Heterologous expression of alkene monooxygenase from Rhodococcus rhodochrous B-276.
Alkene monooxygenase (AMO) from Rhodococcus rhodochrous (formerly Nocardia corallina) B-276 is a three-component enzyme system encoded by the four-gene operon amoABCD. AMO catalyses the stereoselective epoxygenation of aliphatic alkenes, yielding primarily R enantiomers. The presumed site of alkene oxygenation is a dinuclear iron centre similar to that in the soluble methane monooxygenases of methanotrophic bacteria, to which AMO exhibits a significant degree of amino acid sequence identity. The AMO complex was not expressed in Escherichia coli, at least partly because that host did not produce all of the AMO polypeptides. Expression of AMO was achieved in Streptomyces lividans by cloning the AMO genes into the thiostrepton-inducible expression plasmid pIJ6021. No background of AMO activity was detected in S. lividans cells without amoABCD and expression of AMO activity, at a level comparable to that from wild-type R. rhodochrous B-276, coincided with appearance of the AMO subunits. Recombinant AMO activity in cell-free extracts of S. lividans was stimulated by the addition of NADH and produced R-epoxypropane with comparable enantiomeric excess to AMO purified from the original organism. Although the whole AMO complex could not be expressed in E. coli, the functional coupling protein (AmoB) and reductase (AmoD) were expressed individually in E. coli as fusions with glutathione S-transferase. The expression systems described here now allow structure/function studies on AMO to be carried out by site-directed mutagenesis.
Smith
T J
TJ
Department of Biological Sciences, University of Warwick, Coventry, UK.
Lloyd
J S
JS
Gallagher
S C
SC
Fosdike
W L
WL
Murrell
J C
JC
Dalton
H
H
D001483:Base Sequence; D003001:Cloning, Molecular; D015971:Gene Expression Regulation, Enzymologic; D005818:Genetic Engineering; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009876:Operon; D010105:Oxygenases / Q000096:biosynthesis*; D012240:Rhodococcus / Q000201:enzymology*; D013302:Streptomyces
D010105:Oxygenases; C072883:alkene monooxygenase