Structural basis for thermostability revealed through the identification and characterization of a highly thermostable phosphotriesterase-like lactonase from Geobacillus stearothermophilus

Abstract

A new enzyme homologous to phosphotriesterase was identified from the bacterium Geobacillus stearothermophilus (GsP). This enzyme belongs to the amidohydrolase family and possesses the ability to hydrolyze both lactone and organophosphate (OP) compounds, making it a phosphotriesterase-like lactonase (PLL). GsP possesses higher OP-degrading activity than recently characterized PLLs, and it is extremely thermostable. GsP is active up to 100°C with an energy of activation of 8.0 kcal/mol towards ethyl paraoxon, and it can withstand an incubation temperature of 60°C for two days. In an attempt to understand the thermostability of PLLs, the X-ray structure of GsP was determined and compared to those of existing PLLs. Based upon a comparative analysis, a new thermal advantage score and plot was developed and reveals that a number of different factors contribute to the thermostability of PLLs.

Isolation and Characterization ofN-Acylhomoserine Lactonase from the Thermophilic Bacterium,Geobacillus caldoxylosilyticusYS-8

“…(AidH) and GsP being respectively dependent on Mn 2þ and Co 2þ for their lactonase activity. 26,27) More interesting is that the sequence similarity was high between GsP and GKL, even despite the distinct difference of metal effect, for which reason is not currently clear. 23) It should be noted that there is a difference in metal dependence between the GKL and AHL lactonases from G. caldoxylosilyticus YS-8, despite their similar substrate specificity and thermostability.…”

“…Our data therefore suggest that the AHL-degrading enzyme from G. caldosylosilyticus YS-8 could be regarded as thermostable when comparing with AiiM from M. testaceum which showed over 80% of its maximum activity over a wide range of temperature from 15 C to 60 C. 25) Focusing on the thermostability of lactonases, the phosphotriesterase-like lactonase from G. steartothermophilus (GsP) showed extreme thermostability, retaining its full activity even after incubating at 100 C for 15 min with methyl paraoxon as the substrate. 26) However, it exhibited relatively low AHL lactonase activity towards HHL with a k cat /K m value of 0.27 min À1 mM À1 , although it showed high corresponding values towards methyl paraoxon (1.74 min À1 mM À1 ) and even -undecanoic lactone (924 min À1 mM À1 ), suggesting that this GsP can hardly be completely regarded as an AHL lactonase.…”

Directed Evolution of a Thermostable Quorum-quenching Lactonase from the Amidohydrolase Superfamily

“…GKL was found to retain full activity toward ␦-nonalactone after prolonged periods of heat exposure (a plot of the activity against duration of heat treatment is detailed in supplemental Fig. S4; the increase in GKL activity upon heat treatment could be attributed to the activation of the enzyme by heat; GKL is an enzyme from a thermophile, and reports of heat activation of other thermophilic enzymes had been similarly described (22)). GKL was also found to retain quorumquenching lactonase activity toward 3-oxo-C8 HSL after heat treatment for 15 min at 60 and 90°C ( Table 2).…”

Crystallization and preliminary X-ray diffraction analysis of the lactonaseVmoLac fromVulcanisaeta moutnovskia

“…PLLs hydrolyze organophosphate compounds with poor to moderate efficiency (Hiblot et al, 2012b;Hawwa, Larsen et al, 2009;Zhang et al, 2012) compared with PTEs (Omburo et al, 1992;Jackson et al, 2006;Donarski et al, 1989). Some PLL representatives, however, offer interesting biotechnological potentialities for engineering an efficient organophosphate biodecontaminant because of their high thermal stability (Hiblot et al, 2012a,b;Hawwa, Larsen et al, 2009;Hawwa, Aikens et al, 2009), as illustrated by several engineering studies on these enzymes (Merone et al, 2010;Hawwa, Larsen et al, 2009;Xue et al, 2013;Chow et al, 2010).…”