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Probing the role of copper in the biosynthesis of the molybdenum cofactor in Escherichia coli and Rhodobacter sphaeroides

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Abstract

The crystal structure of Cnx1G, an enzyme involved in the biosynthesis of the molybdenum cofactor (Moco) in Arabidopsis thaliana, revealed the remarkable feature of a copper ion bound to the dithiolene unit of a molybdopterin intermediate (Kuper et al. Nature 430:803–806, 2004). To characterize further the role of copper in Moco biosynthesis, we examined the in vivo and/or in vitro activity of two Moco-dependent enzymes, dimethyl sulfoxide reductase (DMSOR) and nitrate reductase (NR), from cells grown under a variety of copper conditions. We found the activities of DMSOR and NR were not affected when copper was depleted from the media of either Escherichia coli or Rhodobacter sphaeroides. These data suggest that while copper may be utilized during Moco biosynthesis when it is available, copper does not appear to be strictly required for Moco biosynthesis in these two organisms.

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Notes

  1. Briefly, Kuper et al. purified Cnx1E from A. thaliana with its MPT-AMP intermediate and incubated it with MgCl2 and Na2MoO4 in either the presence or the absence of CuCl2. They then used the nit-1 assay (the in vitro activity of NR from Neurospora crassa [18]) as a probe to infer the ability of copper to inhibit the insertion of molybdenum into MPT to form Moco.

  2. Optical densities were obtained at the end of the growth period (Fig. S5). The OD600 value was essentially unaltered by the presence (standard Sistrom’s medium) or absence (treated with 100 μM BCS) of copper in the growth medium.

  3. The fact that ubiquinol oxidase and cytochrome c oxidase still had measurable (albeit very low) activities despite the extraordinary lengths we went to in order to remove it highlights the difficulty of truly removing trace copper.

Abbreviations

BCS:

Bathocuproine disulfonic acid

DMS:

Dimethyl sulfide

DMSO:

Dimethyl sulfoxide

DMSOR:

Dimethyl sulfoxide reductase

GC:

Gas chromatography

ICP:

Inductively coupled plasma

LB:

Luria–Bertani

MDH:

Malate dehydrogenase

MGD:

Molybdopterin guanine dinucleotide

Moco:

Molybdenum cofactor

MPT:

Molybdopterin

MPT-AMP:

Adenylylated molybdopterin

NR:

Nitrate reductase

WT:

Wild-type

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Acknowledgements

We thank Robert Gennis (University of Illinois), Joel Weiner (University of Alberta), Samuel Kaplan, (University of Texas Medical School), and Jonathan Hosler (University of Mississippi Medical Center) for the bacterial strains and plasmids listed in the “Materials and methods.” We also thank Dennis Winge (University of Utah) and Matthew Sigman (University of Utah) for use of the ICP spectrophotometer and gas chromatograph system, respectively. Finally, we thank Ralf Mendel (Technical University of Braunschweig) for many helpful discussions and comments. Financial support was provided by the National Institutes of Health (grant GM66236) and by the Research Corporation (grant CS0890). E.L.H. is a Cotrell Scholar of Research Corporation

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Authors and Affiliations

  1. Department of Chemistry, University of Utah, 315 South 1400 East, Salt Lake City, UT, 84112, USA

    M. Scott Morrison

  2. Departments of Medicine and Biochemistry, University of Utah Health Science Center, Salt Lake City, UT, 84132, USA

    Paul A. Cobine

  3. Department of Biochemistry and Molecular Biology, Michigan State University, 510 Biochemistry Building, East Lansing, MI, 48824-1319, USA

    Eric L. Hegg

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  1. M. Scott Morrison
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Correspondence to Eric L. Hegg.

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Morrison, M.S., Cobine, P.A. & Hegg, E.L. Probing the role of copper in the biosynthesis of the molybdenum cofactor in Escherichia coli and Rhodobacter sphaeroides . J Biol Inorg Chem 12, 1129–1139 (2007). https://doi.org/10.1007/s00775-007-0279-x

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