A Further Biochemical Characterization of DrPLL the Thermophilic Lactonase from Deinococcus radiodurans

Author(s): Luigi Mandrich, Spartaco Di Gennaro, Achille Palma, Giuseppe Manco.

Journal Name: Protein & Peptide Letters

Volume 20 , Issue 1 , 2013

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Abstract:

Recently, the cloning of the ORF Dr0930 from Deinococcus radiodurans displaying, as primary activity, a lactonase activity and a promiscuous phosphotriesterase activity was reported. The crystal structure of the resulting recombinant enzyme has been solved, and many mutants have been generated in order to increase the phosphotriesterase activity, with the aim to reach the level of activity of the related pPTE from Pseudomonas diminuta. In this paper we report an additional biochemical characterization of DrPLL and show that this enzyme has an optimal temperature for catalysis of 85 °C and possesses promiscuous carboxylesterase, phophodiesterase and thioesterase activities which were not previously described. A metal analysis was performed on the purified protein by inductively coupled plasma mass spectrometry (ICP-MS ELAN DRC-e), which confirmed the presence of Ni2+ as a main metal in the recombinant protein. Surprisingly, the specificity constants (s=kcat/KM) for the pNP-decanoate and pNP-dodecanoate esters were only one order of magnitude lower than that for the lactone substrate thio-buthyl-γ-butyric-lactone (TBBL), and the KM value for TBBL was more than ten-fold higher than those for the esters. We named this enzyme DrPLL, based on its structural and biochemical features it belongs to the Phosphotriesterase Like Lactonase group, a small protein family belonging to the amidohydrolase superfamily.

Keywords: Amidohydrolase superfamily, deinococcus radiodurans, lactonases, phophotriesterases, promiscuous activities, thermophilic enzymes, crystal structure, mutants, carboxylesterase, thioesterase

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Article Details

VOLUME: 20
ISSUE: 1
Year: 2013
Page: [36 - 44]
Pages: 9
DOI: 10.2174/0929866511307010036

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