Abstract
Chimeric cGMP phosphodiesterases (PDEs) have been constructed using components of the cGMP-binding PDE (PDE5) and cone photoreceptor phosphodiesterase (PDE6alpha') in order to study structure and function of the photoreceptor enzyme. A fully functional chimeric PDE6alpha'/PDE5 enzyme containing the PDE6alpha' noncatalytic cGMP-binding sites, and the PDE5 catalytic domain has been efficiently expressed in the baculovirus/High Five cell system. The catalytic properties of this chimera were practically indistinguishable from those of PDE5, whereas the noncatalytic cGMP binding was similar to that of native purified PDE6alpha'. The inhibitory gamma subunit of PDE6 (Pgamma) enhanced the affinity of cGMP binding at noncatalytic sites of native PDE6alpha' by approximately 6-fold. The polycationic region of Pgamma, Pgamma-24-45, was mainly responsible for this effect, while the inhibitory domain of Pgamma, Pgamma-63-87, was ineffective. On the contrary, Pgamma failed to inhibit catalytic activity of the chimeric PDE6alpha'/PDE5 or to modulate its noncatalytic cGMP binding. Substitutions of Ala residues for the conserved Asn, Asn193 or Asn402, in the two N(K/R)XD-like motifs of the chimeric PDE noncatalytic cGMP-binding sites, each led to a loss of the noncatalytic cGMP binding. Our data suggest that both putative noncatalytic sites of PDE6alpha' are important for binding of cGMP, and that the two binding sites are coupled. Furthermore, mutation Asn402 --> Ala resulted in an approximately 10-fold increase of the Km value for cGMP, indicating that occupation of the noncatalytic cGMP- binding sites of PDE6alpha' may regulate catalytic properties of the enzyme.
Highlights
Substitutions of Ala residues for the conserved Asn, Asn193 or Asn402, in the two N(K/R)XD-like motifs of the chimeric PDE noncatalytic cGMP-binding sites, each led to a loss of the noncatalytic cGMP binding
Expression of PDE6␣Ј and PDE6␣Ј/PDE5 Chimeras in the baculovirus/High Five Cell System—The native PDE6␣Ј is known to form a catalytic homodimer, so it was thought that the baculovirus-expressed protein might be more stable and active than would the rod PDE6␣ and  co-expressed in the same system [21]
PDE5 is similar to PDE6␣Ј with respect to formation of a homodimer, and the PDE5 has been successfully expressed in the baculovirus/insect cell system [22]
Summary
Substitutions of Ala residues for the conserved Asn, Asn193 or Asn402, in the two N(K/R)XD-like motifs of the chimeric PDE noncatalytic cGMP-binding sites, each led to a loss of the noncatalytic cGMP binding. A fully functional chimeric PDE6␣/PDE5 enzyme containing the PDE6␣ noncatalytic cGMP-binding sites, and the PDE5 catalytic domain has been efficiently expressed in the baculovirus/ High Five cell system. The catalytic properties of this chimera were practically indistinguishable from those of PDE5, whereas the noncatalytic cGMP binding was similar to that of native purified PDE6␣.
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