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The structure of the C-C bond hydrolase MhpC provides insights into its catalytic mechanism

The structure of the C-C bond hydrolase MhpC provides insights into its catalytic mechanism
The structure of the C-C bond hydrolase MhpC provides insights into its catalytic mechanism
2-Hydroxy-6-ketonona-2,4-diene-1,9-dioic acid 5,6-hydrolase (MhpC) is a 62 kDa homodimeric enzyme of the phenylpropionate degradation pathway of Escherichia coli. The 2.1 Å resolution X-ray structure of the native enzyme determined from orthorhombic crystals confirms that it is a member of the ?/? hydrolase fold family, comprising eight ?-strands interconnected by loops and helices. The 2.8 Å resolution structure of the enzyme co-crystallised with the non-hydrolysable substrate analogue 2,6-diketo-nona-1,9-dioic acid (DKNDA) confirms the location of the active site in a buried channel including Ser110, His263 and Asp235, postulated contributors to a serine protease-like catalytic triad in homologous enzymes. It appears that the ligand binds in two separate orientations. In the first, the C6 keto group of the inhibitor forms a hemi-ketal adduct with the Ser110 side-chain, the C9 carboxylate group interacts, via the intermediacy of a water molecule, with Arg188 at one end of the active site, while the C1 carboxylate group of the inhibitor comes close to His114 at the other end. In the second orientation, the C1 carboxylate group binds at the Arg188 end of the active site and the C9 carboxylate group at the His114 end. These arrangements implicated His114 or His263 as plausible contributors to catalysis of the initial enol/keto tautomerisation of the substrate but lack of conservation of His114 amongst related enzymes and mutagenesis results suggest that His263 is the residue involved. Variability in the quality of the electron density for the inhibitor amongst the eight molecules of the crystal asymmetric unit appears to correlate with alternative positions for the side-chain of His114. This might arise from half-site occupation of the dimeric enzyme and reflect the apparent dissociation of approximately 50% of the keto intermediate from the enzyme during the catalytic cycle.
C–C bond hydrolase, a/b hydrolase, non-nucleophilic serine, hemi-ketal adduct, crystal structure
0022-2836
253-265
Dunn, G.
eaa5c04d-7f2a-4aca-a981-e13ba1061c5a
Montgomery, M.G.
d81e6781-e087-4a10-b4cc-bd8efcb14467
Mohammed, F.
e6dd5a98-bea5-484a-873a-da499c034594
Coker, A.
c64a7d9b-3a0d-4e79-a3eb-aaa64920116a
Cooper, J.B.
d9f0f6a8-1260-48fc-aa5c-3dbc650e3ec0
Robertson, T.
82d76ba0-9d7c-4341-b5af-19c7c8288d50
Garcia, J.L.
364ca6ce-a842-4e2a-9e6e-4e86f067fd8b
Bugg, T.D.H.
5b6bfe9f-6631-4055-91d2-185236ff9b19
Wood, S.P.
430faabf-7f5c-4cf6-9bcc-5955f5e09566
Dunn, G.
eaa5c04d-7f2a-4aca-a981-e13ba1061c5a
Montgomery, M.G.
d81e6781-e087-4a10-b4cc-bd8efcb14467
Mohammed, F.
e6dd5a98-bea5-484a-873a-da499c034594
Coker, A.
c64a7d9b-3a0d-4e79-a3eb-aaa64920116a
Cooper, J.B.
d9f0f6a8-1260-48fc-aa5c-3dbc650e3ec0
Robertson, T.
82d76ba0-9d7c-4341-b5af-19c7c8288d50
Garcia, J.L.
364ca6ce-a842-4e2a-9e6e-4e86f067fd8b
Bugg, T.D.H.
5b6bfe9f-6631-4055-91d2-185236ff9b19
Wood, S.P.
430faabf-7f5c-4cf6-9bcc-5955f5e09566

Dunn, G., Montgomery, M.G., Mohammed, F., Coker, A., Cooper, J.B., Robertson, T., Garcia, J.L., Bugg, T.D.H. and Wood, S.P. (2005) The structure of the C-C bond hydrolase MhpC provides insights into its catalytic mechanism. Journal of Molecular Biology, 346 (1), 253-265. (doi:10.1016/j.jmb.2004.11.033).

Record type: Article

Abstract

2-Hydroxy-6-ketonona-2,4-diene-1,9-dioic acid 5,6-hydrolase (MhpC) is a 62 kDa homodimeric enzyme of the phenylpropionate degradation pathway of Escherichia coli. The 2.1 Å resolution X-ray structure of the native enzyme determined from orthorhombic crystals confirms that it is a member of the ?/? hydrolase fold family, comprising eight ?-strands interconnected by loops and helices. The 2.8 Å resolution structure of the enzyme co-crystallised with the non-hydrolysable substrate analogue 2,6-diketo-nona-1,9-dioic acid (DKNDA) confirms the location of the active site in a buried channel including Ser110, His263 and Asp235, postulated contributors to a serine protease-like catalytic triad in homologous enzymes. It appears that the ligand binds in two separate orientations. In the first, the C6 keto group of the inhibitor forms a hemi-ketal adduct with the Ser110 side-chain, the C9 carboxylate group interacts, via the intermediacy of a water molecule, with Arg188 at one end of the active site, while the C1 carboxylate group of the inhibitor comes close to His114 at the other end. In the second orientation, the C1 carboxylate group binds at the Arg188 end of the active site and the C9 carboxylate group at the His114 end. These arrangements implicated His114 or His263 as plausible contributors to catalysis of the initial enol/keto tautomerisation of the substrate but lack of conservation of His114 amongst related enzymes and mutagenesis results suggest that His263 is the residue involved. Variability in the quality of the electron density for the inhibitor amongst the eight molecules of the crystal asymmetric unit appears to correlate with alternative positions for the side-chain of His114. This might arise from half-site occupation of the dimeric enzyme and reflect the apparent dissociation of approximately 50% of the keto intermediate from the enzyme during the catalytic cycle.

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Submitted date: 24 September 2004
Published date: 11 February 2005
Keywords: C–C bond hydrolase, a/b hydrolase, non-nucleophilic serine, hemi-ketal adduct, crystal structure
Organisations: Biological Sciences

Identifiers

Local EPrints ID: 24091
URI: https://eprints.soton.ac.uk/id/eprint/24091
ISSN: 0022-2836
PURE UUID: 0808cfc2-4cd0-4861-88cc-babd49c0ec14

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Date deposited: 22 Mar 2006
Last modified: 17 Jul 2017 16:15

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Contributors

Author: G. Dunn
Author: M.G. Montgomery
Author: F. Mohammed
Author: A. Coker
Author: J.B. Cooper
Author: T. Robertson
Author: J.L. Garcia
Author: T.D.H. Bugg
Author: S.P. Wood

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