Structural characterization and reversal of the natural organophosphate resistance of a D-type esterase, Saccharomyces cerevisiae S-formylglutathione hydrolase

Biochemistry
Patricia M LeglerCharles B Millard

Abstract

Saccharomyces cerevisiae expresses a 67.8 kDa homodimeric serine thioesterase, S-formylglutathione hydrolase (SFGH), that is 39.9% identical with human esterase D. Both enzymes possess significant carboxylesterase and S-formylglutathione thioesterase activity but are unusually resistant to organophosphate (OP) inhibitors. We determined the X-ray crystal structure of yeast (y) SFGH to 2.3 A resolution by multiwavelength anomalous dispersion and used the structure to guide site-specific mutagenesis experiments addressing substrate and inhibitor reactivity. Our results demonstrate a steric mechanism of OP resistance mediated by a single indole ring (W197) located in an enzyme "acyl pocket". The W197I substitution enhances ySFGH reactivity with paraoxon by >1000-fold ( k i (W197I) = 16 +/- 2 mM (-1) h (-1)), thereby overcoming natural OP resistance. W197I increases the rate of OP inhibition under pseudo-first-order conditions but does not accelerate OP hydrolysis. The structure of the paraoxon-inhibited W197I variant was determined by molecular replacement (2.2 A); it revealed a stabilized sulfenic acid at Cys60. Wild-type (WT) ySFGH is inhibited by thiol reactive compounds and is sensitive to oxidation; thus, the cysteine sulfenic...Continue Reading

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Citations

Sep 5, 2015·Free Radical Research·Nelmi O Devarie-BaezCristina M Furdui
Aug 22, 2012·Archives of Biochemistry and Biophysics·Patricia M LeglerCharles B Millard
Aug 5, 2009·Protein Science : a Publication of the Protein Society·Karin E van StraatenDavid A R Sanders
Oct 30, 2013·Proteins·Russell SpitzerAjay N Jain
Oct 10, 2013·Mass Spectrometry Reviews·Cristina M Furdui, Leslie B Poole
Oct 13, 2018·Protein Science : a Publication of the Protein Society·Polytimi S DimitriouKonstantin Denessiouk
Jan 8, 2009·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Dong WuZhi-Jie Liu
Apr 20, 2020·Biochemical Pharmacology·Kayla D BouknightPatricia M Legler
May 18, 2020·Progress in Lipid Research·Crystall M D SwarbrickJade K Forwood
Apr 17, 2021·Free Radical Biology & Medicine·Inah HwangJihye Paik
Sep 16, 2021·The Journal of Physical Chemistry. B·Bailu YanZhongyue J Yang
Dec 19, 2021·Protein Science : a Publication of the Protein Society·Benjamin T CaswellDavid C Cantu

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