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 betaAsp-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-betaAsp-128 O-2 distance is approximately 3.7A, in contrast to the distance of approximately 2.7A predicted for the intermediate formed with the corresponding primary amine substrate. This difference of approximately 1.0 A is due to an unexpected conformation of the substrate moiety, which is supported by molecular dynamic simulations and reflected in the approximately 10(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.
Highlights
Aromatic amine dehydrogenase (AADH)4 and the related methylamine dehydrogenase (MADH) are inducible periplasmic quinoproteins produced by some Gram-negative bacteria
Using kinetic crystallography approaches with the AADH/ tryptamine system, we report in this paper the discovery of an unusual covalent intermediate that can be explained by assuming reaction with indol-3-aminoethanol, the tryptamine-derived carbinolamine
In contrast to the generally fast reaction rates observed for primary amines, the inherently slow reaction rates observed for carbinolamines allow us to address those questions on the AADH amine oxidation mechanism described in the previous paragraph
Summary
Materials—BisTris propane buffer, DCIP (2,6-dichlorophenolindophenol; sodium salt), PES (phenazine ethosulfate; N-ethyldibenzopyrazine ethyl sulfate salt), -phenylethylamine, tryptamine, benzylamine, phenylacetaldehyde, formaldehyde, and indole-3-acetaldehyde-sodium bisulfite addition compound were obtained from Sigma. IIIb) were produced by soaking the form A crystals in a stabilizing solution containing 25% PEG MME 2K, 120 mM ammonium sulfate, 100 mM sodium cacodylate, pH 7.5, and 1 mM phenylacetaldehyde for 15 min. Crystals of the phenylacetamide and formamide adducts of AADH (intermediate Vc) were produced by soaking the form A crystals in a stabilizing solution that included either 2 mM phenylacetaldehyde or 5 mM formaldehyde for 36 h. Aldehyde-dependent Reduction of AADH and MADH—Aldehydes were added to a 1-ml solution containing 5 M oxidized AADH or MADH, 10 mM BisTris propane buffer, pH 7.5, and 50 mM ammonium sulfate (unless stated otherwise). Reaction mixtures with the enzyme preincubated in 1 mM phenylacetaldehyde and 50 mM ammonium sulfate typically contained 25 nM (for -phenylethylamine and tryptamine-dependent reactions) or 50 nM AADH (for benzylamine-dependent reactions), 0.04 mM DCPIP, 5 mM PES, and 500 M substrate.
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