Directed evolution of a quorum-quenching lactonase from Mycobacterium avium subsp. paratuberculosis K-10 in the amidohydrolase superfamily.
The PLL(PTE-like lactonase)-group of enzymes within the amidohydrolase superfamily hydrolyze N-acyl-homoserine lactones (AHLs) that are involved in bacterial quorum-sensing pathways. These enzymes possess the (beta/alpha)(8)-barrel fold and serve as attractive templates for in vitro evolution and engineering of quorum-quenching biological molecules that can serve as antivirulence therapeutic agents. Using a quorum-quenching lactonase from Mycobacterium avium subsp. paratuberculosis K-10 (GI: 41409766) as the initial template for in vitro evolution experiments, we enhanced the catalytic efficiency and increased the substrate range of the wild-type enzyme through a single point mutation on the loop at the C-terminal end of the eighth beta-strand. This N266Y mutant had an increased value of k(cat)/K(M) of 30- and 32-fold toward 3-oxo-N-octanoyl-l-homoserine lactone and N-hexanoyl-l-homoserine lactone, respectively; the evolved mutant also exhibited lactonase activity toward 3-oxo-N-hexanoyl-l-homoserine lactone and N-butyryl-l-homoserine lactone, AHLs that were previously not hydrolyzed by the wild-type enzyme. This article reinforces the evolutionary potential of the (beta/alpha)(8)-barrel fold and highlights the possibility of using quorum-quenching lactonases in the amidohydrolase superfamily as templates for engineering biomolecules of therapeutic use.
Chow
Jeng Yeong
JY
Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, 8 Medical Drive, Singapore.
Wu
Long
L
Yew
Wen Shan
WS
D000595:Amino Acid Sequence; D000619:Aminohydrolases / Q000378:metabolism*; D001681:Biological Assay; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000378:metabolism; D019143:Evolution, Molecular; D015723:Gene Library; D007700:Kinetics; D007783:Lactones / Q000737:chemistry*; D008670:Metals / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009162:Mycobacterium avium / Q000235:genetics / Q000378:metabolism*; D053038:Quorum Sensing / Q000235:genetics*; D017386:Sequence Homology, Amino Acid
D007783:Lactones; D008670:Metals; D000619:Aminohydrolases