Structural elements responsible for conversion of streptavidin to a pseudoenzyme.
Avidin enhances the hydrolysis of biotinyl p-nitrophenyl ester (BNP) under mild alkaline conditions, whereas streptavidin prevents hydrolysis of BNP up to pH 12. Recently, we imposed hydrolytic activity on streptavidin by rational mutagenesis, based on the molecular elements responsible for the hydrolysis by avidin. Three mutants were designed, whereby the desired features, the distinctive L124R point mutation (M1), the L3,4 loop replacement (M2), and the combined mutation (M3), were transferred from avidin to streptavidin. The crystal structures of the mutants, in complex with biotinyl p-nitroanilide (BNA), the stable amide analogue of BNP, were determined. The results demonstrate that the point mutation alone has little effect on hydrolysis, and BNA exhibits a conformation similar to that of streptavidin. Substitution of a lengthier L3,4 loop (from avidin to streptavidin), resulted in an open conformation, thus exposing the ligand to solvent. Moreover, the amide bond of BNA was flipped relative to that of the streptavidin and M1 complexes, thus deflecting the nitro group toward Lys-121. Consequently, the leaving group potential of the nitrophenyl group of BNP is increased, and M2 hydrolyzes BNP at pH values >8.5. To better emulate the hydrolytic potential of avidin, M3 was required. The combination of loop replacement and point mutation served to further increase the leaving group potential by interaction of the nitro group with Arg-124 and Lys-121. The information derived from this study may provide insight into the design of enzymes and transfer of desired properties among homologous proteins.
Eisenberg-Domovich
Yael
Y
Department of Biological Chemistry, Institute of Life Sciences, The Wolfson Centre for Applied Structural Biology, Hebrew University of Jerusalem, Givat Ram, Jerusalem 91904, Israel.
Pazy
Yael
Y
Nir
Orit
O
Raboy
Bilha
B
Bayer
Edward A
EA
Wilchek
Meir
M
Livnah
Oded
O
D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D017354:Point Mutation; D011487:Protein Conformation; D019809:Streptavidin / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014961:X-Ray Diffraction
D019809:Streptavidin