SlyD, A Ni(II) Metallochaperone for [NiFe]-hydrogenase Biosynthesis in Escherichia coli

SlyD is a protein involved in [NiFe]-hydrogenase enzyme maturation and, together with HypB and HypA proteins, contributes to the nickel delivery step. To understand the molecular details of this in vivo function, the nickel-binding activity of SlyD was investigated in vitro. SlyD is a monomeric pr...

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Main Author: Kaluarachchi, Harini
Other Authors: Zamble, Deborah
Language:en_ca
Published: 2011
Subjects:
Online Access:http://hdl.handle.net/1807/31797
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spelling ndltd-TORONTO-oai-tspace.library.utoronto.ca-1807-317972013-11-15T04:03:58ZSlyD, A Ni(II) Metallochaperone for [NiFe]-hydrogenase Biosynthesis in Escherichia coliKaluarachchi, HariniSlyDMetal homeostasis[NiFe]-hydrogenaseMetallochaperone0487SlyD is a protein involved in [NiFe]-hydrogenase enzyme maturation and, together with HypB and HypA proteins, contributes to the nickel delivery step. To understand the molecular details of this in vivo function, the nickel-binding activity of SlyD was investigated in vitro. SlyD is a monomeric protein that can chelate up to 7 nickel ions with an affinity in the sub-nanomolar range. By truncation and mutagenesis studies we show that the unique C-terminal metal-binding domain of this protein is required for Ni(II) binding and that the protein coordinates this metal non-cooperatively. This activity of SlyD supports the proposed in vivo role of SlyD in nickel homeostasis. In addition to nickel, SlyD can bind a variety of other types of transition metals. Therefore it was feasible that the protein contributes to homeostasis of metals other than nickel. To test this hypothesis, the metal selectivity of the protein was examined. The preference of SlyD for the metals examined could be ordered as follows, Mn(II), Fe(II) < Co(II) < Ni(II) ~ Zn(II) << Cu(I) indicating that the affinity of SlyD for the different metals follows the Irving-Williams series of metal-complex stabilities. Although the protein is unable to overcome the large thermodynamic preference in vitro for Cu(I) and exclude Zn(II) chelation, in vivo studies suggest a Ni(II)-specific function for the protein. To understand the function of SlyD as a metallochaperone, its interaction with HypB was investigated. This investigation revealed that SlyD plays a role in Ni(II) storage in E. coli and can function as a Ni(II)-donor to HypB. This study also revealed that SlyD can modulate the metal-binding as well as the GTPase activities of HypB. Based on the experimental data, a role for the HypB-SlyD complex in [NiFe]-hydrogenase biosynthesis is presented.Zamble, Deborah2011-112012-01-10T15:10:46ZNO_RESTRICTION2012-01-10T15:10:46Z2012-01-10Thesishttp://hdl.handle.net/1807/31797en_ca
collection NDLTD
language en_ca
sources NDLTD
topic SlyD
Metal homeostasis
[NiFe]-hydrogenase
Metallochaperone
0487
spellingShingle SlyD
Metal homeostasis
[NiFe]-hydrogenase
Metallochaperone
0487
Kaluarachchi, Harini
SlyD, A Ni(II) Metallochaperone for [NiFe]-hydrogenase Biosynthesis in Escherichia coli
description SlyD is a protein involved in [NiFe]-hydrogenase enzyme maturation and, together with HypB and HypA proteins, contributes to the nickel delivery step. To understand the molecular details of this in vivo function, the nickel-binding activity of SlyD was investigated in vitro. SlyD is a monomeric protein that can chelate up to 7 nickel ions with an affinity in the sub-nanomolar range. By truncation and mutagenesis studies we show that the unique C-terminal metal-binding domain of this protein is required for Ni(II) binding and that the protein coordinates this metal non-cooperatively. This activity of SlyD supports the proposed in vivo role of SlyD in nickel homeostasis. In addition to nickel, SlyD can bind a variety of other types of transition metals. Therefore it was feasible that the protein contributes to homeostasis of metals other than nickel. To test this hypothesis, the metal selectivity of the protein was examined. The preference of SlyD for the metals examined could be ordered as follows, Mn(II), Fe(II) < Co(II) < Ni(II) ~ Zn(II) << Cu(I) indicating that the affinity of SlyD for the different metals follows the Irving-Williams series of metal-complex stabilities. Although the protein is unable to overcome the large thermodynamic preference in vitro for Cu(I) and exclude Zn(II) chelation, in vivo studies suggest a Ni(II)-specific function for the protein. To understand the function of SlyD as a metallochaperone, its interaction with HypB was investigated. This investigation revealed that SlyD plays a role in Ni(II) storage in E. coli and can function as a Ni(II)-donor to HypB. This study also revealed that SlyD can modulate the metal-binding as well as the GTPase activities of HypB. Based on the experimental data, a role for the HypB-SlyD complex in [NiFe]-hydrogenase biosynthesis is presented.
author2 Zamble, Deborah
author_facet Zamble, Deborah
Kaluarachchi, Harini
author Kaluarachchi, Harini
author_sort Kaluarachchi, Harini
title SlyD, A Ni(II) Metallochaperone for [NiFe]-hydrogenase Biosynthesis in Escherichia coli
title_short SlyD, A Ni(II) Metallochaperone for [NiFe]-hydrogenase Biosynthesis in Escherichia coli
title_full SlyD, A Ni(II) Metallochaperone for [NiFe]-hydrogenase Biosynthesis in Escherichia coli
title_fullStr SlyD, A Ni(II) Metallochaperone for [NiFe]-hydrogenase Biosynthesis in Escherichia coli
title_full_unstemmed SlyD, A Ni(II) Metallochaperone for [NiFe]-hydrogenase Biosynthesis in Escherichia coli
title_sort slyd, a ni(ii) metallochaperone for [nife]-hydrogenase biosynthesis in escherichia coli
publishDate 2011
url http://hdl.handle.net/1807/31797
work_keys_str_mv AT kaluarachchiharini slydaniiimetallochaperonefornifehydrogenasebiosynthesisinescherichiacoli
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