Backbone Dynamics of Fusarium solani pisi Cutinase Probed by Nuclear Magnetic Resonance:  The Lack of Interfacial Activation Revisited

Abstract

The backbone dynamics of Fusarium solani pisi cutinase has been studied by a variety of nuclear magnetic resonance experiments to probe internal motions on different time scales. The core of cutinase appears to be highly rigid. The binding site, including the oxyanion hole, is mobile on the microsecond to millisecond time scale, in contrast to the well-defined active site and preformed oxyanion hole elucidated by X-ray crystallography [Martinez, C., de Geus, P., Lauwereys, M., Matthyssens, G., and Cambillau, C. (1992) Nature 356, 615-618]. In this crystal structure, cutinase has a rather open conformation, in which the hydrophobic binding site is exposed. The observed mobility in solution most likely represents the interconversion between open and more closed conformations, like in a true lipase. The opening and closing motions are on a time scale which corresponds with the kinetics of the hydrolysis reaction, i.e., the millisecond range, which suggests that these conformational rearrangements form the rate-limiting step in catalysis. We conclude that the crystal structure probably represents one of the multiple conformations present in solution, which fortuitously is the active conformation. The implications of our findings are discussed with particular reference to the explanation of the lack of interfacial activation as found for cutinase.

Cutinase: From molecular level to bioprocess development

“…This also explains that cutinase, having an exposed active-site serine, can behave like interfacial activated lipases (Egmond and van Bemmel, 1997). Recently, the lack of interfacial activation on cutinase was confirmed by nuclear magnetic resonance (NMR) methods (Prompers et al, 1999a).…”

“…Definition of the number of binding sites Prangé et al, 1998 NMR Resonance assignments and structure in solution Prompers et al, 1997Prompers et al, , 1999a Structure-function relationships EPR Analysis of the labeled active-site mobility in microemulsions Papadimitriou et al, 1996Papadimitriou et al, , 1997 Use of specific inhibitors (mainly phosphonates)…”

Catalysis by Glomerella cingulata Cutinase Requires Conformational Cycling between the Active and Inactive States of Its Catalytic Triad

“…Abbreviations used: E600, diethyl p-nitrophenyl phosphate; PETFP, 3-phenethylthio-1,1,1-trifluoropropan-2-one; LR-HSQC, long-range 15 N heteronuclear single quantum coherence. esterases and lipases.…”

Purification and Characterization of Mycobacterial Phospholipase A: an Activity Associated with Mycobacterial Cutinase

“…The cutinase catalytic triad is a serine, an asparagine, and a histidine in an oxyanion hole. Unlike phospholipases, they do not exhibit interfacial activation, though they are stabilized by detergents (28). Though mycobacteria have open reading frames annotated as putative cutinases based on motif searches, there is no published research on them.…”