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

Zhu, Zhenqi; Davidson, L. Victor

doi:10.1042/bj3290175

Cited by 13 publications

(13 citation statements)

References 29 publications

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“…However, we demonstrated that this was not possible owing to rapid reduction of the TTQ cofactor by the buffer components. Also, as noted previously, univalent cations stimulate spectral changes in AADH, particularly at higher pH values [20] and our own studies revealed a similar trend at higher pH values with various univalent cations (data not shown). Thus, owing to a rapid loss in absorbance at 456 nm (attributed to chemical modification of the TTQ to form an hydroxide adduct) [20], it was not feasible to perform pH-dependent studies of TTQ reduction in the presence of added salt (although studies performed in the presence of 100 mm NaCl at lower pH values yielded results comparable with those obtained in the absence of NaCl).…”

Section: Ph Dependence Of Ttq Reduction With Benzylamine and Deuteratsupporting

confidence: 88%

Tryptophan tryptophylquinone cofactor biogenesis in the aromatic amine dehydrogenase of Alcaligenes faecalis

et al. 2005

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Aromatic amine dehydrogenase (AADH) is a tryptophan tryptophylquinone (TTQ)-dependent quinoprotein that catalyses the oxidative deamination of a wide range of amines to their corresponding aldehydes and ammonia [1]. Electrons released upon substrate oxidation are transferred to the TTQ cofactor ( Fig. 1) and then to the physiological electron acceptor, azurin, which mediates electron transfer from the dehydro- The heterologous expression of tryptophan trytophylquinone (TTQ)-dependent aromatic amine dehydrogenase (AADH) has been achieved in Paracoccus denitrificans. The aauBEDA genes and orf-2 from the aromatic amine utilization (aau) gene cluster of Alcaligenes faecalis were placed under the regulatory control of the mauF promoter from P. denitrificans and introduced into P. denitrificans using a broad-host-range vector. The physical, spectroscopic and kinetic properties of the recombinant AADH were indistinguishable from those of the native enzyme isolated from A. faecalis. TTQ biogenesis in recombinant AADH is functional despite the lack of analogues in the cloned aau gene cluster for mauF, mauG, mauL, mauM and mauN that are found in the methylamine utilization (mau) gene cluster of a number of methylotrophic organisms. Steady-state reaction profiles for recombinant AADH as a function of substrate concentration differed between 'fast' (tryptamine) and 'slow' (benzylamine) substrates, owing to a lack of inhibition by benzylamine at high substrate concentrations. A deflated and temperature-dependent kinetic isotope effect indicated that C-H ⁄ C-D bond breakage is only partially rate-limiting in steady-state reactions with benzylamine. Stopped-flow studies of the reductive half-reaction of recombinant AADH with benzylamine demonstrated that the KIE is elevated over the value observed in steady-state turnover and is independent of temperature, consistent with (a) previously reported studies with native AADH and (b) breakage of the substrate C-H bond by quantum mechanical tunnelling. The limiting rate constant (k lim ) for TTQ reduction is controlled by a single ionization with pK a value of 6.0, with maximum activity realized in the alkaline region. Two kinetically influential ionizations were identified in plots of k lim ⁄ K d of pK a values 7.1 and 9.3, again with the maximum value realized in the alkaline region. The potential origin of these kinetically influential ionizations is discussed.Abbreviations AADH, aromatic amine dehydrogenase; aau, aromatic amine utilization; DCPIP, dichlorophenol indophenol; KIE, kinetic isotope effect; MADH, methylamine dehydrogenase; mau, methylamine utilization; ORF, open reading frame; PES, phenazine ethosulfate; TTQ, tryptophan tryptophylquinone.

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Section: Ph Dependence Of Ttq Reduction With Benzylamine and Deuteratsupporting

confidence: 88%

Tryptophan tryptophylquinone cofactor biogenesis in the aromatic amine dehydrogenase of Alcaligenes faecalis

et al. 2005

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“…Type I causes a red shift of the oxidized spectrum of MADH, whereas type II causes a bleaching of the visible spectrum and an increased absorbance in the near ultraviolet, similar to that observed during reduction of the enzyme by methylamine. Analogous spectral perturbations have been observed with another TTQcontaining enzyme, aromatic amine dehydrogenase (21). The type I binding site is specific for larger cations such as Cs ϩ , Rb ϩ , and NH 4 ϩ as well as di-, tri-, and tetramethylammonium ions.…”

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confidence: 54%

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

Labesse

Ferrari²,

Chen

et al. 1998

Journal of Biological Chemistry

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Various monovalent cations influence the enzymatic activity and the spectroscopic properties of methylamine dehydrogenase (MADH). Here, we report the structure determination of this tryptophan tryptophylquinone-containing enzyme from Methylobacterium extorquens AM1 by high resolution x-ray crystallography (1.75 A). This first MADH crystal structure at low ionic strength is compared with the high resolution structure of the related MADH from Paracoccus denitrificans recently reported. We also describe the first structures (at 1.95 to 2.15 A resolution) of an MADH in the substrate-reduced form and in the presence of trimethylamine and of cesium, two competitive inhibitors. Polarized absorption microspectrophotometry was performed on single crystals under various redox, pH, and salt conditions. The results show that the enzyme is catalytically active in the crystal and that the cations cause the same spectral perturbations as are observed in solution. These studies lead us to propose a model for the entrance and binding of the substrate in the active site.

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“…MADH and amicyanin concentrations were calculated from known extinction coefficients (3,14) in 10 mM phosphate buffer, pH 7.5. The extinction coefficients of the different redox forms of MADH vary with pH (20) and ionic composition of the buffer (21). Purifications of AADH (7) and azurin (8,22) from A. faecalis (IFO 14479) were as described previously, and protein concentrations were calculated from previously determined extinction coefficients (7,23).…”

Section: Methodsmentioning

confidence: 99%

“…The only difference was that the rate of the disproportionation reaction of AADH was faster than that of MADH. The extinction coefficients for the different redox states of MADH and AADH vary with pH in the presence of monovalent cations (20,21). For this reason, BTP buffer was used.…”

Section: Figmentioning

confidence: 99%

Redox Properties of Tryptophan Tryptophylquinone Enzymes

Zhu

Davidson

1998

Journal of Biological Chemistry

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The pH dependence of the redox potentials for the oxidized/reduced couples of methylamine dehydrogenase (MADH) and aromatic amine dehydrogenase (AADH) were determined. For each enzyme, a change of ؊30 mV/pH unit was observed, indicating that the two-electron transfer is linked to the transfer of a single proton. This result differs from what was obtained from redox studies of a tryptophan tryptophylquinone (TTQ) model compound for which the two-electron couple is linked to the transfer of two protons. This result also distinguishes the redox properties of the enzyme-bound TTQ from those of the membrane-bound quinone components of respiratory and photosynthetic electron transfer chains that transfer two protons per two electrons. This difference is attributed to the accessibility of TTQ to solvent in the enzymes. One of the quinol hydroxyls is shielded from solvent and thus is not protonated. The unusual property of TTQ enzymes of stabilizing the anionic form of the reduced quinol is important for the reaction mechanism of MADH because it allows stabilization of physiologically important reaction intermediates. Examination of the extent to which disproportionation of the MADH and AADH semiquinones occurred as a function of pH revealed that the equilibrium concentration of semiquinone increased with pH. This indicates that the proton transfer is linked to the semiquinone/quinol couple. Therefore, the quinol is singly protonated, and the semiquinone is unprotonated and anionic. It was also shown that the oxidation-reduction midpoint potential for AADH is 20 mV less positive than that of MADH over the range of pH values that was studied and that the TTQ semiquinone of AADH was less stable than that of MADH. This may be explained by differences in the active site environments of the two enzymes, which modulate their respective redox properties.Redox reactions involving proteins are ubiquitous processes that are fundamental to respiration, photosynthesis, and reactions of intermediary metabolism. Enzyme-bound quinones and flavins are important cofactors of oxidoreductases that function in biological catalysis and electron transfer. These prosthetic groups, as well as membrane-bound components of respiratory electron transfer chains, are particularly important because they are able to couple the two-electron oxidation of substrates to single electron carriers during metabolism and respiration.Methylamine dehydrogenase (MADH; reviewed in Ref. 1) 1 catalyzes the oxidation of methylamine in the periplasm of many methylotrophic and autotrophic bacteria to form ammonia and formaldehyde concomitant with the two-electron reduction of its redox cofactor (Reaction 1). MADH isan H 2 L 2 heterotetramer with subunit molecular masses of 47 and 15 kDa. The tryptophan tryptophylquinone (TTQ; Fig. 1) (2) prosthetic group, which is located on each small subunit, is derived from two tryptophan side chains that are covalently cross-linked and contain an orthoquinone function through a post-translational modification. In autotrophic bacte...

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

Cited by 13 publications

References 29 publications

Tryptophan tryptophylquinone cofactor biogenesis in the aromatic amine dehydrogenase of Alcaligenes faecalis

Tryptophan tryptophylquinone cofactor biogenesis in the aromatic amine dehydrogenase of Alcaligenes faecalis

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

Redox Properties of Tryptophan Tryptophylquinone Enzymes

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