5AI1

Crystal structure of ketosteroid isomerase containing Y32F, D40N, Y57F and Y119F mutations in the equilenin-bound form

Summary for 5AI1

• Entry DOI: 10.2210/pdb5ai1/pdb
• Descriptor: KETOSTEROID ISOMERASE, EQUILENIN (3 entities in total)
• Functional Keywords: isomerase, ketosteroid isomerase, pseudomonas putida, equilenin
• Biological source: PSEUDOMONAS PUTIDA
• Total number of polymer chains: 1
• Total formula weight: 14765.85

Authors

Cha, H.J.,Jeong, J.H.,Kim, Y.G. (deposition date: 2015-02-11, release date: 2015-05-20, Last modification date: 2024-01-10)

Primary citation

Jang, D.S.,Choi, G.,Cha, H.J.,Shin, S.,Hong, B.H.,Lee, H.J.,Lee, H.C.,Choi, K.Y.
Contribution of a Low-Barrier Hydrogen Bond to Catalysis is not Significant in Ketosteroid Isomerase.
Mol.Cells, 38:409-, 2015

PubMed Abstract: Low-barrier hydrogen bonds (LBHBs) have been proposed to have important influences on the enormous reaction rate increases achieved by many enzymes. Δ(5)-3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of Δ(5)-3-ketosteroid to its conjugated Δ(4)-isomers at a rate that approaches the diffusion limit. Tyr14, a catalytic residue of KSI, has been hypothesized to form an LBHB with the oxyanion of a dienolate steroid intermediate generated during the catalysis. The unusual chemical shift of a proton at 16.8 ppm in the nuclear magnetic resonance spectrum has been attributed to an LBHB between Tyr14 Oη and C3-O of equilenin, an intermediate analogue, in the active site of D38N KSI. This shift in the spectrum was not observed in Y30F/Y55F/D38N and Y30F/Y55F/Y115F/D38N mutant KSIs when each mutant was complexed with equilenin, suggesting that Tyr14 could not form LBHB with the intermediate analogue in these mutant KSIs. The crystal structure of Y30F/Y55F/Y115F/D38N-equilenin complex revealed that the distance between Tyr14 Oη and C3-O of the bound steroid was within a direct hydrogen bond. The conversion of LBHB to an ordinary hydrogen bond in the mutant KSI reduced the binding affinity for the steroid inhibitors by a factor of 8.1-11. In addition, the absence of LBHB reduced the catalytic activity by only a factor of 1.7-2. These results suggest that the amount of stabilization energy of the reaction intermediate provided by LBHB is small compared with that provided by an ordinary hydrogen bond in KSI.

PubMed: 25947291
DOI: 10.14348/MOLCELLS.2015.2266

Experimental method

X-RAY DIFFRACTION (2.103 Å)