3T8U: Crystal Structure Of Ketosteroid Isomerase Y14ay55fd99a From Pseudomonas Testosteroni

Prior site-directed mutagenesis studies in bacterial ketosteroid isomerase (KSI) reported that substitution of both oxyanion hole hydrogen bond donors gives a 10(5)- to 10(8)-fold rate reduction, suggesting that the oxyanion hole may provide the major contribution to KSI catalysis. But these seemingly conservative mutations replaced the oxyanion hole hydrogen bond donors with hydrophobic side chains that could lead to suboptimal solvation of the incipient oxyanion in the mutants, thereby potentially exaggerating the apparent energetic benefit of the hydrogen bonds relative to water-mediated hydrogen bonds in solution. We determined the functional and structural consequences of substituting the oxyanion hole hydrogen bond donors and several residues surrounding the oxyanion hole with smaller residues in an attempt to create a local site that would provide interactions more analogous to those in aqueous solution. These more drastic mutations created an active-site cavity estimated to be approximately 650 A(3) and sufficient for occupancy by 15-17 water molecules and led to a rate decrease of only approximately 10(3)-fold for KSI from two different species, a much smaller effect than that observed from more traditional conservative mutations. The results underscore the strong context dependence of hydrogen bond energetics and suggest that the oxyanion hole provides an important, but moderate, catalytic contribution relative to the interactions in the corresponding solution reaction.
PDB ID: 3T8UDownload
MMDB ID: 95253
PDB Deposition Date: 2011/8/1
Updated in MMDB: 2011/12
Experimental Method:
x-ray diffraction
Resolution: 2.5  Å
Source Organism:
Similar Structures:
Biological Unit for 3T8U: dimeric; determined by author and by software (PISA)
Molecular Components in 3T8U
Label Count Molecule
Proteins (2 molecules)
Steroid Delta-isomerase
Molecule annotation
Chemicals (3 molecules)
* Click molecule labels to explore molecular sequence information.

Citing MMDB