Mn 2+ modulates the kinetic properties of an archaeal member of the PLL family Elena Porzio a , Spartaco Di Gennaro b , Achille Palma b , Giuseppe Manco a,⇑ a Istituto di Biochimica delle Proteine, Consiglio Nazionale delle Ricerche, Via P. Castellino 111, 80131 Naples, Italy b Metapontum Agrobios, S.s. Jonica, 75010, Metaponto di Bernalda, Italy article info Article history: Available online 19 November 2012 Keywords: Organophosphates Phosphotriesterase-like lactonases Metals Stability Kinetic parameters abstract Recently we reported on the characterization of an archaeal member of the amidohydrolase superfamily, namely Sulfolobus acidocaldarius lactonase, showing low but significant and extremely thermostable paraoxonase activity. This enzyme, that we have named SacPox, is a member of the new described family of phosphotriesterase-like lactonases (PLLs). In this family the binuclear metal centre, which is involved in the catalytic machinery, has been poorly studied up to now. In this work we describe the expression of the protein in presence of different metals showing Mn 2+ to support the higher activity. The enzyme has been over-expressed, purified and characterized as a Mn 2+ -containing enzyme by inductive plasma cou- pled mass spectrometry (ICP-MS), showing also surprising kinetic differences in comparison with the cadmium-containing enzyme. The Mn 2+ containing enzyme was about 30-fold more efficient with parao- xon as substrate and more stable than the Cd 2+ counterpart, even though the Mn 2+ affinity for the binu- clear metal centre is apparently lower. These results increase our knowledge of the biochemical characteristics of SacPox mainly with regard to the metal-ions modulation of function. Ó 2012 Elsevier Ireland Ltd. All rights reserved. 1. Introduction In the last few years the environmental decontamination of organophosphates (OPs) has attracted an overwhelming interest. OPs are toxic compounds for vertebrates because they irreversibly inhibit acetylcholinesterase, a key enzyme of the nervous system. They have been distributed globally since the end of World War II and their toxic properties have also been exploited for the devel- opment of chemical warfare agents such as sarin, soman and VX other than for the production of agricultural insecticides [1,2]. Enzymatic detoxification of OPs has become the subject of many studies because alternative methods of removing them, such as bleach treatments and incineration are unfeasible, due to high costs or environmental concerns [3 and references therein]. For this application, bacterial OP hydrolases (OPH) or phosphotriester- ases (PTEs; EC 3.1.8.1) are more interesting due to their broader substrate specificity and higher catalytic rate. In fact, the genes for OP hydrolyses, have been found in Pseudomonas diminuta [4], Flavobacterium sp.s [5], Agrobacterium radiobacter [6]. Naturally occurring enzymes with cognate or promiscuous organophosphate hydrolysis activity have been evolved to target some organophos- phate compounds [7,8]. A related family of enzymes encompassing Bacteria and Archaea is the newly described phosphotriesterase- like lactonase (PLL) family. This family was discovered in an at- tempt to decipher the evolutionary origins of bacterial PTEs. Genes showing 26–35% amino acid identity to PTEs turned out to encode lactonases and have thus been named PLLs [9]. Indeed, the first family member SsoPox from Sulfolobus solfataricus was biochemi- cally characterized as paraoxonase [10]. Additional members of the PLL family have been discovered [11–14], including members whose substrates are lactones other than acyl homoserine lactones (HSLs) [15]. The SsoPox 3D structure has been solved as the free form and in complex with the competitive inhibitor N-decanoyl L-homocysteine thiolactone [16]. Structurally it is a homodimeric (b/a)8-barrel with a binuclear metal center located at the C-termi- nal end of the barrel, as reported for Pseudomonas PTE [17–19]. The catalytic centre is composed of two closely spaced divalent cations ligated to the protein via direct interactions with four histidines, one aspartate and a carboxylated lysine residue. Interestingly, it has been found that in the binuclear metal center there is an iron ion (Fe 2+ as inferred from literature data) at the a inner position and a Co 2+ at the b external position. It has been observed that Sso- Pox catalyzes the hydrolysis of paraoxon and other pesticides with a low proficiency, but with K M value similar to PTE. Similarly to the Pseudomonas PTE its activity depends on the presence of metal ions, and the higher activity has been observed with Cd 2+ [10]. Sso- Pox has been evolved in order to increase its paraoxonase activity [20]. An alternative approach is to look for new natural genes. From this viewpoint we characterized the homologous enzyme from Sulfolobus acidocaldarius, SacPox [21]. Metal ions in enzyme active sites of PTE have been found to affect greatly the activity and 0009-2797/$ - see front matter Ó 2012 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.cbi.2012.11.003 ⇑ Corresponding author. Mailing address: Institute of Protein Biochemistry, CNR, Via P. Castellino 111, 80131 Naples, Italy. Tel.: +39 081 6132296; fax: +39 081 6132248. E-mail address: g.manco@ibp.cnr.it (G. Manco). Chemico-Biological Interactions 203 (2013) 251–256 Contents lists available at SciVerse ScienceDirect Chemico-Biological Interactions journal homepage: www.elsevier.com/locate/chembioint