Cloning, sequencing, expression, and regulation of the structural gene for the copper/topa quinone-containing methylamine oxidase from Arthrobacter strain P1, a gram-positive facultative methylotroph.
Deoxyoligonucleotides corresponding to amino acid sequences of methylamine oxidase and polyclonal anti-methylamine oxidase antibodies were used to probe Arthrobacter strain P1 plasmid and chromosomal DNA libraries. Two open reading frames, maoxI and maoxII, which are greater than 99% homologous, were cloned from the chromosomal library. The deduced amino acid sequences of the coding regions are identical except for two residues near the C termini. On the other hand, the 5'- and 3'-flanking regions of maoxI and maoxII are quite different. While either gene could code for methylamine oxidase, the dissimilarity in the 5'-flanking regions indicates that the genes are differently regulated. It was determined that maoxII alone encodes methylamine oxidase. The tyrosyl residue which is converted to topa quinone in the mature enzyme was located by comparison with amino acid sequences at the cofactor sites in other copper/topa quinone-containing amine oxidase. Transcriptional start sites and possible regulatory elements were identified in the 5' region of maoxI and maoxII, and stem-loop structures were found in the 3'-flanking regions. High levels of methylamine oxidase are produced when Arthrobacter strain P1 is grown on methylamine alone or on glucose plus methylamine, but growth on LB medium plus methylamine resulted in very low production of the enzyme. Expression of maoxII from its own promoter in Escherichia coli grown on glucose or LB medium with or without methylamine gave the same level of production of methylamine oxidase.
Zhang
X
X
Department of Veterans Affairs Medical Center, San Francisco, California 94121.
Fuller
J H
JH
McIntire
W S
WS
D000595:Amino Acid Sequence; D000818:Animals; D001173:Arthrobacter / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D015153:Blotting, Western; D003001:Cloning, Molecular; D003300:Copper / Q000737:chemistry; D004269:DNA, Bacterial; D004295:Dihydroxyphenylalanine / Q000031:analogs & derivatives / Q000737:chemistry; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D014158:Transcription, Genetic
D004269:DNA, Bacterial; D004295:Dihydroxyphenylalanine; C067626:6-hydroxydopa quinone; D003300:Copper; C021567:methylamine dehydrogenase; D000587:Oxidoreductases Acting on CH-NH Group Donors