Download MendeleyMutations of key hydrophobic surface residues of 11 beta-hydroxysteroid dehydrogenase type 1 increase solubility and monodispersity in a bacterial expression system
Lawson, A.J., Walker, E.A., White, S.A., Dafforn, T.R., Stewart, P.M., Ride, J.P.(2009) Protein Sci 18: 1552-1563
- PubMed: 19507261
- DOI: https://doi.org/10.1002/pro.150
- Primary Citation of Related Structures:
3DWF - PubMed Abstract:
11 beta-Hydroxysteroid dehydrogenase type 1 (11 beta-HSD1) is a key enzyme in the conversion of cortisone to the functional glucocorticoid hormone cortisol. This activation has been implicated in several human disorders, notably the metabolic syndrome where 11 beta-HSD1 has been identified as a novel target for potential therapeutic drugs. Recent crystal structures have revealed the presence of a pronounced hydrophobic surface patch lying on two helices at the C-terminus. The physiological significance of this region has been attributed to facilitating substrate access by allowing interactions with the endoplasmic reticulum membrane. Here, we report that single mutations that alter the hydrophobicity of this patch (I275E, L266E, F278E, and L279E in the human enzyme and I275E, Y266E, F278E, and L279E in the guinea pig enzyme) result in greatly increased yields of soluble protein on expression in E. coli. Kinetic analyses of both reductase and dehydrogenase reactions indicate that the F278E mutant has unaltered K(m) values for steroids and an unaltered or increased k(cat). Analytical ultracentrifugation shows that this mutation also decreases aggregation of both the human and guinea pig enzymes, resulting in greater monodispersity. One of the mutants (guinea pig F278E) has proven easy to crystallize and has been shown to have a virtually identical structure to that previously reported for the wild-type enzyme. The human F278E enzyme is shown to be a suitable background for analyzing the effects of naturally occurring mutations (R137C, K187N) on enzyme activity and stability. Hence, the F278E mutants should be useful for many future biochemical and biophysical studies of the enzyme.
Organizational Affiliation:
School of Biosciences, University of Birmingham, Birmingham B15 2TT, United Kingdom.
