Dataset Information

Mechanistic Insights into the Hydrolysis of Organophosphorus Compounds by Paraoxonase-1: Exploring the Limits of Substrate Tolerance in a Promiscuous Enzyme.

ABSTRACT: We designed, synthesized and screened a library of analogs of the organophosphate pesticide metabolite paraoxon against a recombinant variant of human serum paraoxonase-1. Alterations of both the aryloxy leaving group and the retained alkyl chains of paraoxon analogs resulted in substantial changes to binding and hydrolysis, as measured directly by spectrophotometric methods or in competition experiments with paraoxon. Increases or decreases in the steric bulk of the retained groups generally reduced the rate of hydrolysis, while modifications of the leaving group modulated both binding and turnover. Studies on the hydrolysis of phosphoryl azide analogs as well as amino-modified paraoxon analogs, the former being developed as photo-affinity labels, found enhanced tolerance of structural modifications, when compared with O-alkyl substituted molecules. Results from computational modeling predict a predominant active site binding mode for these molecules which is consistent with several proposed catalytic mechanisms in the literature, and from which a molecular-level explanation of the experimental trends is attempted. Overall, the results of this study suggest that while paraoxonase-1 is a promiscuous enzyme, there are substantial constraints in the active site pocket, which may relate to both the leaving group and the retained portion of paraoxon analogs.

SUBMITTER: Muthukrishnan S

PROVIDER: S-EPMC3740977 | biostudies-literature | 2012 Dec

REPOSITORIES: biostudies-literature

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Mechanistic Insights into the Hydrolysis of Organophosphorus Compounds by Paraoxonase-1: Exploring the Limits of Substrate Tolerance in a Promiscuous Enzyme.

Muthukrishnan Sivaramakrishnan S Shete Vivekanand S VS Sanan Toby T TT Vyas Shubham S Oottikkal Shameema S Porter Lauren M LM Magliery Thomas J TJ Hadad Christopher M CM

Journal of physical organic chemistry 20121201 12

We designed, synthesized and screened a library of analogs of the organophosphate pesticide metabolite paraoxon against a recombinant variant of human serum paraoxonase-1. Alterations of both the aryloxy leaving group and the retained alkyl chains of paraoxon analogs resulted in substantial changes to binding and hydrolysis, as measured directly by spectrophotometric methods or in competition experiments with paraoxon. Increases or decreases in the steric bulk of the retained groups generally re ...[more]

PMID: 23946555

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Project description:Promiscuous enzymes are generally considered to be starting points in the evolution of offspring enzymes with more specific or even novel catalytic activities, which is the molecular basis of producing new biological functions. Mhg, a typical ?/? fold hydrolase, was previously reported to have both ?-lactamase and perhydrolase activities. However, despite having high structural similarity to and sharing an identical catalytic triad with an extensively studied esterase from Pseudomonas fluorescens, this enzyme did not show any esterase activity. Molecular docking and sequence analysis suggested a possible role for the entry of the binding pocket in blocking the entrance tunnel, preventing the ester compounds from entering into the pocket. By engineering the entrance tunnel with only one or two amino acid substitutions, we successfully obtained five esterase variants of Mhg. The variants exhibited a very broad substrate acceptance, hydrolyzing not only the classical p-nitrophenol esters but also various types of chiral esters, which are widely used as drug intermediates. Site 233 at the entrance tunnel of Mhg was found to play a pivotal role in modulating the three catalytic activities by adjusting the size and shape of the tunnel, with different amino acid substitutions at this site facilitating different activities. Remarkably, the variant with the L233G mutation was a very specific esterase without any ?-lactamase and perhydrolase activities. Considering the amino acid conservation and differentiation, this site could be a key target for future protein engineering. In addition, we demonstrate that engineering the entrance tunnel is an efficient strategy to regulate enzyme catalytic capabilities. IMPORTANCE:Promiscuous enzymes can act as starting points in the evolution of novel catalytic activities, thus providing a molecular basis for the production of new biological functions. In this study, we identified a critical amino acid residue (Leu233) at the entry of the substrate tunnel of a promiscuous enzyme, Mhg. We found that substitution of this residue with smaller amino acids such as Gly, Ala, Ser, or Pro endowed the enzyme with novel esterase activity. Different amino acids at this site can facilitate different catalytic activities. These findings exhibited universal significance in this subset of ?/? fold hydrolases, including Mhg. Furthermore, we demonstrate that engineering the entrance tunnel is an efficient strategy to evolve new enzyme catalytic capabilities. Our study has important implications for the regulation of enzyme catalytic promiscuity and development of protein engineering methodologies.

Dataset Information

Mechanistic Insights into the Hydrolysis of Organophosphorus Compounds by Paraoxonase-1: Exploring the Limits of Substrate Tolerance in a Promiscuous Enzyme.

Publications

Mechanistic Insights into the Hydrolysis of Organophosphorus Compounds by Paraoxonase-1: Exploring the Limits of Substrate Tolerance in a Promiscuous Enzyme.

Similar Datasets

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