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MAD analyses of yeast 5-aminolaevulinate dehydratase: their use in structure determination and in defining the metal-binding sites

MAD analyses of yeast 5-aminolaevulinate dehydratase: their use in structure determination and in defining the metal-binding sites
MAD analyses of yeast 5-aminolaevulinate dehydratase: their use in structure determination and in defining the metal-binding sites
MAD experiments attempting to solve the structure of 5--aminolaevulinic acid dehydratase using Zn and Pb edges are described. The data obtained proved insufficient for a complete structure solution but were invaluable in subsequent identification of metal-binding sites using anomalous difference Fourier analyses once the structure of the enzyme had been solved. These sites include the highly inhibitory substitution of an enzymic cofactor Zn(2+) ion by Pb(2+) ions, which represents a major contribution towards understanding the molecular basis of lead poisoning. The MAD data collected at the Pb edge were also used with isomorphous replacement data from the same Pb co-crystal and a Hg co-crystal to provide the first delineation of the enzyme's quaternary structure. In this MADIR analysis, the Hg co-crystal data were treated as native data. Anomalous difference Fouriers were again used, revealing that Hg(2+) had substituted for the same Zn(2+) cofactor ion as had Pb(2+), a finding of fundamental importance for the understanding of mercury poisoning. In addition, Pt(2+) ions were found to bind at the same place in the structure. The refined structures of the Pb- and the Hg-complexed enzymes are presented at 2.5 and 3.0 A resolution, respectively.
0907-4449
421-430
Erskine, P.T.
c77b60c5-b80c-4e6a-a103-bf57ecfcbcf6
Duke, E.M.H.
413ba196-b811-48f8-899e-7cd43e0f34db
Tickle, I.J.
7d0714ba-37d0-4b7d-8680-8a4b89ea0a37
Senior, N.M.
2686c820-bc36-4f5f-95f5-bf3f309e6676
Warren, M.J.
6dc30bb6-c290-490e-8aab-44ad27c7c609
Cooper, J.B.
d9f0f6a8-1260-48fc-aa5c-3dbc650e3ec0
Erskine, P.T.
c77b60c5-b80c-4e6a-a103-bf57ecfcbcf6
Duke, E.M.H.
413ba196-b811-48f8-899e-7cd43e0f34db
Tickle, I.J.
7d0714ba-37d0-4b7d-8680-8a4b89ea0a37
Senior, N.M.
2686c820-bc36-4f5f-95f5-bf3f309e6676
Warren, M.J.
6dc30bb6-c290-490e-8aab-44ad27c7c609
Cooper, J.B.
d9f0f6a8-1260-48fc-aa5c-3dbc650e3ec0

Erskine, P.T., Duke, E.M.H., Tickle, I.J., Senior, N.M., Warren, M.J. and Cooper, J.B. (2000) MAD analyses of yeast 5-aminolaevulinate dehydratase: their use in structure determination and in defining the metal-binding sites. Acta Crystallographica Section D: Biological Crystallography, 56 (4), 421-430. (doi:10.1107/S0907444900000597).

Record type: Article

Abstract

MAD experiments attempting to solve the structure of 5--aminolaevulinic acid dehydratase using Zn and Pb edges are described. The data obtained proved insufficient for a complete structure solution but were invaluable in subsequent identification of metal-binding sites using anomalous difference Fourier analyses once the structure of the enzyme had been solved. These sites include the highly inhibitory substitution of an enzymic cofactor Zn(2+) ion by Pb(2+) ions, which represents a major contribution towards understanding the molecular basis of lead poisoning. The MAD data collected at the Pb edge were also used with isomorphous replacement data from the same Pb co-crystal and a Hg co-crystal to provide the first delineation of the enzyme's quaternary structure. In this MADIR analysis, the Hg co-crystal data were treated as native data. Anomalous difference Fouriers were again used, revealing that Hg(2+) had substituted for the same Zn(2+) cofactor ion as had Pb(2+), a finding of fundamental importance for the understanding of mercury poisoning. In addition, Pt(2+) ions were found to bind at the same place in the structure. The refined structures of the Pb- and the Hg-complexed enzymes are presented at 2.5 and 3.0 A resolution, respectively.

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Published date: 1 April 2000

Identifiers

Local EPrints ID: 56696
URI: https://eprints.soton.ac.uk/id/eprint/56696
ISSN: 0907-4449
PURE UUID: d529c4ed-491c-45f6-9561-a422accaa220

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Date deposited: 06 Aug 2008
Last modified: 13 Mar 2019 20:34

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Contributors

Author: P.T. Erskine
Author: E.M.H. Duke
Author: I.J. Tickle
Author: N.M. Senior
Author: M.J. Warren
Author: J.B. Cooper

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