Mutational and Structural Studies Uncover Crucial Amino Acids Determining Activity and Stability of 17β-HSD14.

•Five new variants of 17β-HSD14 characterized by crystallography and enzyme kinetics.•Tyr253′ from the adjacent monomer ties the two monomers together.•Disulfide bridge formed by Cys255 and Cys255′ is not crucial for dimer stabilization.•Gln148 restricts in size the access tunnel of the substrate.