INVOLVEMENT OF A FLAVIN IMINOQUINONE METHIDE IN THE FORMATION OF 6-HYDROXYFLAVIN MONONUCLEOTIDE IN TRIMETHYLAMINE DEHYDROGENASE - A RATIONALE FOR THE EXISTENCE OF 8-ALPHA-METHYL AND C6-LINKED COVALENT FLAVOPROTEINS
M. Mewies et al., INVOLVEMENT OF A FLAVIN IMINOQUINONE METHIDE IN THE FORMATION OF 6-HYDROXYFLAVIN MONONUCLEOTIDE IN TRIMETHYLAMINE DEHYDROGENASE - A RATIONALE FOR THE EXISTENCE OF 8-ALPHA-METHYL AND C6-LINKED COVALENT FLAVOPROTEINS, Biochemistry, 36(23), 1997, pp. 7162-7168
In trimethylamine dehydrogenase, substrate is bound in the active site
via cation-pi bonding to three aromatic residues (Tyr-60, Trp-264, an
d Trp-355). Mutation of one of these residues (Trp-355 --> Leu, mutant
W355L) influences the chemistry of the flavin mononucleotide in the a
ctive site, enabling derivatization to 6-hydroxy-FMN. The W355L mutant
is purified as a mixture of deflavo, natural 6-S-cysteinyl-FMN, and i
nactive 6-hydroxy-FMN forms, and the enzyme is severely compromised in
its ability to oxidatively demethylate trimethylamine. Analysis of sa
mples of the native and recombinant wild-type trimethylamine dehydroge
nases also revealed the presence of B-hydroxy-FMN, but at much reduced
levels compared with that of the W355L enzyme. Unlike that for a C30A
mutant of trimethylamine dehydrogenase, addition of substrate to the
W355L trimethylamine dehydrogenase is not required for the production
of 6-hydroxy-FMN. A mechanism is proposed for the 6-hydroxylation of F
MN in trimethylamine dehydrogenase that involves an electrophilic flav
in iminoquinone methide. The proposed mechanism involving the flavin i
minoquinone methide could apply to the flavinylation of trimethylamine
dehydrogenase at the C6 position but also to the flavinylation of enz
ymes via the 8 alpha position, thus providing a rationale for the evol
ution of covalent flavoproteins in general. Covalent linkage at C6 or
the 8 alpha-methyl prevents 6-hydroxylation by direct modification at
the C6 atom or by preventing formation of the flavin iminoquinone meth
ide, respectively.