Evidence for a methylammonium-binding site on methylamine dehydrogenase of Thiobacillus versutus

Abstract

The nonconvertible substrate analogues di-, tri-, and tetramethylammonium are bound with fairly high affinity to oxidized methylamine dehydrogenase (MADHox) from Thiobacillus versutus and induce the same red-shift in the optical absorbance spectrum of MADHox as do the monovalent cations Cs+, Rb+, and NH4+. Like the monovalent cations, trimethylamine also competitively inhibits the reduction of MADHox by methylamine. Rapid-scan experiments show that within the first few milliseconds of the reaction between MADHox and methylamine a red-shifted intermediate is formed as well. Taken together these experiments demonstrate the existence of a common binding site on MADHox for the substrate CH3NH3+, the substrate analogues (CH3)2NH2+, (CH3)3NH+, and (CH3)4N+, and the monovalent cations Cs+, Rb+, and NH4+. Therefore we conclude that, prior to conversion, methylamine is noncovalently bound to MADHox as a cation. The resonance Raman spectra of MADHox in the absence and presence of Cs+, NH4+, and (CH3)3NH+ are very similar, except for the C=O stretching frequencies of the o-quinone carbonyls of the tryptophyltryptophanquinone (TTQ) active center, which show 5-30 cm-1 downshifts. From these Raman results and the X-ray crystal structure, we conclude that the CH3NH3+ binding site is in close proximity to the O6 carbonyl oxygen of the TTQ.

Kinetic and chemical mechanisms for the effects of univalent cations on the spectral properties of aromatic amine dehydrogenase

“…It must be noted that for MADH from Thiobacillus ersutus, resonance Raman spectroscopy studies [10] indicated that the influence of NH % + was due to its proximity to TTQ and not covalent modification. Addition of NH % + to T. ersutus MADH caused a red shift of the absorption maximum of its UV-visible spectrum from 440 nm to 453 nm, which is similar to the change caused by addition of Cs + [19]. AADH and MADH are similar in that the spectral changes induced by NH % + \NH $ are different from those induced by metallic cations.…”

“…It has been shown for MADH that univalent cations affect the stability of the TTQ semiquinone [19]. Recent studies show that univalent cations enhance the rate of the proton-transfer reaction that gates the electron-transfer reaction from the substratereduced aminoquinol TTQ of MADH to amicyanin (G. R.…”

Crystallographic and Spectroscopic Studies of Native, Aminoquinol, and Monovalent Cation-bound Forms of Methylamine Dehydrogenase from Methylobacterium extorquens AM1

“…The later study demonstrates a primary kinetic isotope effect of approximately 9 -17 (at 30°C), considerably higher than the semiclassical limit for proton abstraction (16). This binding site has the same affinity and the specificity as the type I site of the oxidized MADH (23,24). We postulate that the water molecule may be replaced by the cation that may partially occupy the proximal position.…”

“…The type I cations act as competitive inhibitors of MADH reduction by substrate (22,23). A transient, red-shifted intermediate is also observed in the absorption spectrum of MADH prior to its reduction by methylamine (23). From these results, the binding site for these monovalent cations was predicted to be located at the active site in the vicinity of the cofactor atom O-6 and to mimic the first step of the reaction.…”

“…The type I cations act as competitive inhibitors of MADH reduction by substrate (22,23). A transient, red-shifted intermediate is also observed in the absorption spectrum of MADH prior to its reduction by methylamine (23).…”