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Active site of mercuric reductase resides at the subunit interface and requires Cys_(135) and Cys_(140) from one subunit and Cys_(558) and Cys_(559) from the adjacent subunit: evidence from in vivo and in vitro heterodimer formation

Distefano, Mark D. and Moore, Melissa J. and Walsh, Christopher T. (1990) Active site of mercuric reductase resides at the subunit interface and requires Cys_(135) and Cys_(140) from one subunit and Cys_(558) and Cys_(559) from the adjacent subunit: evidence from in vivo and in vitro heterodimer formation. Biochemistry, 29 (11). pp. 2703-2713. ISSN 0006-2960. https://resolver.caltech.edu/CaltechAUTHORS:20180212-134729330

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Abstract

Mercuric reductase catalyzes the two-electron reduction of Hg(I1) to Hg(0) using NADPH as the reductant; this reaction constitutes the molecular basis for detoxification of Hg(I1) by bacteria. The enzyme is an α_2 homodimer and possesses two pairs of cysteine residues, Cys_(135) and Cys_(140) (redox-active pair) and cys_(558) and Cys_(559) (C-terminal pair), which are known to be essential for catalysis. In the present study, we have obtained evidence for an intersubunit active site, consisting of a redox-active cysteine pair from one subunit and a C-terminal pair from the adjacent subunit, by reconstituting catalytic activity both in vivo and in vitro starting with two inactive, mutant enzymes, Ala_(l35)Ala_(l40)CyS_(558)CyS_(559) (AACC) and Cys_(135)Cys_(140)Ala_(558)(Ala_(559)(CCAA). Genetic complementation studies were used to show that coexpression of AACC and CCAA in the same cell yielded an Hg^R phenotype, some 10^4-fold more resistant than cells expressing only one mutant. Purification and catalytic characterization of a similarly coexpressed protein mixture showed the mixture to have activity levels ca. 25% those of wild type; this is the same as that statistically anticipated for a CCAA-AACC heterodimeric/homodimeric mixture with only one functional active site per heterodimer. Actual physical evidence for the formation of active mutant heterodimers was obtained by chaotrope-induced subunit interchange of inactive pure CCAA and AACC homodimers in vitro followed by electrophoretic separation of heterodimers from homodimers. Taken together, these data provide compelling evidence that the active site in mercuric reductase resides at the subunit interface and contains cysteine residues originating from separate polypeptide chains.


Item Type:Article
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http://dx.doi.org/10.1021/bi00463a013DOIArticle
https://pubs.acs.org/doi/10.1021/bi00463a013PublisherArticle
Additional Information:© 1990 American Chemical Society. Published in print 20 March 1990. This work was supported by NIH Grant GM 21643. We thank Dr. Anne Summers, University of Georgia, for advice with the in vivo efficiency of plating experiments and Dr. Stan Tabor for advice concerning the pGP1-4 vector.
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Funding AgencyGrant Number
NIHGM 21643
Issue or Number:11
Record Number:CaltechAUTHORS:20180212-134729330
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20180212-134729330
Official Citation:Active site of mercuric reductase resides at the subunit interface and requires Cys135 and Cys140 from one subunit and Cys558 and Cys559 from the adjacent subunit: evidence from in vivo and in vitro heterodimer formation Mark D. Distefano, Melissa J. Moore, and Christopher T. Walsh Biochemistry 1990 29 (11), 2703-2713 DOI: 10.1021/bi00463a013
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:84794
Collection:CaltechAUTHORS
Deposited By: Ruth Sustaita
Deposited On:13 Feb 2018 21:10
Last Modified:03 Oct 2019 19:22

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