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-gen e operon amoABCD. AMO catalyses the stereoselective epoxygenation of alipha tic alkenes, yielding primarily R enantiomers. The presumed site of alkene oxygenation is a dinuclear iron centre similar to that in the soluble metha ne monooxygenases of methanotrophic bacteria, to which AMO exhibits a signi ficant degree of amino acid sequence identity, The AMO complex was not expr essed in Escherichia coli, at least partly because that host did not produc e all of the AMO polypeptides. Expression of AMO was achieved in Streptomyc es lividans by cloning the AMO genes into the thiostrepton-inducible expres sion plasmid pIJ6021. No background of AMO activity was detected in S, livi dans cells without amoABCD and expression of AMO activity at a level compar able 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 orga nism. Although the whole AMO complex could not be expressed in E. coli, the functional coupling protein (AmoB) and reductase (AmoD) were expressed ind ividually in E. coli as fusions with glutathione S-transferase. The express ion systems described here now allow structure/function studies on AMO to b e carried out by site-directed mutagenesis.