Publications

• Crystallization and properties of aromatic amine dehydrogenase from Pseudomonas sp.

  Iwaki M., Yagi T., Horiike K., Saeki Y., Ushijima T., Nozaki M.

  An amine dehydrogenase was purified to homogeneity from an extract of a bacterium of the genus Pseudomonas grown in a medium containing beta-phenylethylamine as a sole carbon source and obtained in a crystalline form with about 100-fold purification. The purified enzyme catalyzed the oxidative dea ... >> More

  An amine dehydrogenase was purified to homogeneity from an extract of a bacterium of the genus Pseudomonas grown in a medium containing beta-phenylethylamine as a sole carbon source and obtained in a crystalline form with about 100-fold purification. The purified enzyme catalyzed the oxidative deamination of various aromatic amines as well as some aliphatic amines to a lesser extent. An artificial electron acceptor such as phenazine methosulfate was required for the catalysis. The molecular weight determined by sedimentation equilibrium was 103,000 and the molecule seemed to be composed of two pairs of two nonidentical subunits (Mr 46,000 and 8000). The enzyme had a dull yellow-green color with an absorption maximum at 445 nm and this chromophore appeared to be involved in the catalytic action of the enzyme. << Less

  Arch Biochem Biophys 220:253-262(1983) [PubMed] [EuropePMC]

• Gated and ungated electron transfer reactions from aromatic amine dehydrogenase to azurin.

  Hyun Y.L., Zhu Z., Davidson V.L.

  Interprotein electron transfer (ET) occurs between the tryptophan tryptophylquinone (TTQ) prosthetic group of aromatic amine dehydrogenase (AADH) and copper of azurin. The ET reactions from two chemically distinct reduced forms of TTQ were studied: an O-quinol form that was generated by reduction ... >> More

  Interprotein electron transfer (ET) occurs between the tryptophan tryptophylquinone (TTQ) prosthetic group of aromatic amine dehydrogenase (AADH) and copper of azurin. The ET reactions from two chemically distinct reduced forms of TTQ were studied: an O-quinol form that was generated by reduction by dithionite, and an N-quinol form that was generated by reduction by substrate. It was previously shown that on reduction by substrate, an amino group displaces a carbonyl oxygen on TTQ, and that this significantly alters the rate of its oxidation by azurin (Hyun, Y-L., and Davidson V. L. (1995) Biochemistry 34, 12249-12254). To determine the basis for this change in reactivity, comparative kinetic and thermodynamic analyses of the ET reactions from the O-quinol and N-quinol forms of TTQ in AADH to the copper of azurin were performed. The reaction of the O-quinol exhibited values of electronic coupling (H(AB)) of 0.13 cm(-1) and reorganizational energy (lambda) of 1.6 eV, and predicted an ET distance of approximately 15 A. These results are consistent with the ET event being the rate-determining step for the redox reaction. Analysis of the reaction of the N-quinol by Marcus theory yielded an H(AB) which exceeded the nonadiabatic limit and predicted a negative ET distance. These results are diagnostic of a gated ET reaction. Solvent deuterium kinetic isotope effects of 1.5 and 3.2 were obtained, respectively, for the ET reactions from O-quinol and N-quinol AADH indicating that transfer of an exchangeable proton was involved in the rate-limiting reaction step which gates ET from the N-quinol, but not the O-quinol. These results are compared with those for the ET reactions from another TTQ enzyme, methylamine dehydrogenase, to amicyanin. The mechanism by which the ET reaction of the N-quinol is gated is also related to mechanisms of other gated interprotein ET reactions. << Less

  J Biol Chem 274:29081-29086(1999) [PubMed] [EuropePMC]

• Tryptophan tryptophylquinone cofactor biogenesis in the aromatic amine dehydrogenase of Alcaligenes faecalis. Cofactor assembly and catalytic properties of recombinant enzyme expressed in Paracoccus denitrificans.

  Hothi P., Khadra K.A., Combe J.P., Leys D., Scrutton N.S.

  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 ... >> More

  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. << Less

  FEBS J. 272:5894-5909(2005) [PubMed] [EuropePMC]

• Electron transfer reactions between aromatic amine dehydrogenase and azurin.

  Hyun Y.L., Davidson V.L.

  Binding and electron transfer reactions between the tryptophan tryptophylquinone (TTQ) enzyme, aromatic amine dehydrogenase (AADH), and the type I copper protein azurin have been characterized. In steady-state kinetic assays using azurin as an electron acceptor, it was observed that the apparent K ... >> More

  Binding and electron transfer reactions between the tryptophan tryptophylquinone (TTQ) enzyme, aromatic amine dehydrogenase (AADH), and the type I copper protein azurin have been characterized. In steady-state kinetic assays using azurin as an electron acceptor, it was observed that the apparent Km for azurin decreased with increasing ionic strength. These results are the opposite of what was observed for the reaction between the TTQ enzyme methylamine dehydrogenase (MADH) and amicyanin, despite the fact that in both cases the pairs of redox proteins are each acidic proteins. It was further demonstrated that azurin does not function as an effective electron acceptor for MADH, and that amicyanin does not function as an effective electron acceptor for AADH. Thus, while the two TTQ enzymes each use type I copper proteins as physiologic electron acceptors, there is a strong specificity for which copper protein serves as a redox partner. The kinetic parameters for the electron transfer reactions from reduced AADH to oxidized azurin were determined by stopped-flow spectroscopy. Different results were obtained depending upon whether AADH was reduced chemically with dithionite or with the substrate tyramine. The values for the limiting first-order apparent electron transfer rate constant (kET) at 30 degrees C were 4 and 102 s-1, respectively. Kinetically determined values of Kd also differed by a factor of 2.4. These data suggest that the incorporation of the substrate-derived amino group into the reduced TTQ of AADH significantly increases the apparent kET. The interaction between AADH and azurin was also quantitated using an ultrafiltration binding assay. This yielded a Kd of 300 microM for the AADH--azurin complex.(ABSTRACT TRUNCATED AT 250 WORDS) << Less

  Biochemistry 34:12249-12254(1995) [PubMed] [EuropePMC]

• Crystal structure of an electron transfer complex between aromatic amine dehydrogenase and azurin from Alcaligenes faecalis.

  Sukumar N., Chen Z.-W., Ferrari D., Merli A., Rossi G.L., Bellamy H.D., Chistoserdov A.Y., Davidson V.L., Mathews F.S.

  The crystal structure of an electron transfer complex of aromatic amine dehydrogenase (AADH) and azurin is presented. Electrons are transferred from the tryptophan tryptophylquinone (TTQ) cofactor of AADH to the type I copper of the cupredoxin azurin. This structure is compared with the complex of ... >> More

  The crystal structure of an electron transfer complex of aromatic amine dehydrogenase (AADH) and azurin is presented. Electrons are transferred from the tryptophan tryptophylquinone (TTQ) cofactor of AADH to the type I copper of the cupredoxin azurin. This structure is compared with the complex of the TTQ-containing methylamine dehydrogenase (MADH) and the cupredoxin amicyanin. Despite significant similarities between the two quinoproteins and the two cupredoxins, each is specific for its respective partner and the ionic strength dependence and magnitude of the binding constant for each complex are quite different. The AADH-azurin interface is largely hydrophobic, covering approximately 500 A(2) of surface on each molecule, with one direct hydrogen bond linking them. The closest distance from TTQ to copper is 12.6 A compared with a distance of 9.3 A in the MADH-amicyanin complex. When the MADH-amicyanin complex is aligned with the AADH-azurin complex, the amicyanin lies on top of the azurin but is oriented quite differently. Although the copper atoms differ in position by approximately 4.7 A, the amicyanin bound to MADH appears to be rotated approximately 90 degrees from its aligned position with azurin. Comparison of the structures of the two complexes identifies features of the interface that dictate the specificity of the protein-protein interaction and determine the rate of interprotein electron transfer. << Less

  Biochemistry 45:13500-13510(2006) [PubMed] [EuropePMC]

• Identification of reaction products and intermediates of aromatic-amine dehydrogenase by 15N and 13C NMR.

  Bishop G.R., Zhu Z., Whitehead T.L., Hicks R.P., Davidson V.L.

  13C- and 15N-NMR studies of the reaction of aromatic amine dehydrogenase (AADH) with methylamine demonstrated that the products of the reductive half-reaction are an equivalent of formaldehyde hydrate and a reduced aminoquinol form of the tryptophan tryptophylquinone (TTQ) cofactor which contains ... >> More

  13C- and 15N-NMR studies of the reaction of aromatic amine dehydrogenase (AADH) with methylamine demonstrated that the products of the reductive half-reaction are an equivalent of formaldehyde hydrate and a reduced aminoquinol form of the tryptophan tryptophylquinone (TTQ) cofactor which contains covalently bound substrate-derived N. These data are consistent with the Ping Pong kinetic mechanism and aminotransferase-type chemical reaction mechanism which have been previously proposed for AADH. Comparison of the 15N-NMR spectra of the aminoquinol TTQ intermediates of AADH and methylamine dehydrogenase (MADH) revealed that the substrate-derived aminoquinol N of AADH and MADH exhibited distinct 15N chemical shifts which are separated by approx. 7 p.p.m. In each case, the signal for the substrate-derived aminoquinol N appears optimally with short pulse delay and exhibits a relaxation time and chemical shift which are consistent with 15N covalently bound to an aromatic ring (i.e. aminoquinol) which is attached to a rigid protein matrix. The aminoquinol of AADH is less stable against reoxidation than that of MADH. These data suggest that differences in the active-site mediated electrostatic environments of the aminoquinol N in the respective enzymes may influence both the observed 15N chemical shift and the relative reactivities of the TTQ aminoquinols towards oxygen. These data also demonstrate the utility of 13C- and 15N-NMR spectroscopy as a tool for monitoring the intermediates and products of enzyme-catalysed transformations. << Less

  Biochem. J. 330:1159-1163(1998) [PubMed] [EuropePMC]