Cloning of the maoA gene that encodes aromatic amine oxidase of Escherichia coli W3350 and characterization of the overexpressed enzyme.
The mao operon of Escherichia coli W3350, which comprises the genes maoC and maoA, was cloned and appeared to be similar to that of Klebsiella aerogenes [Sugino, H., Sasaki, M., Azakami, H., Yamashita, M. & Murooka, Y. (1992) J. Bacteriol. 174, 2485-2492]. The gene that encodes aromatic amine oxidase (maoA) was isolated, sequenced, and expressed in E. coli TG2. The purified enzyme exhibited properties characteristic of a copper/topaquinone(TPQ)-containing amine oxidase with respect to the optical absorption and EPR spectra, the size of the subunits, and the optical absorption spectra obtained upon derivatization with hydrazines. However, high-resolution anion-exchange chromatography revealed that the preparation was heterogeneous. The enzyme preparation appeared to consist of at least four enzyme species with different specific activities, A474nm/A340nm ratios and TPQ/subunit ratios. Since the overall properties of the overexpressed enzyme and the authentic enzyme were similar and the separated enzyme species had identical N-terminal amino acid sequences, the heterogeneity does not seem to be caused by improper expression of the gene in the recombinant strain but by factors that interfere with the processing of the specific tyrosine in the precursor enzyme to functional TPQ. Although other causes cannot be excluded, the spectral data and TPQ/subunit ratios reported in the literature for other amine oxidases suggest that suboptimal synthesis of functional TPQ also occurs in other organisms.
Steinebach
V
V
Kluyver Laboratory of Biotechnology, Department of Microbiology & Enzymology, Delft University of Technology, The Netherlands.
Benen
J A
JA
Bader
R
R
Postma
P W
PW
De Vries
S
S
Duine
J A
JA
D000595:Amino Acid Sequence; D001483:Base Sequence; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004295:Dihydroxyphenylalanine / Q000031:analogs & derivatives / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D015870:Gene Expression; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D008970:Molecular Weight; D008995:Monoamine Oxidase / Q000737:chemistry / Q000235:genetics*; D009876:Operon; D010659:Phenylhydrazines; D010957:Plasmids / Q000235:genetics; D013053:Spectrophotometry
D004269:DNA, Bacterial; D010659:Phenylhydrazines; D004295:Dihydroxyphenylalanine; C067626:6-hydroxydopa quinone; D008995:Monoamine Oxidase