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The heterologous expression of tryptophan trytophylquinone (TTQ)-dependent aromatic amine dehydrogenase (AADH) has been achieved in Paracoccus denitrificans. The aauBEDAgenes andorf-2from the aromatic amine utilization (aau) gene cluster of Alcaligenes faecaliswere placed under the regulatory control of themauFpromoter fromP. denitrificans and introduced intoP. denitrificansusing a broad-host-range vector. | iFEBS Journal Tryptophan tryptophylquinone cofactor biogenesis in the aromatic amine dehydrogenase of Alcaligenes faecalis Cofactor assembly and catalytic properties of recombinant enzyme expressed in Paracoccus denitrificans Parvinder Hothi Khalid Abu Khadra Jonathan P. Combe David Leys and Nigel S. Scrutton Manchester Interdisciplinary Biocentre and Faculty of Life Sciences University of Manchester UK Keywords amine oxidation aromatic amine dehydrogenase cofactor biogenesis stopped-flow spectroscopy tryptophan tryptophylquinone Correspondence N. S. Scrutton Manchester Interdisciplinary Biocentre and Faculty of Life Sciences University of Manchester Stopford Building Oxford Road Manchester M13 9PT UK Fax 44 161275 5586 Tel 44 161275 5632 E-mail nigel.scrutton@manchester.ac.uk Received 15 August 2005 revised 19 September 2005 accepted 22 September 2005 doi 10.1111 j.1742-4658.2005.04990.x 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 .
