New insights into the reductive half-reaction mechanism of aromatic amine dehydrogenase revealed by reaction with carbinolamine substrates

J Biol Chem. 2007 Aug 17;282(33):23766-77. doi: 10.1074/jbc.M700677200. Epub 2007 May 1.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Alcaligenes faecalis / enzymology
  • Bacterial Proteins
  • Crystallography, X-Ray
  • Half-Life
  • Hydrolysis
  • Kinetics
  • Methylamines
  • Oxidation-Reduction
  • Oxidoreductases Acting on CH-NH Group Donors / chemistry
  • Oxidoreductases Acting on CH-NH Group Donors / metabolism*


  • Bacterial Proteins
  • Methylamines
  • Oxidoreductases Acting on CH-NH Group Donors
  • aromatic amine dehydrogenase

Associated data

  • PDB/2I0R
  • PDB/2I0S
  • PDB/2I0T
  • PDB/2OIZ
  • PDB/2OJY
  • PDB/2OK4
  • PDB/2OK6