Assembly of redox centers in the trimethylamine dehydrogenase of bacterium W3A1. Properties of the wild-type enzyme and a C30A mutant expressed from a cloned gene in Escherichia coli.
In trimethylamine dehydrogenase, the enzyme-bound FMN is covalently linked to Cys-30 by a 6-S-cysteinyl FMN bond. The role played by this bond in catalysis has been investigated using a recombinant wild-type trimethylamine dehydrogenase and a Cys-30 to Ala-30 mutant, both expressed from a cloned gene (tmd) in the heterologous host Escherichia coli. The recombinant wild-type and C30A enzymes were found to be quantitatively associated with the 4Fe-4S center and ADP which are both present in the enzyme isolated from bacterium W3A1. In contrast to the enzyme isolated from bacterium W3A1, however, both recombinant proteins contained less than stoichiometric amounts of flavin and were refractory to reconstitution by FMN. The FMN in the recombinant wild-type enzyme was shown to be covalently linked to the protein, and the enzyme possessed catalytic properties similar to its counterpart isolated from bacterium W3A1. It is envisaged that flavinylation proceeds via a nucleophilic attack by the thiolate of Cys-30 at C-6 of the isoalloxazine ring of enzyme-bound FMN. The C30A mutant was found to bind FMN noncovalently and to also catalyze the demethylation of trimethylamine. The major effect of removing the 6-S-cysteinyl FMN bond is to raise the apparent Km for trimethylamine by 2 orders of magnitude and to diminish the apparent kcat for the reaction by only a factor of 2. Therefore, the 6-S-cysteinyl FMN bond is not essential for catalysis, but it is required for efficient functioning of the enzyme at micromolar concentrations of substrate.
Scrutton
N S
NS
Department of Biochemistry, University of Cambridge, United Kingdom.
Packman
L C
LC
Mathews
F S
FS
Rohlfs
R J
RJ
Hille
R
R
D000244:Adenosine Diphosphate / Q000378:metabolism; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004578:Electron Spin Resonance Spectroscopy; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D005486:Flavin Mononucleotide; D005415:Flavins / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010084:Oxidation-Reduction; D010089:Oxidoreductases, N-Demethylating / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010957:Plasmids; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D005415:Flavins; D011994:Recombinant Proteins; D000244:Adenosine Diphosphate; D005486:Flavin Mononucleotide; D010089:Oxidoreductases, N-Demethylating; C023186:trimethylamine dehydrogenase