The cytochrome subunit is necessary for covalent FAD attachment to the flavoprotein subunit of p-cresol methylhydroxylase.
When p-cresol methylhydroxylase (PCMH) is expressed in its natural host Pseudomonas putida, or when the genes of the alpha and beta subunits of the enzyme are expressed together in the heterologous host Escherichia coli, flavin-adenine dinucleotide (FAD) is covalently attached to Tyr384 of the alpha subunit and the correct alpha 2 beta 2 form of the enzyme is assembled. The apoflavoprotein has been expressed in E. coli in the absence of the beta cytochrome c subunit and purified. While noncovalent FAD binding to apoflavoprotein in the absence of the cytochrome subunit could not be directly demonstrated, circumstantial evidence suggests that this indeed occurs. Covalent flavinylation requires one molecule each of FAD and cytochrome for each flavoprotein subunit. The flavinylation process leads to the 2-electron-reduced form of covalently bound FAD, and the resulting alpha 2 beta 2 enzyme is identical to wild-type PCMH. This work presents clear evidence that covalent flavinylation occurs by a self-catalytic mechanism; an external enzyme or chaperon is not required, nor is prior chemical activation of FAD or of the protein. This work is the first to define the basic chemistry of covalent flavinylation of an enzyme to produce the normal, active species, and confirms a long standing, postulated chemical mechanism of this process. It also demonstrates, for the first time, the absolute requirement for a partner subunit in the post-translational modification of a protein. It is proposed that the covalent FAD bond to Tyr384 and the phenolic portion of this Tyr are part of the essential electron transfer path from FAD to heme.
Department of Veterans Affairs Medical Center, San Francisco, California 94121, USA.
J
J
Kim
J H
JH
Fuller
V
V
Kuusk
L
L
Cunane
Z W
ZW
Chen
F S
FS
Mathews
W S
WS
McIntire
D000595:Amino Acid Sequence; D001665:Binding Sites; D003580:Cytochromes / Q000737:chemistry / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D005420:Flavoproteins / Q000378:metabolism*; D006418:Heme / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D008969:Molecular Sequence Data; D016958:Pseudomonas putida / Q000201:enzymology
D003580:Cytochromes; D005420:Flavoproteins; D005182:Flavin-Adenine Dinucleotide; D006418:Heme; D006899:Mixed Function Oxygenases; C049502:4-cresol dehydrogenase (hydroxylating)