Isolation and characterization of PDE9A, a novel human cGMP-specific phosphodiesterase

Abstract

We have cloned and characterized the first human isozyme in a new family of cyclic nucleotide phosphodiesterases, PDE9A. By sequence homology in the catalytic domain, PDE9A is almost equidistant from all eight known mammalian PDE families but is most similar to PDESA (34% amino acid identity) and least like PDE5A (28% amino acid identity). We report the cloning of human cDNA encoding a full-length protein of 593 amino acids, including a 261-amino acid region located near the C terminus that is homologous to the ~270-amino acid catalytic domain of other PDEs. PDE9A is expressed in all eight tissues examined as a ~2.0-kilobase mRNA, with highest levels in spleen, small intestine, and brain. The full-length PDE9A was expressed in baculovirus fused to an N-terminal 9-amine acid FLAG tag. Kinetic analysis of the baculovirus-expressed enzyme shows it to be a very high affinity cGMP- specific PDE with a K(m) of 170 nM for cGMP and 230 μ for cAMP. The K(m) for cGMP makes PDE9A one of the highest affinity PDEs known. The V(max) for cGMP (4.9 nmol/min/μg recombinant enzyme) is about twice as fast as that of PDE4 for cAMP. The enzyme is about twice as active in vitro in 1-10 mM Mn2+ than in the same concentration of Mg2+ or Ca2+. PDE9A is insensitive (up to 100 μM) to a variety of PDE inhibitors including rolipram, vinpocetine, SKF- 94120, dipyridamole, and 3-isobutyl-1-methyl-xanthine but is inhibited (IC50 = 35 μM) by zaprinast, a PDE5 inhibitor. PDE9A lacks a region homologous to the allosteric cGMP-binding regulatory regions found in the cGMP-binding PDEs: PDE2, PDES, and PDE6.