Identification of a residue involved in transition-state stabilization in the ATPase reaction of DNA gyrase

Biochemistry
C V Smith, A Maxwell

Abstract

Examination of the X-ray crystal structure of the 43 kDa N-terminal domain of the DNA gyrase B protein (GyrB) shows that the majority of the interactions with bound ATP are made with subdomain 1 (residues 2-220). However, two residues from subdomain 2, Gln335 and Lys337, interact with the gamma-phosphate of ATP. The proposed roles for these residues include nucleotide binding, transition-state stabilization, and triggering protein conformational changes. We have used site-directed mutagenesis to convert Gln335 to Asn and Ala and Lys337 to Gln and Ala in the N-terminal domain of GyrB. Two of the resultant mutant proteins, GyrB43(Q335A) and GyrB43(K337Q), were shown to be correctly folded, and their interactions with ATP have been analyzed in detail. The Q335A protein is apparently unchanged with regard to nucleotide binding and hydrolysis, whereas the K337Q protein shows a modest decrease in nucleotide binding and a drastic reduction in ATPase activity. This is manifested by a approximately 10(3)-fold decrease in kcat. When the two mutations were moved into full-length GyrB, the Q335A mutation again showed little or no effect on activity, whereas the K337Q mutation had undetectable supercoiling and ATPase activities. We conclude...Continue Reading

References

Jan 1, 1991·Critical Reviews in Biochemistry and Molecular Biology·R J Reece, A Maxwell

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Citations

Jan 19, 2000·Trends in Biochemical Sciences·R Dutta, M Inouye
Jan 16, 2013·Quarterly Reviews of Biophysics·Shina C L KamerlinArieh Warshel
Apr 3, 2010·PloS One·Katherine M Evans-RobertsAnthony Maxwell
Sep 10, 2003·Proceedings of the National Academy of Sciences of the United States of America·Scott ClassenJames M Berger
Jan 5, 2005·Archives of Biochemistry and Biophysics·Alejandro Tovar-MéndezDouglas D Randall
Jun 9, 2005·Structure·Kevin D Corbett, James M Berger
May 14, 2004·Annual Review of Biophysics and Biomolecular Structure·Kevin D Corbett, James M Berger
Aug 30, 2016·European Journal of Medicinal Chemistry·Kavita ChaudhariHarun M Patel
Mar 10, 2001·Journal of Dairy Science·J BewleyD B Jackson-Smith
Feb 19, 2002·The Journal of Biological Chemistry·Valérie LamourDino Moras
Mar 12, 2004·The Journal of Biological Chemistry·Jerzy MajkaPeter M J Burgers
Jul 18, 2019·Communications Biology·Lars C PedersenNatalie D Shaw
Nov 3, 2017·Frontiers in Plant Science·Aline KochKarl-Heinz Kogel
Aug 16, 2005·The Journal of Biological Chemistry·Hua WeiGregory L Verdine
Jan 12, 2020·Communications Biology·Lars C PedersenNatalie D Shaw
Aug 1, 2009·EcoSal Plus·Katherine Evans-Roberts, Anthony Maxwell
Oct 6, 2015·EcoSal Plus·Natassja G BushAnthony Maxwell

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