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

Abstract

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...

Incorporating Fast Protein Dynamics into Enzyme Design: A Proposed Mutant Aromatic Amine Dehydrogenase

“…We studied the enzyme aromatic amine dehydrogenase (AADH) whose mechanism of catalysis is well-understood. − AADH is a tryptophan tryptophylquinone (TTQ)-dependent quinoprotein that catalyzes proton transfer. It oxidatively deaminates aromatic primary amines to form the corresponding aldehyde.…”

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

“…[8] The enzyme reaction cycle is completed by long-range electron transfer to the type 1 copper protein azurin following assembly of an AADH-azurin electron transfer complex. [9][10][11] Insight into the reaction cycle has come from detailed analysis of the crystal structures of reaction intermediates, [8,9,12] computational simulations of the reaction chemistry [8,13,14] and isotope analysis of the proton transfer step using fast reaction stopped-flow methods. [5,15,16] These studies have provided a detailed appreciation of the reaction chemistry, whilst emphasizing the importance of proton transfer by quantum tunnelling mechanisms (for example, with tryptamine substrate).…”

“…[12] Nevertheless, in the case of both tryptamine and the p-substituted phenylethylamine substrates, changes in the position of the key N1, C1, and C2 substrate derived atoms during catalysis appear not to significantly affect the position of the aromatic moiety. This contrasts markedly with the case for p-substituted benzylamine substrates, in which the C2 atom is part of the aromatic moiety itself and significant reorientation of the benzyl side chain (as well as several amino acid side chains) is required during catalysis.…”

New insights into the multi-step reaction pathway of the reductive half-reaction catalysed by aromatic amine dehydrogenase: a QM/MM study

“…3 Using ''time-resolved'' X-ray crystallography, several intermediates in the reductive half-reaction were identified. 4,5 Nevertheless, there are significant gaps in our understanding of the chemistry of the catalytic cycle of this important model system for studying hydrogen tunnelling, 4,6,7 and there is a need for new insight derived from computational methods.…”

“…Hence, we increased the size of the QM partition to 104 atoms to include residues which form hydrogen bonds with the carboxylate group of Asp128b and the iminoquinone. With the size of QM partition extended, the height of the barrier increases to 8.1 AE 0.9 kcal mol À1 with mechanical Scheme 1 The proposed reaction mechanism 4,5 for the reductive half-reaction of AADH with tryptamine. when zero-point energy is included) with electronic embedding (EE) (Fig.…”