Tryptophan tryptophylquinone cofactor biogenesis in the aromatic amine dehydrogenase of <i>Alcaligenes faecalis</i>

Hothi, Parvinder; Khadra, Khalid Abu; Combe, Jonathan P.; Leys, D.; Scrutton, Nigel S.

doi:10.1111/j.1742-4658.2005.04990.x

Cited by 10 publications

(12 citation statements)

References 30 publications

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“…TTQ reduction is ϳ80 times more rapid with tryptamine and the KIE value (ϳ55) is substantially inflated compared with the KIE (ϳ12) measured with phenylethylamine (8,14,23). The conformation of intermediate IIIa with tryptamine is distinct …”

Section: Qm/mm Simulations Of the Aadh/r-phenylaminoethanol Iminium Cmentioning

confidence: 88%

“…We have been able to reject the first mechanism by carefully measuring the number of electrons released per amine substrate molecule under standard assay conditions (two in all cases tested, data not shown) and by the fact that amide formation and hydrolysis are exceedingly slow (see below). Consequently, amide formation and hydrolysis cannot be an integral part of the steady-state mechanism for reaction with amines (14,23). We have demonstrated that soaking oxidized crystals of AADH with phenylacetaldehyde and ammonia for prolonged periods leads to AADH reduction.…”

Section: Reaction With Carbinolamines Leads To Formation Of Amide-ttqmentioning

confidence: 92%

“…Purification of Enzymes-AADH was purified from A. faecalis IFO 14479 as described previously (14). MADH was purified from Methylophilus methylotrophus (sp.…”

Section: Methodsmentioning

confidence: 99%

“…Steady-state Analysis-Steady-state kinetic measurements were performed with a 1-cm light path in 10 mM BisTris propane buffer, pH 7.5, at 25°C in a total volume of 1 ml. Enzyme activity was measured using a PES/DCPIP-coupled assay system (14). Reduction of DCPIP was followed at 600 nm using a PerkinElmer Lambda 5 UV-visible spectrophotometer.…”

Section: Methodsmentioning

confidence: 99%

“…The rate of reduction of the TTQ center by R-phenylaminoethanol is decreased by ϳ10 7 -fold when compared with the reduction rate with primary amines (8,14,23). This is despite the iminium-TTQ adduct (IIIb) being the predominant species in solution on mixing R-phenylaminoethanol with AADH, implying that the geometry for C-H bond cleavage in IIIb is suboptimal (e.g.…”

mentioning

confidence: 98%

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New Insights into the Reductive Half-reaction Mechanism of Aromatic Amine Dehydrogenase Revealed by Reaction with Carbinolamine Substrates

Roujeinikova

Hothi

Masgrau

et al. 2007

Journal of Biological Chemistry

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Aromatic amine dehydrogenase uses a tryptophan tryptophylquinone (TTQ) cofactor to oxidatively deaminate primary aromatic amines. In the reductive half-reaction, a proton is transferred from the substrate C1 to ␤Asp-128 O-2, in a reaction that proceeds by H-tunneling. Using solution studies, kinetic crystallography, and computational simulation we show that the mechanism of oxidation of aromatic carbinolamines is similar to amine oxidation, but that carbinolamine oxidation occurs at a substantially reduced rate. This has enabled us to determine for the first time the structure of the intermediate prior to the H-transfer/reduction step. The proton-␤Asp-128 O-2 distance is ϳ3.7 Å , in contrast to the distance of ϳ2.7 Å predicted for the intermediate formed with the corresponding primary amine substrate. This difference of ϳ1.0 Å is due to an unexpected conformation of the substrate moiety, which is supported by molecular dynamic simulations and reflected in the ϳ10 7 -fold slower TTQ reduction rate with phenylaminoethanol compared with that with primary amines. A water molecule is observed near TTQ C-6 and is likely derived from the collapse of the preceding carbinolamine TTQ-adduct. We suggest this water molecule is involved in consecutive proton transfers following TTQ reduction, and is ultimately repositioned near the TTQ O-7 concomitant with protein rearrangement. For all carbinolamines tested, highly stable amide-TTQ adducts are formed following proton abstraction and TTQ reduction. Slow hydrolysis of the amide occurs after, rather than prior to, TTQ oxidation and leads ultimately to a carboxylic acid product. Aromatic amine dehydrogenase (AADH)4 and the related methylamine dehydrogenase (MADH) are inducible periplasmic quinoproteins produced by some Gram-negative bacteria. These enzymes allow growth on primary amines as a source of carbon and nitrogen (1, 2). In Alcaligenes faecalis, AADH is specific for phenylethylamines, but also reacts to a lesser extent with primary aliphatic amines (1, 4). On the other hand, MADH is highly specific for smaller amines such as methylamine and ethylamine (3). Both enzymes exhibit an ␣ 2 ␤ 2 heterotetrameric structure (␣ molecular mass 40 kDa, ␤ 12 kDa), with each ␤-subunit possessing a covalently bound redox-active tryptophan tryptophylquinone (TTQ) cofactor (4). The oxidative deamination reaction proceeds in two steps. In the reductive half-reaction, the TTQ is reduced by substrate leading to incorporation of the substrate-derived amino group into the TTQ cofactor, resulting in conversion of the TTQ from a quinone to N-quinol form (Fig. 1) (4, 5). In the oxidative half-reaction, the TTQ cofactor is subsequently reoxidized by electron transfer to either the periplasmic type I blue copper protein azurin (AADH) or amicyanin (MADH) (6, 7).Recently, we reported the crystal structures of key intermediates in both the AADH reductive and oxidative half-reactions (8, 9). We have shown that substrate oxidation is dominated by H-tunneling (intermediate IIIa to IVa in Fig. 1) and o...

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Section: Qm/mm Simulations Of the Aadh/r-phenylaminoethanol Iminium Cmentioning

confidence: 88%

Section: Reaction With Carbinolamines Leads To Formation Of Amide-ttqmentioning

confidence: 92%

“…Purification of Enzymes-AADH was purified from A. faecalis IFO 14479 as described previously (14). MADH was purified from Methylophilus methylotrophus (sp.…”

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 98%

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New Insights into the Reductive Half-reaction Mechanism of Aromatic Amine Dehydrogenase Revealed by Reaction with Carbinolamine Substrates

Roujeinikova

Hothi

Masgrau

et al. 2007

Journal of Biological Chemistry

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Driving Force Analysis of Proton Tunnelling Across a Reactivity Series for an Enzyme‐Substrate Complex

et al. 2008

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Quantitative structure-activity relationships are widely used to probe C-H bond breakage by quinoprotein enzymes. However, we showed recently that p-substituted benzylamines are poor reactivity probes for the quinoprotein aromatic amine dehydrogenase (AADH) because of a requirement for structural change in the enzyme-substrate complex prior to C-H bond breakage. This rearrangement is partially rate limiting, which leads to deflated kinetic isotope effects for p-substituted benzylamines. Here we report reactivity (driving force) studies of AADH with p-substituted phenylethylamines for which the kinetic isotope effect (approximately 16) accompanying C-H/C-(2)H bond breakage is elevated above the semi-classical limit. We show bond breakage occurs by quantum tunnelling and that within the context of the environmentally coupled framework for H-tunnelling the presence of the p-substituent places greater demand on the apparent need for fast promoting motions. The crystal structure of AADH soaked with phenylethylamine or methoxyphenylethylamine indicates that the structural change identified with p-substituted benzylamines should not limit the reaction with p-substituted phenylethylamines. This is consistent with the elevated kinetic isotope effects measured with p-substituted phenylethylamines. We find a good correlation in the rate constant for proton transfer with bond dissociation energy for the reactive C-H bond, consistent with a rate that is limited by a Marcus-like tunnelling mechanism. As the driving force becomes larger, the rate of proton transfer increases while the Marcus activation energy becomes smaller. This is the first experimental report of the driving force perturbation of H-tunnelling in enzymes using a series of related substrates. Our study provides further support for proton tunnelling in AADH.

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Protein Biosynthesis Interference in Disease

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

Cited by 10 publications

References 30 publications

New Insights into the Reductive Half-reaction Mechanism of Aromatic Amine Dehydrogenase Revealed by Reaction with Carbinolamine Substrates

New Insights into the Reductive Half-reaction Mechanism of Aromatic Amine Dehydrogenase Revealed by Reaction with Carbinolamine Substrates

Driving Force Analysis of Proton Tunnelling Across a Reactivity Series for an Enzyme‐Substrate Complex

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