
Author(s):  
Hugo F. Azurmendi ◽  
Susan C. Wang ◽  
Michael A. Massiah ◽  
Gerrit J. Poelarends ◽  
Christian P. Whitman ◽  
...  
Keyword(s):  
Active Site ◽  
Catalytic Mechanism ◽  
Mutational Analysis ◽  
Active Site Residues ◽  
Chloroacrylic Acid Dehalogenase

scholarly journals Mutational Analysis of Active Site Residues Essential for Sensing of Organic Hydroperoxides by Bacillus subtilis OhrR

2007 ◽  
Vol 189 (19) ◽  
pp. 7069-7076 ◽  
Author(s):  
Sumarin Soonsanga ◽  
Mayuree Fuangthong ◽  
John D. Helmann
Keyword(s):  
Hydrogen Bond ◽  
Bacillus Subtilis ◽  
Conformational Changes ◽  
Active Site ◽  
Mutational Analysis ◽  
High Sensitivity ◽  
Oxidative Modification ◽  
Active Site Residues

ABSTRACT Bacillus subtilis OhrR is the prototype for the one-Cys family of organic peroxide-sensing regulatory proteins. Mutational analyses indicate that the high sensitivity of the active site cysteine (C15) to peroxidation requires three Tyr residues. Y29 and Y40 from the opposing subunit of the functional dimer hydrogen bond with the reactive Cys thiolate, and substitutions at these positions reduce or eliminate the ability of OhrR to respond to organic peroxides. Y19 is also critical for peroxide sensing, and the Ala substitution mutant (OhrR Y19A) is less susceptible to oxidation at the active site C15 in vivo. The Y19A protein also displays decreased sensitivity to peroxide-mediated oxidation in vitro. Y19 is in van der Waals contact with two residues critical for protein function, F16 and R23. The latter residue makes critical contact with the DNA backbone in the OhrR-operator complex. These results indicate that the high sensitivity of the OhrR C15 residue to oxidation requires interactions with the opposed Tyr residues. Oxidative modification of C15 likely disrupts the C15-Y29′-Y40′ hydrogen bond network and thereby initiates conformational changes that reduce the ability of OhrR to bind to its operator site.

Mutational Analysis of Active Site Residues in theStaphylococcus aureusTranspeptidase SrtA†

Biochemistry ◽  
2007 ◽  
Vol 46 (24) ◽  
pp. 7269-7278 ◽  
Author(s):  
Brenda A. Frankel ◽  
Yan Tong ◽  
Matthew L. Bentley ◽  
Michael C. Fitzgerald ◽  
Dewey G. McCafferty
Keyword(s):  
Active Site ◽  
Mutational Analysis ◽  
Active Site Residues

scholarly journals Insights into the catalytic properties of bamboo vacuolar invertase through mutational analysis of active site residues

2009 ◽  
Vol 70 (1) ◽  
pp. 25-31 ◽  
Author(s):  
Tai-Hung Chen ◽  
Yu-Chiao Huang ◽  
Chii-Shen Yang ◽  
Chien-Chih Yang ◽  
Ai-Yu Wang ◽  
...  
Keyword(s):  
Active Site ◽  
Mutational Analysis ◽  
Catalytic Properties ◽  
Active Site Residues ◽  
Vacuolar Invertase

A Mixed-Valent, Unsymmetrical FeIIFeIII Complex with a Terminal Phenolato Ligand as a Model for the Active Site of Purple Acid Phosphatases

1994 ◽  
Vol 33 (8) ◽  
pp. 887-889 ◽  
Author(s):  
Elisabeth Bernard ◽  
William Moneta ◽  
Jean Laugier ◽  
Sylvie Chardon-Noblat ◽  
Alain Deronzier ◽  
...  
Keyword(s):  
Active Site ◽  
Acid Phosphatases ◽  
Purple Acid Phosphatases

scholarly journals Structural elements that modulate the substrate specificity of plant purple acid phosphatases: avenues for improved phosphorus acquisition in crops

2019 ◽  
Author(s):  
Daniel Feder ◽  
Ross P. McGeary ◽  
Natasa Mitić ◽  
Thierry Lonhienne ◽  
Agnelo Furtado ◽  
...  
Keyword(s):  
Active Sites ◽  
Crop Yields ◽  
Organic Phosphorus ◽  
Homology Modelling ◽  
Acid Phosphatases ◽  
Phosphorus Use Efficiency ◽  
Active Site Residues ◽  
Docking Simulations ◽  
Wide Range ◽  
Purple Acid Phosphatases

AbstractPhosphate acquisition by plants is an essential process that is directly implicated in the optimization of crop yields. Purple acid phosphatases (PAPs) are ubiquitous metalloenzymes, which catalyze the hydrolysis of a wide range of phosphate esters and anhydrides. While some plant PAPs display a preference for ATP as the substrate, others are efficient in hydrolyzing phytate or 2-phosphoenolpyruvate (PEP). PAP from red kidney bean (rkbPAP) is an efficient ATP- and ADPase, but has no activity towards phytate. The crystal structure of this enzyme in complex with an ATP analogue (to 2.20 Å resolution) provides insight into the amino acid residues that play an essential role in binding this substrate. Homology modelling was used to generate three-dimensional structures for the active sites of PAPs from tobacco (NtPAP) and Arabidopsis thaliana (AtPAP12 and AtPAP26) that are efficient in hydrolyzing phytate and PEP as substrates, respectively. In combination with substrate docking simulations and a phylogenetic analysis of 49 plant PAP sequences (including the first PAP sequences reported from Eucalyptus), several active site residues were identified that are important in defining the substrate specificities of plant PAPs. These results may inform bioengineering studies aimed at identifying and incorporating suitable plant PAP genes into crops to improve phosphorus use efficiency. Organic phosphorus sources increasingly supplement or replace inorganic fertilizer, and efficient phosphorus use of crops will lower the environmental footprint of agriculture while enhancing food production.

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