Tài liệu Báo cáo khoa học: Tryptophan tryptophylquinone cofactor biogenesis in the aromatic amine

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

Aromatic amine dehydrogenase (AADH) is a trypto￾phan tryptophylquinone (TTQ)-dependent quinopro￾tein that catalyses the oxidative deamination of a wide

range of amines to their corresponding aldehydes and

ammonia [1]. Electrons released upon substrate oxida￾tion are transferred to the TTQ cofactor (Fig. 1) and

then to the physiological electron acceptor, azurin,

which mediates electron transfer from the dehydro￾Keywords

amine oxidation; aromatic amine

dehydrogenase; cofactor biogenesis;

stopped-flow spectroscopy; tryptophan

tryptophyl quinone

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: [email protected]

(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 concen￾tration differed between ‘fast’ (tryptamine) and ‘slow’ (benzylamine) sub￾strates, 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 reduc￾tive 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 (klim) for TTQ

reduction is controlled by a single ionization with pKa value of 6.0, with

maximum activity realized in the alkaline region. Two kinetically influential

ionizations were identified in plots of klim ⁄ Kd of pKa values 7.1 and 9.3,

again with the maximum value realized in the alkaline region. The poten￾tial 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.

5894 FEBS Journal 272 (2005) 5894–5909 ª 2005 FEBS