Function discovery and structural characterization of a methylphosphonate esterase

Dao Feng Xiang, Yury Patskovsky, Venkatesh V. Nemmara, Rafael Toro, Steven C. Almo, Frank M. Raushel

Research output: Contribution to journalArticle

2 Citations (Scopus)

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). The structure was also determined with manganese and butyrate in the active site (PDB id: 4QSF). Pmi1525 folds as a distorted (β/α)<inf>8</inf>-barrel that is typical for members of the amidohydrolase superfamily and cog1735. 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<inf>cat</inf> and k<inf>cat</inf>/K<inf>m</inf> values of 580 s<sup>-1</sup> and 1.2 × 10<sup>5</sup> M<sup>-1</sup> s<sup>-1</sup>, respectively) and 4-nitrophenyl butyrate (k<inf>cat</inf> and k<inf>cat</inf>/K<inf>m</inf> values of 140 s<sup>-1</sup> and 1.4 × 10<sup>5</sup> M<sup>-1</sup> s<sup>-1</sup>, respectively). Pmi1525 is stereoselective for the hydrolysis of chiral methylphosphonate esters. The enzyme hydrolyzes the (S<inf>P</inf>)-enantiomer of isobutyl 4-nitrophenyl methylphosphonate 14 times faster than the corresponding (R<inf>P</inf>)-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.

Original languageEnglish (US)
Pages (from-to)2919-2930
Number of pages12
JournalBiochemistry
Volume54
Issue number18
DOIs
StatePublished - May 12 2015

Fingerprint

Esterases
Organophosphonates
Amidohydrolases
Enantiomers
Enzymes
Hydrolysis
Catalytic Domain
Esters
Cacodylic Acid
Proteus mirabilis
Detoxification
Butyrates
Substrates
Bioinformatics
Manganese
Computational Biology
Zinc
Screening
methylphosphonic acid
Proteins

ASJC Scopus subject areas

  • Biochemistry

Cite this

Xiang, D. F., Patskovsky, Y., Nemmara, V. V., Toro, R., Almo, S. C., & Raushel, F. M. (2015). Function discovery and structural characterization of a methylphosphonate esterase. Biochemistry, 54(18), 2919-2930. https://doi.org/10.1021/acs.biochem.5b00199

Function discovery and structural characterization of a methylphosphonate esterase. / Xiang, Dao Feng; Patskovsky, Yury; Nemmara, Venkatesh V.; Toro, Rafael; Almo, Steven C.; Raushel, Frank M.

In: Biochemistry, Vol. 54, No. 18, 12.05.2015, p. 2919-2930.

Research output: Contribution to journalArticle

Xiang, DF, Patskovsky, Y, Nemmara, VV, Toro, R, Almo, SC & Raushel, FM 2015, 'Function discovery and structural characterization of a methylphosphonate esterase', Biochemistry, vol. 54, no. 18, pp. 2919-2930. https://doi.org/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. In: Biochemistry. 2015 ; Vol. 54, No. 18. pp. 2919-2930.
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