Purification of intact and nicked forms of a zinc-containing, Mg2+-dependent, low Km cyclic AMP phosphodiesterase from bakers' yeast.

Suoranta, K; Londesborough, John

doi:10.1016/s0021-9258(17)39823-x

Cited by 42 publications

(10 citation statements)

References 32 publications

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“…The substrate specificity of k cat / K M cAMP over k cat / K M cGMP is 1.32 × 10 5 (Table ), indicating that caPDE2 is highly specific for cAMP and does not hydrolyze cGMP under normal physiological conditions. Our K M of 35 nM is basically comparable to the values of 100–200 nM for bakers’ yeast PDE2 ,, but much smaller than the values of 1.5, 0.2, and 1.8 μM of cAMP-specific PDE4D2, PDE7A1, and PDE8A, respectively (Table ). , The explanation was not clear, but the replacement of the tyrosine (Tyr159 of PDE4D2), which is absolutely conserved in human PDEs, with Phe273 of caPDE2 would make tighter hydrophobic interactions with adenosine of cAMP (Figure ) and thus enhance cAMP binding.…”

Section: Resultssupporting

confidence: 69%

“…Both yPDE1 and yPDE2 hydrolyze cAMP and participate in cAMP signaling pathways. Yeast PDE2 was named as a high-affinity cAMP PDE because its K M values were in the range of 0.17–1.0 μM, − whereas yPDE1 has a low affinity with a K M value of 100–150 μM for cAMP. − In contrast with the well-characterized roles of cAMP in the physiological processes of yeast, the cGMP signaling pathway of yeasts has rarely been reported. C. albicans PDE1 was reported to have K M values of 250 and 490 μM and V max values of 0.044 and 1.17 μmol mg –1 min –1 for cGMP and cAMP, respectively, suggesting its inefficiency in the cGMP signaling pathway due to its extremely low V max .…”

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confidence: 99%

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Crystal Structures of Candida albicans Phosphodiesterase 2 and Implications for Its Biological Functions

et al. 2018

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The cAMP signaling system plays important roles in the physiological processes of pathogen yeast Candida albicans, but its functional mechanism has not been well illustrated. Here, we report the enzymatic characterization and crystal structures of C. albicans phosphodiesterase 2 (caPDE2) in the unliganded and 3-isobutyl-1-methylxanthine-complexed forms. caPDE2 is a monomer in liquid and crystal states and specifically hydrolyzes cAMP with a K of 35 nM. It does not effectively hydrolyze cGMP as shown by the 1.32 × 10-fold specificity of cAMP/cGMP. The crystal structure of caPDE2 shows significant differences from those of human PDEs. First, the N-terminal fragment of caPDE2 (residues 1-201) tightly associates with the catalytic domain to form a rigid molecular entity, implying its stable molecular conformation for C. albicans to resist environmental stresses. Second, the M-loop, a critical fragment for binding of the substrate and inhibitors to human PDEs, is not a part of the caPDE2 active site. This feature of caPDE2 may provide a structural basis for the design of selective inhibitors for the treatment of yeast infection.

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Section: Resultssupporting

confidence: 69%

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confidence: 99%

Crystal Structures of Candida albicans Phosphodiesterase 2 and Implications for Its Biological Functions

et al. 2018

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“…Reconstitution by Co 2+ and Ni 2+ has also been observed at structural Zn 2+ sites in metalloenzymes. However, in several of these cases, other metals such as Ca 2+ , Cu 2+ , or Fe 2+ are also capable of restoring significant activity to the enzyme ( , ). This is not the case with LpxC where incubation of the apoenzyme with Ca 2+ , Cu 2+ , Cd 2+ , or Mg 2+ does not stimulate deacetylase activity.…”

Section: Discussionmentioning

confidence: 99%

UDP-3-O-(R-3-Hydroxymyristoyl)-N-acetylglucosamine Deacetylase of Escherichia coli Is a Zinc Metalloenzyme

1999

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The enzyme UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc deacetylase (LpxC) catalyzes the committed step in the biosynthesis of lipid A and is therefore a potential antibiotic target. Inhibition of this enzyme by hydroxamate compounds [Onishi, H. R.; Pelak, B. A.; Gerckens, L. S.; Silver, L. L.; Kahan, F. M.; Chen, M. H.; Patchett, A. A.; Stachula, S. S.; Anderson, M. S.; Raetz, C. R. H. (1996) Science 274, 980-982] suggested the presence of a metal ion cofactor. We have investigated the substrate specificity and metal dependence of the deacetylase using spectroscopic and kinetic analyses. Comparison of the steady-state kinetic parameters for the physiological substrate UDP-3-O-(R-3-hydroxymyristoyl)-GlcNAc and an alternative substrate, UDP-GlcNAc, demonstrates that the ester-linked R-3-hydroxymyristoyl chain increases kcat/KM (5 x 10(6))-fold. Metal-chelating reagents, such as dipicolinic acid (DPA) and ethylenediaminetetraacetic acid, completely inhibit LpxC activity, implicating an essential metal ion. Plasma emission spectroscopy and colorimetric assays directly demonstrate that purified LpxC contains bound Zn2+. This Zn2+ can be removed by incubation with DPA, causing a decrease in the LpxC activity that can be restored by subsequent addition of Zn2+. However, high concentrations of Zn2+ also inhibit LpxC. Addition of Co2+, Ni2+, or Mn2+ to apo-LpxC also activates the enzyme to varying degrees while no additional activity is observed upon the addition of Cd2+, Ca2+, Mg2+, or Cu2+. This is consistent with the profile of metals that substitute for catalytic zinc ions in metalloproteinases. Co2+ ions stimulate LpxC activity maximally at a stoichiometry of 1:1. These data demonstrate that E. coli LpxC is a metalloenzyme that requires bound Zn2+ for optimal activity.

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“…The signaling of cAMP in yeast has been shown to play critical roles in physiological processes, including metabolism, cell wall biosynthesis, cell growth, and mating. − Overexpression of the PDE2 gene enhances the tolerance of yeast to oxidative and ethanol stress for survival. − Both yPDE1 and yPDE2 appear to participate in the cAMP signaling pathway, although yPDE2 shows high affinity with a K M of 0.17–1.0 μM for cAMP, − and yPDE1 has a low affinity with a K M of 100–150 μM. ,, In comparison with numerous publications about the roles of cAMP in the physiological processes of yeast, the cGMP signaling pathway has not been well studied, although it was reported that cGMP activated cAMP-dependent protein kinase of Pichia pastoris yeast . There is only one report showing that yPDE1 from Candida albicans has very weak cGMP activity with a K M of 250 μM and a V max of 0.044 μmol mg –1 min –1 , in comparison with a K M of 490 μM and a V max of 1.17 μmol mg –1 min –1 for cAMP .…”

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confidence: 99%

Dual Specificity and Novel Structural Folding of Yeast Phosphodiesterase-1 for Hydrolysis of Second Messengers Cyclic Adenosine and Guanosine 3′,5′-Monophosphate

et al. 2014

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Cyclic nucleotide phosphodiesterases (PDEs) decompose second messengers cAMP and cGMP that play critical roles in many physiological processes. PDE1 of Saccharomyces cerevisiae has been subcloned and expressed in Escherichia coli. Recombinant yPDE1 has a KM of 110 μM and a kcat of 16.9 s–1 for cAMP and a KM of 105 μM and a kcat of 11.8 s–1 for cGMP. Thus, the specificity constant (kcat/KMcAMP)/(kcat/KMcGMP) of 1.4 indicates a dual specificity of yPDE1 for hydrolysis of both cAMP and cGMP. The crystal structures of unliganded yPDE1 and its complex with GMP at 1.31 Å resolution reveal a new structural folding that is different from those of human PDEs but is partially similar to that of some other metalloenzymes such as metallo-β-lactamase. In spite of their different structures and divalent metals, yPDE1 and human PDEs may share a common mechanism for hydrolysis of cAMP and cGMP.

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Purification of intact and nicked forms of a zinc-containing, Mg2+-dependent, low Km cyclic AMP phosphodiesterase from bakers' yeast.

Cited by 42 publications

References 32 publications

Crystal Structures of Candida albicans Phosphodiesterase 2 and Implications for Its Biological Functions

Crystal Structures of Candida albicans Phosphodiesterase 2 and Implications for Its Biological Functions

UDP-3-O-(R-3-Hydroxymyristoyl)-N-acetylglucosamine Deacetylase of Escherichia coli Is a Zinc Metalloenzyme

Dual Specificity and Novel Structural Folding of Yeast Phosphodiesterase-1 for Hydrolysis of Second Messengers Cyclic Adenosine and Guanosine 3′,5′-Monophosphate

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