Alteration of the stereo- and regioselectivity of alkene monooxygenase based on coupling protein interactions.
Alkene monooxygenase from Xanthobacter autotrophicus Py2 (XAMO) catalyses the asymmetric epoxidation of a broad range of alkenes. As well as the electron transfer components (a NADH-oxidoreductase and a Rieske-type ferredoxin) and the terminal oxygenase containing the binuclear non-haem iron active site, it requires a small catalytic coupling/effector protein, AamD. The effect of changing AamD stoichiometry and substitution with effector protein homologues on the regioselectivity of toluene hydroxylation and stereoselectivity of styrene epoxidation has been studied. At sub-optimal stoichiometries, there was a marked change in regioselectivity, but no significant change in epoxidation stereoselectivity. Recombinant coupling proteins from a number of phylogenetically related oxygenases were investigated for their ability to functionally replace AamD. Substitution of AamD with IsoD, the coupling protein from the closely related isoprene monooxygenase, changed the regioselectivity of toluene hydroxylation and stereoselectivity of styrene epoxidation, although this was accompanied by a high level of uncoupling. This indicates the importance of coupling protein interaction in controlling the catalytic specificity. Sequence analysis suggests that interaction between Asn34 and Arg57 is important for complementation specificity of the coupling proteins, providing a candidate for site-directed mutagenesis studies.
Champreda
Verawat
V
Department of Biological Sciences, Imperial College London, London, SW7 2AZ, UK.
Choi
Young-Jun
YJ
Zhou
Ning-Yi
NY
Leak
David J
DJ
D000595:Amino Acid Sequence; D001120:Arginine / Q000737:chemistry / Q000378:metabolism; D001216:Asparagine / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D006900:Hydroxylation; D008969:Molecular Sequence Data; D010105:Oxygenases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013237:Stereoisomerism; D014050:Toluene / Q000737:chemistry / Q000378:metabolism; D020584:Xanthobacter / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D014050:Toluene; D001216:Asparagine; D001120:Arginine; D010105:Oxygenases; C072883:alkene monooxygenase