A further biochemical characterization of DrPLL the thermophilic lactonase from Deinococcus radiodurans.
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 Ni(2+) as a main metal in the recombinant protein. Surprisingly, the specificity constants (s=k(cat)/K(M)) 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 K(M) 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.
Institute of Protein Biochemistry (IBP), National Research Council (CNR), Naples, Italy. l.mandrich@ibp.cnr.it
Luigi
L
Mandrich
Spartaco
S
Di Gennaro
Achille
A
Palma
Giuseppe
G
Manco
D015107:4-Butyrolactone / Q000031:analogs & derivatives / Q000378:metabolism; D000581:Amidohydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism*; D034301:Deinococcus / Q000201:enzymology / Q000235:genetics; D004795:Enzyme Stability; D006358:Hot Temperature; D013058:Mass Spectrometry; D009532:Nickel / Q000032:analysis; D044345:Phosphoric Triester Hydrolases / Q000378:metabolism; D013054:Spectrophotometry, Atomic; D013379:Substrate Specificity
D009532:Nickel; D002265:Carboxylic Ester Hydrolases; D044345:Phosphoric Triester Hydrolases; D000581:Amidohydrolases; D015107:4-Butyrolactone