1c7h
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CRYSTAL STRUCTURE OF A MUTANT R75A IN KETOSTEROID ISOMERASE FROM PSEDOMONAS PUTIDA BIOTYPE B
OverviewOverview
The aromatic residues Phe-54, Phe-82, and Trp-116 in the hydrophobic, substrate-binding pocket of Delta(5)-3-ketosteroid isomerase from, Pseudomonas putida biotype B have been characterized in their roles in, steroid binding and catalysis. Kinetic and equilibrium binding analyses, were carried out for the mutant enzymes with the substitutions Phe-54 -->, Ala or Leu, Phe-82 --> Ala or Leu, and Trp-116 --> Ala, Phe, or Tyr. The, removal of their bulky, aromatic side chains at any of these three, positions results in reduced k(cat), particularly when Phe-82 or Trp-116, is replaced by Ala. The results are consistent with the binding, interactions of the aromatic residues with the bound steroid contributing, to catalysis. All the mutations except the F82A mutation increase K(m);, the F82A mutation decreases K(m) by ca. 3-fold, suggesting a possibility, that the phenyl ring at position 82 might be unfavorable for substrate, binding. The K(D) values for d-equilenin, an intermediate analogue, suggest that a space-filling hydrophobic side chain at position 54, a, phenyl ring at position 82, and a nonpolar aromatic or small side chain at, position 116 might be favorable for binding the reaction intermediate. In, contrast to the increased K(D) for equilenin, the enzymes with any, substitutions at positions 54 and 116 display a decreased K(D) for, 19-nortestosterone, a product analogue, indicating that Phe-54 and Trp-116, might be unfavorable for product binding. The crystal structure of F82A, determined to 2.1-A resolution reveals that Phe-82 is important for, maintaining the active site geometry. Taken together, our results, demonstrate that Phe-54, Phe-82, and Trp-116 contribute differentially to, the stabilization of steroid species including substrate, intermediate, and product.
About this StructureAbout this Structure
1C7H is a Single protein structure of sequence from Pseudomonas putida. Active as Steroid Delta-isomerase, with EC number 5.3.3.1 Full crystallographic information is available from OCA.
ReferenceReference
Roles of active site aromatic residues in catalysis by ketosteroid isomerase from Pseudomonas putida biotype B., Kim DH, Nam GH, Jang DS, Choi G, Joo S, Kim JS, Oh BH, Choi KY, Biochemistry. 1999 Oct 19;38(42):13810-9. PMID:10529226
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