Structural and biochemical characterization of recombinant wild type and aC30A mutant of trimethylamine dehydrogenase from Methylphilus methylotrophus (sp W(3)A(1))

Trimethylamine dehydrogenase (TMADH) is an iron-sulfur flavoprotein that ca talyzes the oxidative demethylation of trimethylamine to form dimethylamine and formaldehyde. It contains a unique flavin, in the form of a 6-S-cystei nyl FMN, which is bent by similar to 25 degrees along the N5-N10 axis of th e flavin isoalloxazine ring. This unusual conformation is thought to modula te the properties of the flavin to facilitate catalysis, and has been postu lated to be the result of covalent linkage to Cys-30 at the flavin C6 atom. We report here the crystal structures of recombinant wild-type and the C30 A mutant TMADH enzymes, both determined at 2.2 Angstrom resolution. Combine d crystallographic and NMR studies reveal the presence of inorganic phospha te in the FMN binding site in the deflavo fraction of both recombinant wild -type and C30A proteins. The presence of tightly bound inorganic phosphate in the recombinant enzymes explains the inability to reconstitute the defla vo forms of the recombinant wild-type and C30A enzymes that are generated i n vivo. The active site structure and flavin conformation in C30A TMADH are identical to those in recombinant and native TMADH, thus revealing that, c ontrary to expectation, the 6-S-cysteinyl FMN link is not responsible for t he 25 degrees butterfly bending along the N5-N10 axis of the flavin in TMAD H. Computational quantum chemistry studies strongly support the proposed ro le of the butterfly bend in modulating thp redox properties of the flavin. Solution studies reveal major differences in the kinetic behavior of the wi ld-type and C30A proteins. Computational studies reveal a hitherto, unrecog nized, contribution made by the S-gamma atom of Cys-30 to substrate binding , and a role for Cys-SO in the optimal geometrical alignment of substrate w ith the 6-S-cysteinyl FMN in the enzyme active site.