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 μM) compares well with those of the native bovine lung PDE5 (5.6 μM) and full- length wild type recombinant PDE5 (2 ± 0.4 μM). The catalytic fragment and full-length PDE5 have similar IC50 values for the inhibitors 3-isobutyl-1- methylxanthine (20 μM) 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.