Catalysis by N-Acetyl-d-glucosaminylphosphatidylinositol De-N-acetylase (PIG-L) from Entamoeba histolytica

Ashraf, Mohammad; Sreejith, Perinthottathil; Yadav, Usha; Komath, Sneha Sudha

doi:10.1074/jbc.m112.427245

Cited by 4 publications

(9 citation statements)

References 14 publications

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“…Activity is expressed as a percentage relative to the purified OsGLYI-8 without any exogenous addition of metal ions (taken as 100%). requirement for metal ions for catalysis (Ashraf et al, 2013). A possible explanation for this came through mutagenesis studies, which suggested that in the absence of metal ions (primarily Mg 2+ and Mn 2+ ), the active site residues, His and Asp, can participate as a general acid-base pair, therefore effectively abolishing the strict requirement for the metal ion, which can, however, stimulate the activity of PIG-L over the metal-depleted form (Ashraf et al, 2013).…”

Section: Resultsmentioning

confidence: 99%

“…requirement for metal ions for catalysis (Ashraf et al, 2013). A possible explanation for this came through mutagenesis studies, which suggested that in the absence of metal ions (primarily Mg 2+ and Mn 2+ ), the active site residues, His and Asp, can participate as a general acid-base pair, therefore effectively abolishing the strict requirement for the metal ion, which can, however, stimulate the activity of PIG-L over the metal-depleted form (Ashraf et al, 2013). In the case of OsGLYI-8 too, highly similar results were found wherein the enzyme was active in the near absence of metal ions while the activity was stimulated upon exogenous addition of divalent cations post-EDTA treatment.…”

Section: Resultsmentioning

confidence: 99%

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A nuclear‐localized rice glyoxalase I enzyme, OsGLYI‐8, functions in the detoxification of methylglyoxal in the nucleus

et al. 2017

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The cellular levels of methylglyoxal (MG), a toxic byproduct of glycolysis, rise under various abiotic stresses in plants. Detoxification of MG is primarily through the glyoxalase pathway. The first enzyme of the pathway, glyoxalase I (GLYI), is a cytosolic metalloenzyme requiring either Ni or Zn for its activity. Plants possess multiple GLYI genes, of which only some have been partially characterized; hence, the precise molecular mechanism, subcellular localization and physiological relevance of these diverse isoforms remain enigmatic. Here, we report the biochemical properties and physiological role of a putative chloroplast-localized GLYI enzyme, OsGLYI-8, from rice, which is strikingly different from all hitherto studied GLYI enzymes in terms of its intracellular localization, metal dependency and kinetics. In contrast to its predicted localization, OsGLYI-8 was found to localize in the nucleus along with its substrate, MG. Further, OsGLYI-8 does not show a strict requirement for metal ions for its activity, is functional as a dimer and exhibits unusual biphasic steady-state kinetics with a low-affinity and a high-affinity substrate-binding component. Loss of AtGLYI-2, the closest Arabidopsis ortholog of OsGLYI-8, results in severe germination defects in the presence of MG and growth retardation under salinity stress conditions. These defects were rescued upon complementation with AtGLYI-2 or OsGLYI-8. Our findings thus provide evidence for the presence of a GLYI enzyme and MG detoxification in the nucleus.

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

A nuclear‐localized rice glyoxalase I enzyme, OsGLYI‐8, functions in the detoxification of methylglyoxal in the nucleus

et al. 2017

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“…Upon substrate binding, one water molecule is displaced. However, it has been reported that mutation of this general catalytic base does not change the enzymatic activity of the Entamoeba histolytica PIG-L protein in vitro (Ashraf et al, 2013). 4), and a nucleophilic attack takes place after which a tetrahydral oxyanion intermediate is formed.…”

Section: Discussionmentioning

confidence: 99%

“…DnpA is a putative de-N-acetylase Ashraf et al, 2013). The proteins typically display low overall sequence identity (~25%) but share a catalytic site containing a number of conserved residues that are essential for enzymatic activity.…”

Section: Dnpa Does Not Cause Major Lps Modificationsmentioning

confidence: 99%

“…The differentially expressed genes were classified into functional categories according to the PseudoCAP categories for both dnpA mutant and overexpression strain. Full species names and GenBank protein Accession Numbers: Homo sapiens, BAA74775 (Watanabe et al, 1999); Rattus norvegicus, BAA74776 (Watanabe et al, 1999;Urbaniak et al, 2005); Trypanosoma brucei, AAN60997 (Chang et al, 2002); Leishmania major, AAN60998 (Chang et al, 2002); Plasmodium falciparum, CAG25059.2 ; Entamoeba histolytica, XP_654497.1 (Vats et al, 2005;Ashraf et al, 2013); Thermococcus kodakaraensis, Q6F4N1 (Tanaka et al, 2004); Nonomuraea sp. 4 Partial sequence alignment of the active site of DnpA and related N-deacetylases.…”

Section: Transcriptome Analysismentioning

confidence: 99%

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A putative de-N-acetylase of the PIG-L superfamily affects fluoroquinolone tolerance inPseudomonas aeruginosa

et al. 2014

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A major cause of treatment failure of infections caused by Pseudomonas aeruginosa is the presence of antibiotic-insensitive persister cells. The mechanism of persister formation in P. aeruginosa is largely unknown, and so far, only few genetic determinants have been linked to P. aeruginosa persistence. Based on a previous high-throughput screening, we here present dnpA (de-N-acetylase involved in persistence; gene locus PA14_66140/PA5002) as a new gene involved in noninherited fluoroquinolone tolerance in P. aeruginosa. Fluoroquinolone tolerance of a dnpA mutant is strongly reduced both in planktonic culture and in a biofilm model, whereas overexpression of dnpA in the wild-type strain increases the persister fraction. In addition, the susceptibility of the dnpA mutant to different classes of antibiotics is not affected. dnpA is part of the conserved LPS core oligosaccharide biosynthesis gene cluster. Based on primary sequence analysis, we predict that DnpA is a de-N-acetylase, acting on an unidentified substrate. Site-directed mutagenesis suggests that this enzymatic activity is essential for DnpA-mediated persistence. A transcriptome analysis indicates that DnpA primarily affects the expression of genes involved in surface-associated processes. We discuss the implications of these findings for future antipersister therapies targeted at chronic P. aeruginosa infections.

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Molecular dynamics simulations and statistical coupling analysis of GPI12 in L. major: functional co-evolution and conservedness reveals potential drug–target sites

2015

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Catalysis by N-Acetyl-d-glucosaminylphosphatidylinositol De-N-acetylase (PIG-L) from Entamoeba histolytica

Cited by 4 publications

References 14 publications

A nuclear‐localized rice glyoxalase I enzyme, OsGLYI‐8, functions in the detoxification of methylglyoxal in the nucleus

A nuclear‐localized rice glyoxalase I enzyme, OsGLYI‐8, functions in the detoxification of methylglyoxal in the nucleus

A putative de-N-acetylase of the PIG-L superfamily affects fluoroquinolone tolerance inPseudomonas aeruginosa

Molecular dynamics simulations and statistical coupling analysis of GPI12 in L. major: functional co-evolution and conservedness reveals potential drug–target sites

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