Kold, David1; Dauter, Zbigniew6; Laustsen, Anne K6; Brzozowski, Andrzej M6; Turkenburg, Johan P6; Nielsen, Anders D6; Koldsø, Heidi7; Petersen, Evamaria4; Schiøtt, Birgit8; De Maria, Leonardo6; Wilson, Keith S6; Svendsen, Allan6; Wimmer, Reinhard2
1 Department of Chemistry and Bioscience, The Faculty of Engineering and Science, Aalborg University, VBN2 Section of Biotechnology, The Faculty of Engineering and Science, Aalborg University, VBN3 The Faculty of Engineering and Science, Aalborg University, VBN4 Department of Physics and Nanotechnology, The Faculty of Engineering and Science, Aalborg University, VBN5 Microbial Communities, The Faculty of Engineering and Science, Aalborg University, VBN6 unknown7 Interdisciplinær Nanoscience Forskerskole8 Interdisciplinary Nanoscience Center - INANO-Kemi, Langelandsgade
The interaction of lipolytic enzymes with anionic surfactants is of great interest with respect to industrially produced detergents. Here, we report the interaction of cutinase from the thermophilic fungus Humicola insolens with the anionic surfactant SDS, and show the enzyme specifically binds a single SDS molecule under nondenaturing concentrations. Protein interaction with SDS was investigated by NMR, ITC and molecular dynamics simulations. The NMR resonances of the protein were assigned, with large stretches of the protein molecule not showing any detectable resonances. SDS is shown to specifically interact with the loops surrounding the catalytic triad with medium affinity (Ka ≈ 105 M−1). The mode of binding is closely similar to that seen previously for binding of amphiphilic molecules and substrate analogues to cutinases, and hence SDS acts as a substrate mimic. In addition, the structure of the enzyme has been solved by X-ray crystallography in its apo form and after cocrystallization with diethyl p-nitrophenyl phosphate (DNPP) leading to a complex with monoethylphosphate (MEP) esterified to the catalytically active serine. The enzyme has the same fold as reported for other cutinases but, unexpectedly, esterification of the active site serine is accompanied by the ethylation of the active site histidine which flips out from its usual position in the triad.
Protein Science, 2014, Vol 23, Issue 8, p. 1023-1035