skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Function Discovery and Structural Characterization of a Methylphosphonate Esterase

Abstract

Pmi1525, an enzyme of unknown function from Proteus mirabilis HI4320 and the amidohydrolase superfamily, was cloned, purified to homogeneity, and functionally characterized. The three-dimensional structure of Pmi1525 was determined with zinc and cacodylate bound in the active site (PDB id: 3RHG). We also determined the structure with manganese and butyrate in the active site (PDB id: 4QSF). Pmi1525 folds as a distorted (β/α)8-barrel that is typical for members of the amidohydrolase superfamily and cog1735. Moreover, the substrate profile for Pmi1525 was determined via a strategy that marshaled the utilization of bioinformatics, structural characterization, and focused library screening. The protein was found to efficiently catalyze the hydrolysis of organophosphonate and carboxylate esters. The best substrates identified for Pmi1525 are ethyl 4-nitrophenylmethyl phosphonate (k cat and k cat /Km values of 580 s –1 and 1.2 × 10 5 M –1 s –1, respectively) and 4-nitrophenyl butyrate (k cat and k cat /K m values of 140 s –1 and 1.4 × 105 M –1 s –1, respectively). Pmi1525 is stereoselective for the hydrolysis of chiral methylphosphonate esters. The enzyme hydrolyzes the (S P)-enantiomer of isobutyl 4-nitrophenyl methylphosphonate 14 times faster than the corresponding (R P)-enantiomer. The catalytic properties of thismore » enzyme make it an attractive template for the evolution of novel enzymes for the detection, destruction, and detoxification of organophosphonate nerve agents.« less

Authors:
 [1];  [2];  [1];  [2];  [2];  [1]
  1. Texas A & M Univ., College Station, TX (United States)
  2. Einstein College of Medicine, Bronx, NY (United States)
Publication Date:
Research Org.:
Brookhaven National Laboratory (BNL), Upton, NY (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
OSTI Identifier:
1229090
Report Number(s):
BNL-111165-2015-JA
Journal ID: ISSN 0006-2960
DOE Contract Number:  
SC00112704
Resource Type:
Journal Article
Journal Name:
Biochemistry
Additional Journal Information:
Journal Volume: 54; Journal Issue: 18; Journal ID: ISSN 0006-2960
Publisher:
American Chemical Society (ACS)
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Xiang, Dao Feng, Patskovsky, Yury, Nemmara, Venkatesh V., Toro, Rafael, Almo, Steven C., and Raushel, Frank M. Function Discovery and Structural Characterization of a Methylphosphonate Esterase. United States: N. p., 2015. Web. doi:10.1021/acs.biochem.5b00199.
Xiang, Dao Feng, Patskovsky, Yury, Nemmara, Venkatesh V., Toro, Rafael, Almo, Steven C., & Raushel, Frank M. Function Discovery and Structural Characterization of a Methylphosphonate Esterase. United States. doi:10.1021/acs.biochem.5b00199.
Xiang, Dao Feng, Patskovsky, Yury, Nemmara, Venkatesh V., Toro, Rafael, Almo, Steven C., and Raushel, Frank M. Tue . "Function Discovery and Structural Characterization of a Methylphosphonate Esterase". United States. doi:10.1021/acs.biochem.5b00199.
@article{osti_1229090,
title = {Function Discovery and Structural Characterization of a Methylphosphonate Esterase},
author = {Xiang, Dao Feng and Patskovsky, Yury and Nemmara, Venkatesh V. and Toro, Rafael and Almo, Steven C. and Raushel, Frank M.},
abstractNote = {Pmi1525, an enzyme of unknown function from Proteus mirabilis HI4320 and the amidohydrolase superfamily, was cloned, purified to homogeneity, and functionally characterized. The three-dimensional structure of Pmi1525 was determined with zinc and cacodylate bound in the active site (PDB id: 3RHG). We also determined the structure with manganese and butyrate in the active site (PDB id: 4QSF). Pmi1525 folds as a distorted (β/α)8-barrel that is typical for members of the amidohydrolase superfamily and cog1735. Moreover, the substrate profile for Pmi1525 was determined via a strategy that marshaled the utilization of bioinformatics, structural characterization, and focused library screening. The protein was found to efficiently catalyze the hydrolysis of organophosphonate and carboxylate esters. The best substrates identified for Pmi1525 are ethyl 4-nitrophenylmethyl phosphonate (kcat and kcat /Km values of 580 s–1 and 1.2 × 105 M–1 s–1, respectively) and 4-nitrophenyl butyrate (kcat and kcat /Km values of 140 s–1 and 1.4 × 105 M–1 s–1, respectively). Pmi1525 is stereoselective for the hydrolysis of chiral methylphosphonate esters. The enzyme hydrolyzes the (SP)-enantiomer of isobutyl 4-nitrophenyl methylphosphonate 14 times faster than the corresponding (RP)-enantiomer. The catalytic properties of this enzyme make it an attractive template for the evolution of novel enzymes for the detection, destruction, and detoxification of organophosphonate nerve agents.},
doi = {10.1021/acs.biochem.5b00199},
journal = {Biochemistry},
issn = {0006-2960},
number = 18,
volume = 54,
place = {United States},
year = {2015},
month = {5}
}