New insights into the reductive half-reaction mechanism of aromatic amine dehydrogenase revealed by reaction with carbinolamine substrates.
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.
Roujeinikova
Anna
A
Manchester Interdisciplinary Biocenter, University of Manchester, Manchester M17DN, United Kingdom.
Hothi
Parvinder
P
Masgrau
Laura
L
Sutcliffe
Michael J
MJ
Scrutton
Nigel S
NS
Leys
David
D
D042481:Alcaligenes faecalis / Q000201:enzymology; D001426:Bacterial Proteins; D018360:Crystallography, X-Ray; D006207:Half-Life; D006868:Hydrolysis; D007700:Kinetics; D008744:Methylamines; D010084:Oxidation-Reduction; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D008744:Methylamines; D000587:Oxidoreductases Acting on CH-NH Group Donors; C037193:aromatic amine dehydrogenase