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

Abstract

A thermostable quorum-quenching lactonase from Geobacillus kaustophilus HTA426 (GI: 56420041) was used as an initial template for in vitro directed evolution experiments. This enzyme belongs to the phosphotriesterase-like lactonase (PLL) group of enzymes within the amidohydrolase superfamily that hydrolyze N-acylhomoserine lactones (AHLs) that are involved in virulence pathways of quorum-sensing pathogenic bacteria. Here we have determined the N-butyryl-L-homoserine lactone-liganded structure of the catalytically inactive D266N mutant of this enzyme to a resolution of 1.6 Å . Using a tunable, bioluminescence-based quorum-quenching molecular circuit, the catalytic efficiency was enhanced, and the AHL substrate range increased through two point mutations on the loops at the C-terminal ends of the third and seventh ␤-strands. This E101N/R230I mutant had an increased value of k cat /K m of 72-fold toward 3-oxo-N-dodecanoyl-L-homoserine lactone. The evolved mutant also exhibited lactonase activity toward N-butyryl-L-homoserine lactone, an AHL that was previously not hydrolyzed by the wild-type enzyme. Both the purified wild-type and mutant enzymes contain a mixture of zinc and iron and are colored purple and brown, respectively, at high concentrations. The origin of this coloration is suggested to be because of a charge transfer complex involving the ␤-cation and Tyr-99 within the enzyme active site. Modulation of the charge transfer complex alters the lactonase activity of the mutant enzymes and is reflected in enzyme coloration changes. We attribute the observed enhancement in catalytic reactivity of the evolved enzyme to favorable modulations of the active site architecture toward productive geometries required for chemical catalysis.The amidohydrolase superfamily of enzymes comprises members that catalyze hydrolytic reactions on a broad range of substrates bearing ester or amide functional groups with carbon or phosphorus centers (1). These reactions are mediated by a conserved mononuclear or binuclear center within a (␤/␣) 8 -barrel structural scaffold (2) and are initiated by the activation of a water molecule for nucleophilic attack on an activated scissile bond of the substrate for subsequent hydrolysis. The amidohydrolase superfamily was first described by Holm and Sander in 1997 (3) and has since expanded to cover more than 30 reactions involving a diverse range of substrates (2, 4), including quorum-sensing N-acylhomoserine lactones (AHLs) 4 (5). We recently reported the directed evolution of a member of the amidohydrolase superfamily that hydrolyzes AHLs (6); this quorum-quenching enzyme is part of a group of divergently related enzymes within the superfamily, the phosphotriesterase-like lactonases (PLLs), which hydrolyze quorumsensing AHLs. Quorum-sensing is an integral part of microbial interaction and is responsible for virulence or pathogenicity of disease-causing bacteria (7). Modulation and perturbation of this quorum-sensing pathway has been demonstrated in principle to be an effective "anti-microb...

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

“…23) The substrate specificity identified in our study is also consistent with the previous report showing the AHL lactonase from Bacillus sp. with strong catalytic activity against all ten of the AHL substrates.…”

“…In fact, GKL was determined to be a Zn 2þ -dependent AHL lactonase, showing that it had no activity in the presence of Co 2þ which was however found to enhance the AHL lactonase activity of G. caldoxylosilyticus towards DHL. 23) Ni 2þ and Mn 2þ also increased its activity, whereas the presence of Zn 2þ or Cu 2þ had no effect on the activity (data not shown). This is consistent with the AHL lactonase from Ochrobactrum sp.…”

“…23) Although the substrate specificity and thermostability tended to be similar to the AHL lactonase of G. caldoxylosilyticus YS-8, we have recently obtained interesting preliminary results, possibly showing a difference between the two enzymes. In fact, GKL was determined to be a Zn 2þ -dependent AHL lactonase, showing that it had no activity in the presence of Co 2þ which was however found to enhance the AHL lactonase activity of G. caldoxylosilyticus towards DHL.…”

“…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. The mechanisms whereby such positive metals as Co 2þ and Mn 2þ interact with the AHL lactonase of G. caldoxylosilyticus YS-8 need to be determined by a crystallographic investigation.…”

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).…”