ASSEMBLY OF REDOX CENTERS IN THE TRIMETHYLAMINE DEHYDROGENASE OF BACTERIUM W(3)A(1) - 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 li nked to Cys-30 by a 6-S cysteinyl FMN bond. The role played by this bo nd in catalysis has been investigated using a recombinant wild-type tr imethylamine dehydrogenase and a Cys-30 to Ala-30 mutant, both express ed from a cloned gene (tmd) in the heterologous host Escherichia coli. The recombinant wild-type and C30A enzymes were found to be quantitat ively associated with the 4Fe-4S center and ADP which are both present in the enzyme isolated from bacterium W(3)A(1). In contrast to the en zyme isolated from bacterium W(3)A(1), however, both recombinant prote ins contained less than stoichiometric amounts of flavin and were refr actory to reconstitution by FMN. The FMN in the recombinant wild-type enzyme was shown to be covalently linked to the protein, and the enzym e possessed catalytic properties similar to its counterpart isolated f rom bacterium W(3)A(1). It is envisaged that flavinylation proceeds vi a a nucleophilic attack by the thiolate of Cys-30 at C-6 of the isoall oxazine ring of enzyme-bound FMN. The C30A mutant was found to bind FM N noncovalently and to also catalyze the demethylation of trimethylami ne. The major effect of removing the 6-S-cysteinyl FMN bond is to rais e the apparent K-m for trimethylamine by 2 orders of magnitude and to diminish the apparent k(cat) for the reaction by only a factor of 2. T herefore, the 6-S-cysteinyl FMN bond is not essential for catalysis, b ut it is required for efficient functioning of the enzyme at micromola r concentrations of substrate.