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

Abstract

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.

Identification of the cAMP phosphodiesterase CpdA as novel key player in cAMP‐dependent regulation in Corynebacterium glutamicum

“…An ICP-MS analysis revealed the presence of 2.80 6 0.13 zinc atoms per molecule of purified CpdA, which is consistent with the fact that related PDEs of V. fischeri and S. cerevisiae contain zinc as well (Callahan et al, 1995;Tian et al, 2014). The purified CpdA was found to be in dimeric or tetrameric state as determined by size exclusion chromatography.…”

“…Based on amino acid sequence alignments and the presence of the signature motif the protein belongs to class II cAMP phosphodiesterases. These values are considerably higher compared with several other class II PDEs, such as S. cerevisiae PDE1 (110 mM) (Tian et al, 2014), CpdP of V. fischeri (73 mM) (Callahan et al, 1995) or CpdE of Myxococcus xanthus (12 mM) (Kimura et al, 2011). In the crystal structure of PDE1 of S. cerevisiae (Tian et al, 2014), the three histidine residues (H59, H61 and H64 in C. glutamicum CpdA) and the first aspartate residue (D63 in C. glutamicum CpdA) were shown to be involved in the coordination of two Zn 21 ions.…”

“…In contrast, CpdA Cg did not react with AMP representing a phosphomonoester. The PDE1 enzyme of S. cerevisiae also accepts both cAMP and cGMP as substrates (Tian et al, 2014). The phosphodiesterase PdeE of M. xanthus can degrade both cyclic nucleotides and phosphomonoesters such as AMP, although with lower activity (Kimura et al, 2011).…”

“…The motif was originally defined as HxHLDH (Richter, 2002), however, in C. glutamicum CpdA the corresponding sequence is HLHTDH and sequence alignments with CpdA Cg homologs of other Corynebacterium, Mycobacterium, Nocardia and Streptomyces species revealed an extended consensus motif with the sequence (F,L,I)(S,T)H(L,M)HXDH(C,T)XD. In the crystal structure of PDE1 of S. cerevisiae (Tian et al, 2014), the three histidine residues (H59, H61 and H64 in C. glutamicum CpdA) and the first aspartate residue (D63 in C. glutamicum CpdA) were shown to be involved in the coordination of two Zn 21 ions. Based on the detection of 2.8 zinc ions per CpdA Cg monomer a similar function for these residues in actinobacterial CpdA proteins appears likely.…”

“…The known PDEs working on cAMP have been assigned to three classes (Richter, 2002). The histidine and aspartate residues in this motif are involved in the coordination of metal ions which are bound in the catalytic centre of the protein (Tian et al, 2014). The second class covers PDEs of lower eukaryotes such as Saccharomyces cerevisiae (Ma et al, 1999), Candida albicans (Hoyer et al, 1994) and Dictyostelium discoideum (Lacombe et al, 1986) as well as PDEs of bacteria such as Vibrio fischeri (Dunlap and Callahan, 1993;Callahan et al, 1995) or Myxococcus xanthus (Kimura et al, 2011).…”