Resolution of chiral phosphate, phosphonate, and phosphinate esters by an enantioselective enzyme library.
An array of 16 enantiomeric pairs of chiral phosphate, phosphonate, and phosphinate esters was used to establish the breadth of the stereoselective discrimination inherent within the bacterial phosphotriesterase and 15 mutant enzymes. For each substrate, the leaving group was 4-hydroxyacetophenone while the other two groups attached to the phosphorus core consisted of an asymmetric mixture of methyl, methoxy, ethyl, ethoxy, isopropoxy, phenyl, phenoxy, cyclohexyl, and cyclohexoxy substituents. For the wild-type enzyme, the relative rates of hydrolysis for the two enantiomers ranged from 3 to 5.4 x 10(5). Various combinations of site-specific mutations within the active site were used to create modified enzymes with alterations in their enantioselective properties. For the single-site mutant enzyme, G60A, the stereoselectivity is enhanced relative to that of the wild-type enzyme by 1-3 orders of magnitude. Additional mutants were obtained where the stereoselectivity is inverted relative to the wild-type enzyme for 13 of the 16 pairs of enantiomers tested for this investigation. The most dramatic example was obtained for the hydrolysis of 4-acetylphenyl methyl phenyl phosphate. The G60A mutant preferentially hydrolyzes the SP-enantiomer by a factor of 3.7 x 10(5). The I106G/F132G/H257Y mutant preferentially hydrolyzes the RP-enantiomer by a factor of 9.7 x 10(2). This represents an enantioselective discrimination of 3.6 x 10(8) between these two mutants, with a total of only four amino acid changes. The rate differential between the two enantiomers for any given mutant enzyme is postulated to be governed by the degree of nonproductive binding within the enzyme active site and stabilization of the transition state. This hypothesis is supported by computational docking of the high-energy, pentavalent form of the substrates to modeled structures of the mutant enzyme; the energies of the docked transition-state analogues qualitatively capture the enantiomeric preferences of the various mutants for the different substrates. These results demonstrate that the catalytic properties of the wild-type phosphotriesterase can be exploited for the kinetic resolution of a wide range of phosphate, phosphonate, and phosphinate esters and that the active site of this enzyme is remarkably amenable to structural perturbations via amino acid substitution.
Nowlan
Charity
C
Department of Chemistry, Texas A&M University, College Station, Texas 77842-3012, USA.
Li
Yingchun
Y
Hermann
Johannes C
JC
Evans
Timothy
T
Carpenter
Joseph
J
Ghanem
Eman
E
Shoichet
Brian K
BK
Raushel
Frank M
FM
D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D017076:Computer-Aided Design; D004952:Esters / Q000737:chemistry*; D006868:Hydrolysis; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D063065:Organophosphonates / Q000737:chemistry*; D010710:Phosphates / Q000737:chemistry*; D044345:Phosphoric Triester Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity
D001426:Bacterial Proteins; D004952:Esters; D063065:Organophosphonates; D010710:Phosphates; D044345:Phosphoric Triester Hydrolases