It had been previously reported that aromatic amines were not substrates for the bacterial quinoprotein methylamine dehydrogenase. In this study, benzylamine-dependent activity was also not observed in the steady-state assay of this enzyme with the artificial electron acceptor phenazine ethosulfate (PES). Benzylamines did, however, stoichiometrically reduce the protein-bound tryptophan tryptophylquinone (TTQ) prosthetic group and acted as reversible competitive inhibitors of methylamine oxidation when the enzyme was assayed with PES. When methylamine dehydrogenase activity was monitored using a steady-state assay which employed its physiological electron acceptor amicyanin instead of PES, very low but detectable benzylamine-dependent activity was observed. The reactions of a series of para-substituted benzylamines with methylamine dehydrogenase were examined. A Hammett plot of the log of Ki values for the competitive inhibition by these amines against sigma p exhibited a negative slope. Rapid kinetic measurements allowed the determination of values of k3 and Ks for the reduction of TTQ by each of these amines. A Hammett plot of log k3 versus sigma p exhibited a positive slope, which suggests that the oxidation of these amines by methylamine dehydrogenase proceeds through a carbanionic reaction intermediate. A negative slope was observed for the correlation between log Ks and sigma p. Plots of log k3 and log Ks against substituent constants which reflected either resonance or field/inductive parameters for each para substituent indicated that the magnitude of k3 was primarily influenced by field/inductive effects while Ks was primarily influenced by resonance effects. No correlation was observed between either k3 or Ks and the relative hydrophobicity of the para-substituted benzylamines or steric parameters.(ABSTRACT TRUNCATED AT 250 WORDS)

The three-dimensional structure of the quinoprotein methylamine dehydrogenase from Paracoccus dentrificans (PD-MADH) has been determined at 2.8 A resolution by the molecular replacement method combined with map averaging procedures, using data collected from an area detector. The structure of methylamine dehydrogenase from Thio-bacillus versutus, which contains an "X-ray" sequence, was used as the starting search model. MADH consists of 2 heavy (H) and 2 light (L) subunits related by a molecular 2-fold axis. The H subunit is folded into seven four-stranded beta segments, forming a disk-shaped structure, arranged with pseudo-7-fold symmetry. A 31-residue elongated tail exists at the N-terminus of the H subunit in MADH from T. versutus but is partially digested in this crystal form of MADH from P. denitrificans, leaving the H subunit about 18 residues shorter. Each L subunit contains 127 residues arranged into 10 beta-strands connected by turns. The active site of the enzyme is located in the L subunit and is accessible via a hydrophobic channel between the H and L subunits. The redox cofactor of MADH, tryptophan tryptophylquinone is highly unusual. It is formed from two covalently linked tryptophan side chains at positions 57 and 107 of the L subunit, one of which contains an orthoquinone.