Structural and functional impairment of an Old Yellow Enzyme homologue upon affinity tag incorporation

Protein Expression and Purification
Teresa B FitzpatrickPeter Macheroux

Abstract

Recently, it has been reported that the previously uncharacterized YqjM protein from Bacillus subtilis is a true homologue of the physiologically enigmatic yeast Old Yellow Enzyme (OYE). In this study, it was also demonstrated that YqjM is involved in the oxidative stress response of B. subtilis, thus highlighting a novel direction to pursue the role of the OYE family of proteins in the cell. As part of an attempt to pin down the exact physiological role of these enzymes, both a N-terminal glutathione S-transferase and a C-terminal histidine-tagged form of the protein were created to enable "pull-down" assays and identify interacting partners which could aid in the functional definition. However, here we report on a comparison of the biochemical properties of the tagged forms with the native/untagged YqjM, revealing critical differences in the catalytic activities and quaternary structure of the protein forms. UV-visible spectrophotometric features as well as steady state and individual half-reaction kinetic parameters show that the affinity tagged forms are severely impaired both in ligand binding and catalysis. Gel filtration and dynamic light scattering studies show that incorporation of a tag also has major effects on the q...Continue Reading

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Mar 28, 2003·The Journal of Biological Chemistry·Teresa B FitzpatrickPeter Macheroux

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Citations

Sep 16, 2008·Applied and Environmental Microbiology·Pieter van DillewijnJuan-Luis Ramos
Dec 3, 2010·BMC Plant Biology·Silvia DonniniGraziano Zocchi
Mar 28, 2007·Angewandte Chemie·André MüllerBettina Rosche
Nov 11, 2010·Chembiochem : a European Journal of Chemical Biology·Martyn E HulleyNigel S Scrutton
Nov 28, 2009·Chembiochem : a European Journal of Chemical Biology·Björn V AdalbjörnssonNigel S Scrutton
May 14, 2005·The Journal of Biological Chemistry·Karina KitzingTim Clausen
Nov 5, 2019·Biochimica Et Biophysica Acta. Proteins and Proteomics·M C R RauchF Hollmann
Oct 21, 2009·Biochemistry·Olivia SpiegelhauerHolger Dobbek

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