SacPox from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius is a proficient lactonase

Abstract

Background: SacPox, an enzyme from the extremophilic crenarchaeal Sulfolobus acidocaldarius (Sac), was isolated by virtue of its phosphotriesterase (or paraoxonase; Pox) activity, i.e. its ability to hydrolyze the neurotoxic organophosphorus insecticides. Later on, SacPox was shown to belong to the Phosphotriesterase-Like Lactonase family that comprises natural lactonases, possibly involved in quorum sensing, and endowed with promiscuous, phosphotriesterase activity.

Results: Here, we present a comprehensive and broad enzymatic characterization of the natural lactonase and promiscuous organophosphorus hydrolase activities of SacPox, as well as a structural analysis using a model.

Conclusion: Kinetic experiments show that SacPox is a proficient lactonase, including at room temperature. Moreover, we discuss the observed differences in substrate specificity between SacPox and its closest homologues SsoPox and SisLac together with the possible structural causes for these observations.

Figure 1

Chemical structure of SacPox substrates. Chemical structures of (A) phosphotriesters, (B) esters, (C) Acyl-Homoserine Lactones, (D) γ-lactones and (E) δ-lactones are presented. For phosphotriesters, R corresponds to different nature of substituents; LG corresponds to the leaving group. The terminal substituent could be S atom if the molecule is a thionophosphotriester or an O atom if the molecule is an oxonophosphotriester. For esters, R corresponds to different nature of substituent. For AHLs and γ/δ-lactones, R corresponds to different size of acyl chain.

Figure 2

Phylogenetic analysis of the PLL family. A. Phylogenetic tree of PLLs, PTEs, and close homologues. Members of PLL-B are colored in green while within the PLL-As, mesophilic and archaeal PLLs are respectively colored in red and orange. The clades of PHPs, PTEs and RTXs were collapsed for clarity. All the sequences used for this tree are listed in Additional file 1: Table S1. B. Sequence alignment of BdPTE from B. diminuta, SsoPox from S. solfataricus, SacPox from S. acidocaldarius and DrOPH from D. radiodurans. Conserved amino acid residues are highlighted in black and similar residues in grey. Conserved active site residues involved in metals coordination are highlighted by red stars. Secondary structures are represented according to SsoPox structure (with pink arrows depicting β-sheets and red cylinders depicting α-helixes).

Figure 3

Structural model of SacPox. A. Structural superposition of SsoPox structure (2VC5; grey) and the SacPox model (green). Cobalt, iron and the catalytic water molecule are respectively represented by pink, orange and red spheres. Bimetallic center coordinating residues are represented as sticks. B. Active site view of superimposed SsoPox structure (grey) and the SacPox model (green). Several active site residues are represented as sticks. Numbering is made according to SacPox sequence.