Kinetic and physical evidence that the diheme enzyme MauG tightly binds to a biosynthetic precursor of methylamine dehydrogenase with incompletely formed tryptophan tryptophylquinone.

Methylamine dehydrogenase (MADH) contains the protein-derived cofactor tryptophan tryptophylquinone (TTQ) which is generated by the posttranslational modification of two endogenous tryptophan residues. The modifications are incorporation of two oxygens into one tryptophan side chain and the covalent cross-linking of that side chain to a second tryptophan residue. This process requires at least one accessory gene, mauG. Inactivation of mauG in vivo results in production of an inactive 119 kDa tetrameric alpha 2beta 2 protein precursor of MADH with incompletely synthesized TTQ. This precursor can be converted to active MADH with mature TTQ in vitro by reaction with MauG, a 42 kDa diheme enzyme. Steady-state kinetic analysis of the MauG-dependent conversion of the precursor to mature MADH with completely synthesized TTQ yielded values of k cat of 0.20 +/- 0.01 s (-1) and K m of 6.6 +/- 0.6 microM for the biosynthetic precursor protein in an in vitro assay. In the absence of an electron donor to initiate the reaction it was possible to isolate the MauG-biosynthetic precursor (enzyme-substrate) complex in solution using high-resolution size-exclusion chromatography. This stable complex is noncovalent and could be separated into its component proteins by anion-exchange chromatography. In contrast to the enzyme-substrate complex, a mixture of MauG and its reaction product, mature MADH, did not elute as a complex during size-exclusion chromatography. The differential binding of MauG to its protein substrate and protein product of the reaction indicates that significant conformational changes in one or both of the proteins occur during catalysis which significantly affects the protein-protein interactions.