Role of the intrinsic metal in RNA polymerase from Escherichia coli. In vivo substitution of tightly bound zinc with cobalt

Speckhard, David C.; Wu, Felicia Y.-H.; Wu, Cheng‐Wen

doi:10.1021/bi00643a011

Cited by 60 publications

(54 citation statements)

References 29 publications

(26 reference statements)

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“…In vivo Cd-substituted aspartate transcarbamylase retains most of its activity in Zn-deficient E. coli cells (Speckhard et al 1977). Therefore, in E. huxleyi, Cd replacement for Zn or Co in the major Zn or Co metalloproteins probably has two opposite effects: a beneficial effect when the Cd form of the protein retains part of its activity; a toxic effect when the Cd form of the protein loses activity.…”

Section: Resultsmentioning

confidence: 99%

Zinc, cadmium, and cobalt interreplacement and relative use efficiencies in the coccolithophore Emiliania huxleyi

Tang

Shaked

et al. 2007

Limnology & Oceanography

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We investigate the interreplacement of Zn, Cd, and Co in the cosmopolitan coccolithophore Emiliania huxleyi by examining its growth rate, cellular metal quotas, and cellular metal uptake rates under different combinations of Zn, Cd, and Co concentrations in the medium. Co and Zn can fully replace each other, except perhaps for small individual requirements that are met by minute metal concentrations supplied as contaminants. Zn is used at 75% efficiency by E. huxleyi compared with Co. In contrast, Cd can only partially replace Zn or Co and is used at 66% efficiency compared with Co. Up to 50% of the cellular Zn or Co can be replaced by Cd, once the minimum cellular Zn or Co requirement is fulfilled. The limitation by Zn, Cd, and Co and the interreplacement of the three metals occurs over a range of unchelated metal concentrations, 0.2-5 pmol L 21 , that is relevant to surface oceanic waters. Based on our laboratory results and the published metal concentrations in surface oceanic waters, we calculate that interreplacement of Zn, Cd, and Co should occur in regions where Zn is highly depleted.

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Section: Resultsmentioning

confidence: 99%

Zinc, cadmium, and cobalt interreplacement and relative use efficiencies in the coccolithophore Emiliania huxleyi

Tang

Shaked

et al. 2007

Limnology & Oceanography

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“…Cobalt can substitute for Zn in RNA polymerase without a dramatic loss in functionality, as shown in ref. 46. Structural proteins like Zn-β ribbons and Zn fingers also bind Co, although the DNA binding activity of a Co-bound Zn finger is much less efficient relative to the Zn-bound form (47).…”

Section: Emergence Of the Akaryotes Metal Homeostasis And Biogeochementioning

confidence: 99%

History of biological metal utilization inferred through phylogenomic analysis of protein structures

Dupont

Butcher

Valas³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

275

248

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The fundamental chemistry of trace elements dictates the molecular speciation and reactivity both within cells and the environment at large. Using protein structure and comparative genomics, we elucidate several major influences this chemistry has had upon biology. All of life exhibits the same proteome size-dependent scaling for the number of metal-binding proteins within a proteome. This fundamental evolutionary constant shows that the selection of one element occurs at the exclusion of another, with the eschewal of Fe for Zn and Ca being a defining feature of eukaryotic proteomes. Early life lacked both the structures required to control intracellular metal concentrations and the metal-binding proteins that catalyze electron transport and redox transformations. The development of protein structures for metal homeostasis coincided with the emergence of metal-specific structures, which predominantly bound metals abundant in the Archean ocean. Potentially, this promoted the diversification of emerging lineages of Archaea and Bacteria through the establishment of biogeochemical cycles. In contrast, structures binding Cu and Zn evolved much later, providing further evidence that environmental availability influenced the selection of the elements. The late evolving Zn-binding proteins are fundamental to eukaryotic cellular biology, and Zn bioavailability may have been a limiting factor in eukaryotic evolution. The results presented here provide an evolutionary timeline based on genomic characteristics, and key hypotheses can be tested by alternative geochemical methods.Archean-Proterozoic | biogeochemistry | bioinorganic chemistry | evolution | metal homeostasis M etalloproteins contain one or more ions of an inorganic element in their 3D structure and are said to comprise 30% of all proteins (1). Many biological pathways contain at least one metalloenzyme (2) and consequently require Mg, K, Ca, Fe, Mn, and Zn to sustain life (3). Other elements, like Cu, Mo, Ni, Se, and Co, are required by many-though not all-organismal lineages, and the utilization of trace elements varies greatly between species (3). The different elements have a range of affinities for most coordinating environments in the order Mg +2 /Ca +2 < Mn +2 < Fe +2 < Co +2 < Ni +2 < Cu +2 ∼Zn +2 , an equilibrium series known as the Irving-Williams Series (4). This array of outer-sphere chemistry provides significant catalytic diversity, yet has consequences for both biological and environmental chemistry. Within the cell, metals compete for protein binding sites; hence, extensive protein networks involving transporters and metalsensing regulatory proteins are required to maintain the proper subcellular concentrations of each element, and in some cases directly shuttle each metal to its requisite metalloprotein in the proper cellular compartment (1, 5, 6). It has been hypothesized that the establishment of a metal homeostasis system is required for distinct phylotypes of cells to develop (7).Environmentally, for similar fundamental chemical reasons, the...

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“…Previously, we replaced the two intrinsic Zn ions in E. coli RPase in vivo with Co(I1) by growing the bacteria in a Zndepleted Co-enriched medium (Speckhard et al, 1977). The enzyme purified from these cells contains 2 mol of Co/mol of enzyme with concomitant reduction in the Zn content.…”

mentioning

confidence: 99%

“…This was difficult for E. coli RPase because of the extremely tight binding of Zn to the enzyme. These Zn ions could neither be removed from RPase by prolonged dialysis against metal chelators nor be exchanged with other divalent metal ions present in dialysis solutions (Speckhard et al, 1977). In this paper, we describe a simple denaturation-reconstitution method to substitute selectively one of the two Zn ions in E. coli RPase, which is located in the / 3 subunit.…”

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confidence: 99%

Selective substitution in vitro of an intrinsic zinc of Escherichia coli RNA polymerase with various divalent metals

Chatterji¹,

Wu²

1982

Biochemistry

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A simple in vitro substitution method involving a sequential denaturation--reconstitution process was developed to substitute selectively one of the two intrinsic Zn ions in Escherichia coli RNA polymerase with Co, Mn, Ni, or Cu ion. The resultant metal hybrid Co-Zn, Mn-Zn, Ni-Zn, and Cu-Zn RNA polymerases possess 100, 100, 60, and 17% of the enzymatic activity of the reconstituted Zn-Zn enzyme, respectively. The substituted metal was found to be located in the beta subunit of the polymerase which contains the substrate binding site. The biochemical and physical properties of these metal-substituted polymerases were compared with those of the native Zn enzyme. Co-Zn and Ni-Zn core polymerases exhibit characteristic absorption spectra in the near-UV and visible region, while Mn-Zn and Cu-Zn enzymes do not. The Co-Zn enzyme shows two major peaks at 400 nm (epsilon = 3000) and 475 nm (epsilon = 2700), while the Ni-Zn enzyme exhibits a major peak at 462 nm (epsilon = 8000). The difference absorption spectrum of Ni-Zn core polymerase could be perturbed by the addition of substrate ATP but not by UTP in the absence of template and Mg(II) ion. These observations suggest that the substituted metal was located at the initiation site of the enzyme. The various metal hybrid enzymes do not differ appreciably in their abilities to incorporate noncomplementary nucleotide or deoxyribonucleotide into RNA product. It was found, however, that the difference in enzymatic activities of these metal hybrid enzymes resides at least partly in the initiation step of RNA synthesis.

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Role of the intrinsic metal in RNA polymerase from Escherichia coli. In vivo substitution of tightly bound zinc with cobalt

Cited by 60 publications

References 29 publications

Zinc, cadmium, and cobalt interreplacement and relative use efficiencies in the coccolithophore Emiliania huxleyi

Zinc, cadmium, and cobalt interreplacement and relative use efficiencies in the coccolithophore Emiliania huxleyi

History of biological metal utilization inferred through phylogenomic analysis of protein structures

Selective substitution in vitro of an intrinsic zinc of Escherichia coli RNA polymerase with various divalent metals

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