Purification and characterization of hexahistidine-tagged cyclohexanone monooxygenase expressed in Saccharomyces cerevisiae and Escherichia coli.
Cyclohexanone monooxygenase (CMO) is a soluble flavoenzyme originally isolated from Acinetobacter spp. which carries out Baeyer-Villiger reactions with cyclic ketone substrates. In the present study we cloned the Acinetobacter CMO gene and modified it for facile purification from heterologous expression systems by incorporation of a His(6)-tag at its C-terminus. A single purification step employing metal (Ni(2+))-affinity column chromatography provided essentially homogeneous enzyme in yields of 69-72%. The properties of the purified, recombinant enzymes (rCMO) were compared with that of native CMO (nCMO) isolated from Acinetobacter cultures grown in the presence of cyclohexanone. The specific activities of His(6)-tagged rCMO and nCMO toward their index substrate, cyclohexanone, were similar and ranged from 14 to 20 micromol/min/mg. nCMO and rCMO from the Escherichia coli expression system exhibited molecular masses, determined by electrospray mass spectrometry, of 60,800 and 61,615 Da, respectively, an increase for the recombinant enzyme equivalent to the mass of the His(6)-tag. However, rCMO expressed in Saccharomyces cerevisiae consistently exhibited a mass some 50 Da larger than rCMO expressed in bacteria. Edman degradation confirmed that rCMO purified from the E. coli system and nCMO shared the same N-terminal sequence, whereas no sequence information could be obtained for rCMO expressed in yeast. Therefore, the yeast-expressed enzyme possesses an additional posttranslational modification(s), possibly acylation, at the N-terminus. Expression in E. coli is the preferred system for future site-directed mutagenesis studies and crystallization efforts.
Cheesman
M J
MJ
Department of Medicinal Chemistry, University of Washington, Seattle, WA 98195, USA.
Kneller
M B
MB
Kelly
E J
EJ
Thompson
S J
SJ
Yeung
C K
CK
Eaton
D L
DL
Rettie
A E
AE
D000150:Acinetobacter / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli; D006639:Histidine; D007700:Kinetics; D008970:Molecular Weight; D010105:Oxygenases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae; D021241:Spectrometry, Mass, Electrospray Ionization
D011994:Recombinant Proteins; D006639:Histidine; D010105:Oxygenases; C019961:cyclohexanone oxygenase