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Tryptophan tryptophylquinone cofactor biogenesis in the aromatic amine dehydrogenase of Alcaligenes faecalis
Author(s) -
Hothi Parvinder,
Khadra Khalid Abu,
Combe Jonathan P.,
Leys David,
Scrutton Nigel S.
Publication year - 2005
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2005.04990.x
Subject(s) - chemistry , benzylamine , alcaligenes faecalis , paracoccus denitrificans , stereochemistry , biochemistry , enzyme , medicinal chemistry , biology , bacteria , genetics
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 p K 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 p K 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.