Roles for Arg426 and Trp111 in the modulation of NADH oxidase activity of the catalase-peroxidase KatG from Burkholderia pseudomallei inferred from pH-induced structural changes

Biochemistry
Xavier CarpenaIgnacio Fita

Abstract

Crystals of Burkholderia pseudomallei KatG retain their ability to diffract X-rays at high resolution after adjustment of the pH from 5.6 to 4.5, 6.5, 7.5, and 8.5, providing a unique view of the effect of pH on protein structure. One significant pH-sensitive change lies in the appearance of a perhydroxy group attached to the indole nitrogen of the active site Trp111 above pH 7, similar to a modification originally observed in the Ser324Thr variant of the enzyme at pH 5.6. The modification forms rapidly from molecular oxygen in the buffer with 100% occupancy after one minute of soaking of the crystal at room temperature and pH 8.5. The low temperature (4 K) ferric EPR spectra of the resting enzyme, being very sensitive to changes in the heme iron microenvironment, confirm the presence of the modification above pH 7 in native enzyme and variants lacking Arg426 or Met264 and its absence in variants lacking Trp111 or Tyr238. The indole-perhydroxy group is very likely the reactive intermediate of molecular oxygen in the NADH oxidase reaction, and Arg426 is required for its reduction. The second significant pH-sensitive change involves the buried side chain of Arg426 that changes from one predominant conformation at low pH to a seco...Continue Reading

References

May 16, 2000·The Journal of Biological Chemistry·G RegelsbergerC Obinger
Feb 27, 2003·European Journal of Biochemistry·Christa JakopitschChristian Obinger
Mar 12, 2003·Journal of Molecular Biology·Xavi CarpenaIgnacio Fita
Jul 3, 2004·The Journal of Biological Chemistry·Thomas BertrandKatherine A Brown

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Citations

Jun 18, 2010·The Journal of Biological Chemistry·Ben WisemanPeter C Loewen
Nov 28, 2013·Archives of Biochemistry and Biophysics·Olive J NjumaDouglas C Goodwin
Mar 18, 2009·Journal of Biological Inorganic Chemistry : JBIC : a Publication of the Society of Biological Inorganic Chemistry·Ben WisemanPeter C Loewen
Jan 8, 2010·Protein Science : a Publication of the Protein Society·Christine E CadeReza A Ghiladi
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Apr 17, 2008·Biochimica Et Biophysica Acta·Robert L MooreDouglas C Goodwin
Jan 3, 2012·Archives of Biochemistry and Biophysics·Adelaida DíazXavi Carpena
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Mar 21, 2007·FEMS Immunology and Medical Microbiology·Panagoula CharalabousJohn W Smalley
Jan 7, 2018·Archives of Biochemistry and Biophysics·Vanessa Vega-GarcíaWilhelm Hansberg
May 3, 2014·Journal of the American Chemical Society·Peter C LoewenCarme Rovira
Oct 12, 2007·Journal of the American Chemical Society·Pietro VidossichCarme Rovira

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