Expression of an Active, Monomeric Catalytic Domain of the cGMP-binding cGMP-specific Phosphodiesterase (PDE5)

Abstract

Phosphodiesterases (PDEs) comprise a superfamily of phosphohydrolases that degrade 3,5-cyclic nucleotides. All known mammalian PDEs are dimeric, but the functional significance of dimerization is unknown. A deletion mutant of cGMP-binding cGMP-specific PDE (PDE5), encoding the 357 carboxyl-terminal amino acids including the catalytic domain, has been generated, expressed, and purified. The K m of the catalytic fragment for cGMP (5.5 ؎ 0.51 M) compares well with those of the native bovine lung PDE5 (5.6 M) and full-length wild type recombinant PDE5 (2 ؎ 0.4 M). The catalytic fragment and full-length PDE5 have similar IC 50 values for the inhibitors 3-isobutyl-1-methylxanthine (20 M) and sildenafil (Viagra TM )(4 nM). Based on measured values for Stokes radius (29 Å) and sedimentation coefficient (2.9 S), the PDE5 catalytic fragment has a calculated molecular mass of 35 kDa, which agrees well with that predicted by amino acid content (43.3 kDa) and with that estimated using SDS-polyacrylamide gel electrophoresis (39 kDa). The combined data indicate that the recombinant PDE5 catalytic fragment is monomeric, and retains the essential catalytic features of the dimeric, full-length enzyme. Therefore, the catalytic activity of PDE5 holoenzyme requires neither interaction between the catalytic and regulatory domains nor interactions between subunits of the dimer.Intracellular levels of cyclic nucleotide are determined both by their rates of formation by adenylyl cyclases and guanylyl cyclases and their rates of breakdown by cyclic nucleotide phosphodiesterases (PDEs) 1 (1). Both the cyclases and the PDEs are highly regulated (2-8). Mammalian PDEs form a superfamily of enzymes comprised of 10 families of PDEs which differ in amino acid sequences (2, 3, 9 -11), substrate specificities, inhibitor sensitivities, modes of regulation, and tissue distribution. PDEs are chimeric proteins (12-15) which contain specific domains that provide for cyclic nucleotide hydrolysis, interaction with allosteric/regulatory molecules, autoinhibition, subcellular localization, and perhaps for other functions as well (12,16,17). For mammalian PDEs, the regulatory domains appear to reside mainly in the amino-terminal portions of the proteins. The carboxyl-terminal portion of each PDE contains a highly conserved region of ϳ250 amino acids (4), which comprises the catalytic domain.The cGMP-binding cGMP-specific PDE (PDE5 or cG-BPDE), which is the focus of this study, is abundant in lung, platelets, and vascular smooth muscle (18 -23). It is potently inhibited by sildenafil (Viagra TM ), which has proven to be therapeutically efficacious in the treatment of male erectile dysfunction (24). The PDE5 is highly specific for cGMP, both in its catalytic site, and in the two cGMP-binding allosteric sites within the aminoterminal half of the protein (14, 25). The regulation of the PDE5 catalytic activity is not well understood, although evidence has been presented that catalytic function is influenced by elements within the regulatory domain (26,27). Cycl...

Crystal Structures of Phosphodiesterases 4 and 5 in Complex with Inhibitor 3-Isobutyl-1-methylxanthine Suggest a Conformation Determinant of Inhibitor Selectivity

“…The crystal of PDE5A1 contains one catalytic domain in the crystallographic asymmetric unit, which apparently exists as a monomer. This is consistent with the biochemical studies that the monomeric fragment of bovine PDE5A (residues 508 -865) has similar catalytic activity as the full-length PDE5A (36). The electron density showed that residue 778 of PDE5A1 is better modeled with leucine instead of isoleucine in the wild type PDE5A1 sequence.…”

“…A typical batch of purification yielded over 10 mg of PDE5A1 from a 2-liter cell culture. The fragment of human PDE5A1 expressed in E. coli had a catalytic activity of about 2 mol/min/mg, which is comparable with that of the protein expressed in a baculovirus system (36).…”

Structural and Functional Features in Human PDE5A1 Regulatory Domain That Provide for Allosteric cGMP Binding, Dimerization, and Regulation

“…The basis for this effect is not known, but a leucine zipper motif (Ile 312 -X 6 -Leu 318 -X 6 -Glu 324 -X 6 -Leu 331 -X 6 -Ile 337 -X 6 -Leu 344 -X 6 -Ile 351 ) located in the segment of sequence between the GAFs might contribute. Disruption of this motif in PDE5 holoenzyme by site-directed mutagenesis does not produce monomerization (39). However, the high affinity of dimerization imparted by either GAF a or GAF b might mask a weakened interaction in this motif.…”

Mammalian Cyclic Nucleotide Phosphodiesterases: Molecular Mechanisms and Physiological Functions

“…Isolated C domains of PDEs are catalytically active, and some are monomers (103, 161-163, 168, 183, 184, 202, 327, 365, 398, 417, 418). In some cases, their kinetic char-acteristics are indistinguishable from those of the holoenzyme, whereas in other instances there are differences (40,103,297,298). Based on the x-ray crystal structure for a near full-length PDE2, Pandit et al (287) have proposed that autoinhibition is provided by physical constraints imposed by dimer contacts in the C domains and that activation occurs by disruption of that interface (Fig.…”