PDBsum entry 1spu

Oxidoreductase

PDB id: 1spu

Contents

Protein chains

718 a.a. *

Metals

_CA ×4

_CU ×2

Waters ×1064

* Residue conservation analysis

References listed in PDB file

Key reference

Title

Catalytic mechanism of the quinoenzyme amine oxidase from escherichia coli: exploring the reductive half-Reaction.

Authors

C.M.Wilmot, J.M.Murray, G.Alton, M.R.Parsons, M.A.Convery, V.Blakeley, A.S.Corner, M.M.Palcic, P.F.Knowles, M.J.Mcpherson, S.E.Phillips.

Ref.

Biochemistry, 1997, 36, 1608-1620. [DOI no: 10.1021/bi962205j]

PubMed id

9048544

Note In the PDB file this reference is annotated as "TO BE PUBLISHED". The citation details given above were identified by an automated search of PubMed on title and author names, giving a percentage match of 96%.

Abstract

The crystal structure of the complex between the copper amine oxidase from Escherichia coli (ECAO) and a covalently bound inhibitor, 2-hydrazinopyridine, has been determined to a resolution of 2.0 A. The inhibitor covalently binds at the 5 position of the quinone ring of the cofactor, 2,4,5-trihydroxyphenylalaninequinone (TPQ). The inhibitor complex is analogous to the substrate Schiff base formed during the reaction with natural monoamine substrate. A proton is abstracted from a methylene group adjacent to the amine group by a catalytic base during the reaction. The inhibitor, however, has a nitrogen at this position, preventing proton abstraction and trapping the enzyme in a covalent complex. The electron density shows this nitrogen is hydrogen bonded to the side chain of Asp383, a totally conserved residue, identifying it as the probable catalytic base. The positioning of Asp383 is such that the pro-S proton of a substrate would be abstracted, consistent with the stereospecificity of the enzyme determined by 1H NMR spectroscopy. Site-directed mutagenesis and in vivo suppression have been used to substitute Asp383 for 12 other residues. The resulting proteins either lack or, in the case of glutamic acid, have very low enzyme activity consistent with an essential catalytic role for Asp383. The O4 position on the quinone ring is involved in a short hydrogen bond with the hydroxyl of conserved residue Tyr369. The distance between the oxygens is less than 2.5 A, consistent with a shared proton, and suggesting ionization at the O4 position of the quinone ring. The Tyr369 residue appears to play an important role in stabilizing the position of the quinone/inhibitor complex. The O2 position on the quinone ring is hydrogen bonded to the apical water ligand of the copper. The basal water ligand, which lies 2.0 A from the copper in the native structure, is at a distance of 3.0 A in the complex. In the native structure, the active site is completely buried, with no obvious route for entry of substrate. In the complex, the tip of the pyridine ring of the bound inhibitor is on the surface of the protein at the edge of the interface between domains 3 and 4, suggesting this as the entry point for the amine substrate.

Secondary reference #1

Title

Crystal structure of a quinoenzyme: copper amine oxidase of escherichia coli at 2 a resolution.

Authors

M.R.Parsons, M.A.Convery, C.M.Wilmot, K.D.Yadav, V.Blakeley, A.S.Corner, S.E.Phillips, M.J.Mcpherson, P.F.Knowles.

Ref.

Structure, 1995, 3, 1171-1184. [DOI no: 10.1016/S0969-2126(01)00253-2]

PubMed id

8591028

Figure 1.
Figure 1. Pathway for the reductive half-reaction of amine oxidase. The numbering used in describing the TPQ moiety is shown in the first panel. The substrate, shown here as a substituted phenylmethylamine, reacts with the active-site TPQ to form the substrate Schiff base (1). Abstraction of the pro-S proton by the general base results in a carbanionic intermediate (2) leading to the product Schiff base (3). Hydrolysis of this species releases the product aldehyde and leaves the redox cofactor in the reduced aminoquinol form (4). TPQ is regenerated by oxidation by molecular oxygen in the oxidative half-reaction. Figure 1. Pathway for the reductive half-reaction of amine oxidase. The numbering used in describing the TPQ moiety is shown in the first panel. The substrate, shown here as a substituted phenylmethylamine, reacts with the active-site TPQ to form the substrate Schiff base (1). Abstraction of the pro-S proton by the general base results in a carbanionic intermediate (2) leading to the product Schiff base (3). Hydrolysis of this species releases the product aldehyde and leaves the redox cofactor in the reduced aminoquinol form (4). TPQ is regenerated by oxidation by molecular oxygen in the oxidative half-reaction.

Figure 6.
Figure 6. Active site of E. coli amine oxidase. The polypeptide backbone is shown as a continuous coil, coloured white for subunit A and grey for subunit B. Conserved residues are shown in all-atom representation and the copper is shown as a green van der Waals dot surface. The position of TPQ in crystal form I is illustrated in green, coordinated to the copper. The precise location and orientation of the TPQ ring is not completely determined at the resolution of the current studies of crystal form II, and its general location is indicated by a yellow phenyl ring, close to the putative catalytic base Asp383 (red). In crystal form II, TPQ is not a copper ligand and the copper coordination is completed by two water molecules, shown in yellow. Figure 6. Active site of E. coli amine oxidase. The polypeptide backbone is shown as a continuous coil, coloured white for subunit A and grey for subunit B. Conserved residues are shown in all-atom representation and the copper is shown as a green van der Waals dot surface. The position of TPQ in crystal form I is illustrated in green, coordinated to the copper. The precise location and orientation of the TPQ ring is not completely determined at the resolution of the current studies of crystal form II, and its general location is indicated by a yellow phenyl ring, close to the putative catalytic base Asp383 (red). In crystal form II, TPQ is not a copper ligand and the copper coordination is completed by two water molecules, shown in yellow. ([3]Figure 5 and [4]Figure 6 generated using MIDASPLUS [[5]51].)

The above figures are reproduced from the cited reference with permission from Cell Press