Asp-99 donates a hydrogen bond not to Tyr-14 but to the steroid directly in the catalytic mechanism of Delta 5-3-ketosteroid isomerase from Pseudomonas putida biotype B.
Delta 5-3-ketosteroid isomerase (KSI) catalyzes the allylic isomerization of Delta 5-3-ketosteroids at a rate approaching the diffusion limit by an intramolecular transfer of a proton. Despite the extensive studies on the catalytic mechanism, it still remains controversial whether the catalytic residue Asp-99 donates a hydrogen bond to the steroid or to Tyr-14. To clarify the role of Asp-99 in the catalysis, two single mutants of D99E and D99L and three double mutants of Y14F/D99E, Y14F/D99N, and Y14F/D99L have been prepared by site-directed mutagenesis. The D99E mutant whose side chain at position 99 is longer by an additional methylene group exhibits nearly the same kcat as the wild-type while the D99L mutant exhibits ca. 125-fold lower kcat than that of the wild-type. The mutations made at positions 14 and 99 exert synergistic or partially additive effect on kcat in the double mutants, which is inconsistent with the mechanism based on the hydrogen-bonded catalytic dyad, Asp-99 COOH...Tyr-14 OH...C3-O of the steroid. The crystal structure of D99E/D38N complexed with equilenin, an intermediate analogue, at 1.9 A resolution reveals that the distance between Tyr-14 O eta and Glu-99 O epsilon is ca. 4.2 A, which is beyond the range for a hydrogen bond, and that the distance between Glu-99 O epsilon and C3-O of the steroid is maintained to be ca. 2.4 A, short enough for a hydrogen bond to be formed. Taken together, these results strongly support the idea that Asp-99 contributes to the catalysis by donating a hydrogen bond directly to the intermediate.
Choi
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G
Department of Life Sciences, Center for Biofunctional Molecules and School of Environmental Engineering, Pohang University of Science and Technology, Pohang, Kyungbuk, 790-784, Korea.
Ha
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NC
Kim
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SW
Kim
D H
DH
Park
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S
Oh
B H
BH
Choi
K Y
KY
D001216:Asparagine / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites / Q000235:genetics; D002384:Catalysis; D004855:Equilenin / Q000737:chemistry; D018698:Glutamic Acid / Q000235:genetics; D006860:Hydrogen Bonding; D007700:Kinetics; D046911:Macromolecular Substances; D016297:Mutagenesis, Site-Directed; D009277:Nandrolone / Q000378:metabolism; D016958:Pseudomonas putida / Q000201:enzymology* / Q000235:genetics; D013251:Steroid Isomerases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014443:Tyrosine / Q000235:genetics / Q000378:metabolism*
D046911:Macromolecular Substances; D001224:Aspartic Acid; D018698:Glutamic Acid; D014443:Tyrosine; D009277:Nandrolone; D001216:Asparagine; D013251:Steroid Isomerases; C022660:steroid delta-isomerase; D004855:Equilenin