Function Discovery and Structural Characterization of a Methylphosphonate Esterase

Journal Article · Tue May 12 04:00:00 EDT 2015 · Biochemistry

DOI:https://doi.org/10.1021/acs.biochem.5b00199· OSTI ID:1229090

Xiang, Dao Feng ^[1]; Patskovsky, Yury ^[2]; Nemmara, Venkatesh V. ^[1]; Toro, Rafael ^[2]; Almo, Steven C. ^[2]; Raushel, Frank M. ^[1]

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1. Texas A & M Univ., College Station, TX (United States)
2. Einstein College of Medicine, Bronx, NY (United States)

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 × 10⁵ 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 this enzyme make it an attractive template for the evolution of novel enzymes for the detection, destruction, and detoxification of organophosphonate nerve agents.

• Research Organization: Brookhaven National Laboratory (BNL), Upton, NY (United States)
• Sponsoring Organization: USDOE Office of Science (SC), Basic Energy Sciences (BES) (SC-22)
• OSTI ID: 1229090
• Report Number(s): BNL--111165-2015-JA
• Journal Information: Biochemistry, Journal Name: Biochemistry Journal Issue: 18 Vol. 54; ISSN 0006-2960
• Publisher: American Chemical Society (ACS)
• Country of Publication: United States
• Language: English

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59 BASIC BIOLOGICAL SCIENCES