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The YHS-Domain of an Adenylyl Cyclase from Mycobacterium phlei Is a Probable Copper-Sensor Module.

Linder JU - PLoS ONE (2015)

Bottom Line: They are found in a variety of enzymes including copper-transporting ATPases and adenylyl cyclases.With Mg2+ as the probable physiological cofactor of the adenylyl cyclase Cu2+ specifically switches the enzyme from an inactive to an active state.By analogy to TRASH-domains binding of Cu2+ probably occurs via one conserved aspartate and three conserved cysteine-residues in the YHS-domain.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Bayreuth, Bayreuth, Germany.

ABSTRACT
YHS-domains are small protein modules which have been proposed to bind transition-metal ions like the related TRASH-domains. They are found in a variety of enzymes including copper-transporting ATPases and adenylyl cyclases. Here we investigate a class IIIc adenylyl cyclase from Mycobacterium phlei which contains a C-terminal YHS-domain linked to the catalytic domain by a peptide of 8 amino acids. We expressed the isolated catalytic domain and the full-length enzyme in E. coli. The catalytic domain requires millimolar Mn2+ as a cofactor for efficient production of cAMP, is unaffected by low micromolar concentrations of Cu2+ and inhibited by concentrations higher than 10 μM. The full-length enzyme also requires Mn2+ in the absence of an activator. However, 1-10 μM Cu2+ stimulate the M. phlei adenylyl cyclase sixfold when assayed with Mn2+. With Mg2+ as the probable physiological cofactor of the adenylyl cyclase Cu2+ specifically switches the enzyme from an inactive to an active state. Other transition-metal ions do not elicit activity with Mg2+. We favor the view that the YHS-domain of M. phlei adenylyl cyclase acts as a sensor for copper ions and signals elevated levels of the transition-metal via cAMP. By analogy to TRASH-domains binding of Cu2+ probably occurs via one conserved aspartate and three conserved cysteine-residues in the YHS-domain.

No MeSH data available.


Related in: MedlinePlus

Stimulation of full-length MpAC1-233 by Cu2+ assayed with Mn2+ as a cofactor.(A) Effect of Cu2+. Reactions contained 0.4 μM MpAC1-233, 5 mM Mn2+ and 1 mM ATP. (B) Substrate kinetics measured with 5 mM Mn2+ as a cofactor and 10 μM Cu2+. (C) Mn2+-dependance assayed at 1 mM ATP and 10 μM Cu2+.
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pone.0141843.g004: Stimulation of full-length MpAC1-233 by Cu2+ assayed with Mn2+ as a cofactor.(A) Effect of Cu2+. Reactions contained 0.4 μM MpAC1-233, 5 mM Mn2+ and 1 mM ATP. (B) Substrate kinetics measured with 5 mM Mn2+ as a cofactor and 10 μM Cu2+. (C) Mn2+-dependance assayed at 1 mM ATP and 10 μM Cu2+.

Mentions: The hypothesis of the YHS-domain of M. phlei AC being a transition-metal ion binding domain with similarity to the YHS-domains of CuATPases was tested in assays of the untagged full-length MpAC1-233 with micromolar concentrations of Cu2+ and Mn2+ as a cofactor (Fig 4A). Cu2+ maximally stimulated MpAC1-233 sixfold at concentrations of 1 to 10 μM. Because we used an enzyme concentrations of 0.4 μM to assure efficient dimerization of MpAC1-233 (see below), assays with Cu2+ below 1 μM were not conducted. Yet, the data imply that the EC50 for activation by Cu2+ is in the nanomolar range. On the other hand, high concentrations of Cu2+ were inhibitory similar to the results seen with the isolated catalytic domain. Thus, we concluded that the stimulatory effect of low concentrations of Cu2+ on MpAC1-233 is mediated by the YHS-domain. The inhibitory effect of higher concentrations of Cu2+ may be due to general interactions and reactions with protein similar to those exploited in classic protein assays like the Lowry- and the Biuret-method [21]. Kinetic analysis of MpAC1-233 showed that 10 μM Cu2+ led to an increase in vmax and a concomitant decrease of SC50 for ATP (Fig 4B, Table 1). The Hill coefficient of 1.2 ± 0.3 indicated that Cu2+ did not induce pronounced cooperativity for ATP. Furthermore, addition of 10μM Cu2+ resulted in a 10-fold higher affinity for the cofactor Mn2+ with an EC50 of 0.7 ± 0.2 mM for the free ion (Fig 4C). Taken together Cu2+ appears to increase the catalytic efficiency of the enzyme.


The YHS-Domain of an Adenylyl Cyclase from Mycobacterium phlei Is a Probable Copper-Sensor Module.

Linder JU - PLoS ONE (2015)

Stimulation of full-length MpAC1-233 by Cu2+ assayed with Mn2+ as a cofactor.(A) Effect of Cu2+. Reactions contained 0.4 μM MpAC1-233, 5 mM Mn2+ and 1 mM ATP. (B) Substrate kinetics measured with 5 mM Mn2+ as a cofactor and 10 μM Cu2+. (C) Mn2+-dependance assayed at 1 mM ATP and 10 μM Cu2+.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4626032&req=5

pone.0141843.g004: Stimulation of full-length MpAC1-233 by Cu2+ assayed with Mn2+ as a cofactor.(A) Effect of Cu2+. Reactions contained 0.4 μM MpAC1-233, 5 mM Mn2+ and 1 mM ATP. (B) Substrate kinetics measured with 5 mM Mn2+ as a cofactor and 10 μM Cu2+. (C) Mn2+-dependance assayed at 1 mM ATP and 10 μM Cu2+.
Mentions: The hypothesis of the YHS-domain of M. phlei AC being a transition-metal ion binding domain with similarity to the YHS-domains of CuATPases was tested in assays of the untagged full-length MpAC1-233 with micromolar concentrations of Cu2+ and Mn2+ as a cofactor (Fig 4A). Cu2+ maximally stimulated MpAC1-233 sixfold at concentrations of 1 to 10 μM. Because we used an enzyme concentrations of 0.4 μM to assure efficient dimerization of MpAC1-233 (see below), assays with Cu2+ below 1 μM were not conducted. Yet, the data imply that the EC50 for activation by Cu2+ is in the nanomolar range. On the other hand, high concentrations of Cu2+ were inhibitory similar to the results seen with the isolated catalytic domain. Thus, we concluded that the stimulatory effect of low concentrations of Cu2+ on MpAC1-233 is mediated by the YHS-domain. The inhibitory effect of higher concentrations of Cu2+ may be due to general interactions and reactions with protein similar to those exploited in classic protein assays like the Lowry- and the Biuret-method [21]. Kinetic analysis of MpAC1-233 showed that 10 μM Cu2+ led to an increase in vmax and a concomitant decrease of SC50 for ATP (Fig 4B, Table 1). The Hill coefficient of 1.2 ± 0.3 indicated that Cu2+ did not induce pronounced cooperativity for ATP. Furthermore, addition of 10μM Cu2+ resulted in a 10-fold higher affinity for the cofactor Mn2+ with an EC50 of 0.7 ± 0.2 mM for the free ion (Fig 4C). Taken together Cu2+ appears to increase the catalytic efficiency of the enzyme.

Bottom Line: They are found in a variety of enzymes including copper-transporting ATPases and adenylyl cyclases.With Mg2+ as the probable physiological cofactor of the adenylyl cyclase Cu2+ specifically switches the enzyme from an inactive to an active state.By analogy to TRASH-domains binding of Cu2+ probably occurs via one conserved aspartate and three conserved cysteine-residues in the YHS-domain.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry, University of Bayreuth, Bayreuth, Germany.

ABSTRACT
YHS-domains are small protein modules which have been proposed to bind transition-metal ions like the related TRASH-domains. They are found in a variety of enzymes including copper-transporting ATPases and adenylyl cyclases. Here we investigate a class IIIc adenylyl cyclase from Mycobacterium phlei which contains a C-terminal YHS-domain linked to the catalytic domain by a peptide of 8 amino acids. We expressed the isolated catalytic domain and the full-length enzyme in E. coli. The catalytic domain requires millimolar Mn2+ as a cofactor for efficient production of cAMP, is unaffected by low micromolar concentrations of Cu2+ and inhibited by concentrations higher than 10 μM. The full-length enzyme also requires Mn2+ in the absence of an activator. However, 1-10 μM Cu2+ stimulate the M. phlei adenylyl cyclase sixfold when assayed with Mn2+. With Mg2+ as the probable physiological cofactor of the adenylyl cyclase Cu2+ specifically switches the enzyme from an inactive to an active state. Other transition-metal ions do not elicit activity with Mg2+. We favor the view that the YHS-domain of M. phlei adenylyl cyclase acts as a sensor for copper ions and signals elevated levels of the transition-metal via cAMP. By analogy to TRASH-domains binding of Cu2+ probably occurs via one conserved aspartate and three conserved cysteine-residues in the YHS-domain.

No MeSH data available.


Related in: MedlinePlus