Jeng Yeong Chow§,*, Bo Xue‡,*, Kang Hao Lee§, Alvin Tung‡, Long Wu§, Robert C. Robinson‡,2 and Wen Shan Yew§,1.
§ - Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 8 Medical Drive, Singapore 117597.
‡ - Institute of Molecular and Cell Biology, 61 Biopolis Drive, Singapore 138673.
* - Equal contribution
Published in J Biol Chem. 2010 Oct 27 as Manuscript M110.177139
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 PLL (Phosphotriesterase-Like Lactonase)-group of enzymes within the amidohydrolase superfamily that hydrolyzes N-acyl-homoserine 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 3rd and 7th β-strands, respectively. This E101N/R230I mutant had an increased value of k(cat)/K(M) of 72-fold towards 3-oxo-N-dodecanoyl-L-homoserine lactone. The evolved mutant also exhibited lactonase activity towards 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 due to a charge transfer complex involving the β-cation and Tyr99 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.