Crystallization and preliminary X-ray diffraction analysis of the lactonase<i>Vmo</i>Lac from<i>Vulcanisaeta moutnovskia</i>

Hiblot, Julien; Gotthard, Guillaume; Champion, Charlotte; Chabrière, Éric; Elias, Mikael

doi:10.1107/s1744309113024846

Cited by 17 publications

(10 citation statements)

References 40 publications

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“…The Vmo Lac enzyme is a recently discovered PLL-A member from the extremophilic archaeon V. moutnovskia that lives between 60 and 98°C 33 38 . Consequently, Vmo Lac is highly stable and exhibits a maximum of activity at 80°C 34 .…”

Section: Discussionmentioning

confidence: 99%

Crystal structure of VmoLac, a tentative quorum quenching lactonase from the extremophilic crenarchaeon Vulcanisaeta moutnovskia

Hiblot

Bzdrenga

Champion

et al. 2015

Sci Rep

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A new representative of the Phosphotriesterase-Like Lactonases (PLLs) family from the hyperthermophilic crenarchaeon Vulcanisaeta moutnovskia has been characterized and crystallized. VmoLac is a native, proficient lactonase with promiscuous, low phosphotriesterase activity. VmoLac therefore represents an interesting candidate for engineering studies, with the aim of developing an efficient bacterial quorum-quenching agent. Here, we provide an extensive biochemical and kinetic characterization of VmoLac and describe the X-ray structures of the enzyme bound to a fatty acid and to its cognate substrate 3-oxo-C10 AHL (Acyl-Homoserine Lactone). The structures highlight possible structural determinants that may be involved in its extreme thermal stability (Tm = 128°C). Moreover, the structure reveals that the substrate binding mode of VmoLac significantly differs from those of its close homologues, possibly explaining the substrate specificity of the enzyme. Finally, we describe the specific interactions between the enzyme and its substrate, and discuss the possible lactone hydrolysis mechanism of VmoLac.

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Section: Discussionmentioning

confidence: 99%

Crystal structure of VmoLac, a tentative quorum quenching lactonase from the extremophilic crenarchaeon Vulcanisaeta moutnovskia

Hiblot

Bzdrenga

Champion

et al. 2015

Sci Rep

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“…The protein was overproduced in E. coli strain BL21 (DE3)-pGro7/GroEL (Takara) using a similar protocol to previous studies of PLLs (Hiblot, Gotthard, Champion et al, 2013;Hiblot et al, 2012Hiblot et al, , 2015Gotthard et al, 2011). Briefly, the expression of the protein was performed in 2 l ZYP autoinduction medium (100 mg ml À1 ampicillin and 34 mg ml À1 chloramphenicol) inoculated with 10 ml overnight preculture.…”

Section: Cloning Expression and Purification Of Gclmentioning

confidence: 99%

The quorum-quenching lactonase fromGeobacillus caldoxylosilyticus: purification, characterization, crystallization and crystallographic analysis

Bergonzi¹,

Schwab²,

Elias³

2016

Acta Crystallogr F Struct Biol Commun

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Lactonases are enzymes that are capable of hydrolyzing various lactones such as aliphatic lactones or acyl-homoserine lactones (AHLs), with the latter being used as chemical signaling molecules by numerous Gram-negative bacteria. Lactonases therefore have the ability to quench the chemical communication, also known as quorum sensing, of numerous bacteria, and in particular to inhibit behaviors that are regulated by this system, such as the expression of virulence factors or the production of biofilms. A novel representative from the metallo-β-lactamase superfamily, dubbed GcL, was isolated from the thermophilic bacterium Geobacillus caldoxylosilyticus. Because of its thermophilic origin, GcL may constitute an interesting candidate for the development of biocontrol agents. Here, we show that GcL is a thermostable enzyme with a half-life at 75°C of 152.5 ± 10 min. Remarkably, it is also shown that GcL is among the most active lactonases characterized to date, with catalytic efficiencies (kcat/Km) against AHLs of greater than 10(6) M(-1) s(-1). The structure of GcL is expected to shed light on the catalytic mechanism of the enzyme and the molecular determinants for the substrate specificity in this class of lactonases. Here, the expression, purification, characterization, crystallization and X-ray diffraction data collection to 1.6 Å resolution of GcL are reported.

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“…PLLs from extremophilic crenarchaeaon sources [3,4,16,34]. These counterparts exhibit industry-compatible properties ( e.g.…”

Section: Introductionmentioning

confidence: 99%

Sac Pox from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius is a proficient lactonase

et al. 2014

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BackgroundSacPox, 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.ResultsHere, 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.ConclusionKinetic 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.

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

Cited by 17 publications

References 40 publications

Crystal structure of VmoLac, a tentative quorum quenching lactonase from the extremophilic crenarchaeon Vulcanisaeta moutnovskia

Crystal structure of VmoLac, a tentative quorum quenching lactonase from the extremophilic crenarchaeon Vulcanisaeta moutnovskia

The quorum-quenching lactonase fromGeobacillus caldoxylosilyticus: purification, characterization, crystallization and crystallographic analysis

Sac Pox from the thermoacidophilic crenarchaeon Sulfolobus acidocaldarius is a proficient lactonase

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