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Hydrolysis of the non-canonical cyclic nucleotide cUMP by PDE9A: kinetics and binding mode

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Abstract

The non-canonical cyclic nucleotide cUMP and the phosphodiesterase PDE9A both occur in neuronal cells. Using HPLC-coupled tandem mass spectrometry, we characterized the kinetics of PDE9A-mediated cUMP hydrolysis. PDE9A is a low-affinity and high-velocity enzyme for cUMP (Vmax = ~ 6 μmol/min/mg; Km = ~ 401 μM). The PDE9 inhibitor BAY 73-6691 inhibited PDE9A-catalyzed cUMP hydrolysis (Ki = 590 nM). Docking studies indicate two H-bonds between the cUMP uridine moiety and Gln453/Asn405 of PDE9A. By contrast, the guanosine moiety of cGMP forms three H-bonds with Gln453. cCMP is not hydrolyzed at a concentration of 3 μM, but inhibits the PDE9A-catalyzed cUMP hydrolysis at concentrations of 100 μM or more. The probable main reason is that the cytosine moiety cannot act as H-bond acceptor for Gln453. A comparison of PDE9A with PDE7A suggests that the preference of the former for cGMP and cUMP and of the latter for cAMP and cCMP is due to stabilized alternative conformations of the side chain amide of Gln453 and Gln413, respectively. This so-called glutamine switch is known to be involved in the regulation of cAMP/cGMP selectivity of some PDEs.

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Abbreviations

BSA:

Bovine serum albumin

cAMP:

Adenosine 3′,5′-cyclic monophosphate

cCMP:

Cytidine 3′,5′-cyclic monophosphate

cGMP:

Guanosine 3′,5′-cyclic monophosphate

CMP:

Cytidine 5′-monophosphate

cNMP:

Nucleoside 3′,5′-cyclic monophosphate

cUMP:

Uridine 3′,5′-cyclic monophosphate

EDTA:

Ethylenediaminetetraacetic acid

ExoY:

Exotoxin of Pseudomonas aeruginosa with nucleotidyl cyclase properties

GMP:

Guanosine 5′-monophosphate

GST:

Glutathione S-transferase (used as protein tag)

HCN:

Hyperpolarization-activated cyclic nucleotide-gated channel

HPLC-MS/MS:

High-performance liquid chromatography-coupled tandem mass spectrometry

IBMX:

3-Isobutyl-1-methylxanthine

K m :

Michaelis-Menten constant

MRP:

Multidrug resistance-associated protein

NMP:

Nucleoside 5′-monophosphate

PDE:

Phosphodiesterase

PKA:

Protein kinase A

PKG:

Protein kinase G

sAC:

Soluble adenylyl cyclase

SD:

Standard deviation

SEM:

Standard error of the mean

sGC:

Soluble guanylyl cyclase

UMP:

Uridine 5′-monophosphate

V max :

Maximum velocity of an enzymatic reaction under saturating conditions

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Acknowledgements

We thank Prof. Dr. Martin Stangel (Dept. of Clinical Neuroimmunology and Neurochemistry, MHH) and Dr. Sabine Wolter (Institute of Pharmacology, MHH) for the excellent scientific discussions as well as Mrs. Annette Garbe (Research Core Unit Metabolomics, MHH) for the outstanding technical support.

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Authors and Affiliations

  1. Hannover Medical School, Institute of Pharmacology, Carl-Neuberg-Str. 1, 30625, Hannover, Germany

    Jessica Scharrenbroich, Roland Seifert & Erich H. Schneider

  2. Research Core Unit Metabolomics, Hannover Medical School, Institute of Pharmacology, Carl-Neuberg-Str. 1, 30625, Hannover, Germany

    Volkhard Kaever

  3. Department of Medicinal Chemistry II, Institute of Pharmacy, University of Regensburg, 93040, Regensburg, Germany

    Stefan Dove

Authors
  1. Jessica Scharrenbroich
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  2. Volkhard Kaever
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  3. Stefan Dove
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  4. Roland Seifert
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  5. Erich H. Schneider
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Contributions

Participated in research design: Schneider, Scharrenbroich, Seifert

Conducted experiments: Scharrenbroich, Kaever

Performed data analysis: Scharrenbroich, Schneider

Performed docking approaches: Dove

Wrote or contributed to the writing of the manuscript: Scharrenbroich, Schneider, Dove, Kaever, Seifert

Corresponding author

Correspondence to Erich H. Schneider.

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Scharrenbroich, J., Kaever, V., Dove, S. et al. Hydrolysis of the non-canonical cyclic nucleotide cUMP by PDE9A: kinetics and binding mode. Naunyn-Schmiedeberg's Arch Pharmacol 392, 199–208 (2019). https://doi.org/10.1007/s00210-018-1582-5

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  • DOI: https://doi.org/10.1007/s00210-018-1582-5

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