PDBsum entry 2ok4

Oxidoreductase

PDB id: 2ok4

Contents

Protein chains

110 a.a.

122 a.a.

360 a.a.

Ligands

HY1 ×2

Waters ×1051

References listed in PDB file

Key reference

Title

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

Authors

A.Roujeinikova, P.Hothi, L.Masgrau, M.J.Sutcliffe, N.S.Scrutton, D.Leys.

Ref.

J Biol Chem, 2007, 282, 23766-23777. [DOI no: 10.1074/jbc.M700677200]

PubMed id

17475620

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.

Figure 3.
FIGURE 3. Schematic overview of the proposed mechanism for AADH R-carbinolamine oxidation. For ease of comparison with the previously proposed amine oxidation mechanism, a similar notation is used according to Fig. 1. For clarity, only part of the TTQ cofactor is represented, whereas the side chain of the different R-carbinolamines is indicated by an R. Refer to supplementary Scheme 1 for a full description of the substrate-TTQ-enzyme adduct. The active site water molecule (or ammonia in case of a steady state mechanism, see Ref. 24) is denoted W1. Whether a conformational equilibrium between IIIb-A and IIIb-B occurs depends on the nature of the R side chain.

Figure 5.
FIGURE 5. Top, comparison of IIIb-A conformation as observed in the crystals (phenylacetaldehyde derived carbon atoms in cyan) with a more optimal configuration (IIIb-B) based on the modeled intermediate IIIa (Fig. 1) during tryptamine reduction (Ref. 8; phenylacetaldehyde-derived carbon atoms in yellow). Putative hydrogen bonding interactions made between IIIb-B conformation and active site residues are shown by dotted lines. Key active site residues and TTQ cofactor are displayed with green carbons. B, overlay of crystal structures of IIIb-A (with green carbons) and Vc (with cyan carbons) for phenylacetaldehyde and ammonia as substrates. The active site water molecule situated close to C-6 and O-7 is shown as a red sphere (labeled W-1[ox]and W-1[red], respectively).

The above figures are reprinted by permission from the ASBMB: J Biol Chem (2007, 282, 23766-23777) copyright 2007.