Conjoined NO-sensitive guanylyl cyclases

Background

NO-sensitive guanylyl cyclases (NO-GC’s) that catalyze the reaction of GTP to the second messenger molecule cGMP are heterodimeric enzymes consisting of an α and a β₁ subunit. The two prevalent isoforms in humans (α₁/β₁, α₂/β₁) are time-tested targets for drugs that release nitric oxide (NO) and new compounds that either sensitize the enzyme for activation by NO or activate the enzyme independently of NO. Homologous NO-GC subunits consist of an amino-terminal HNOX domain followed by a PAS- and a coiled coil domain and the carboxy-terminal catalytic domain.

Results

Measurement of FRET between fluorescent proteins tagged to the amino- and carboxy-terminus of the subunits indicated close proximity between the amino-terminal HNOX domains and the carboxy-terminal catalytic domains of the enzyme. On the basis of these results we constructed conjoined NO-GC’s by fusion of the α amino-terminus to the β₁ carboxy-terminus leading to a monomeric enzyme complex. Surprisingly but in accordance with the FRET results these conjoined NO-GC’s (β₁α₁, β₁α₂) showed specific enzyme activity and stimulation by NO and various modulators of GC activity.

Conclusion

These obligate enzyme variants faithfully reproduced the pharmacological properties of the heterodimeric enzymes including an isoform specific differential activation of the NO-independent drug cinaciguat. Novel applications of conjoined NO-GC’s including adenoviral gene transfer will be discussed.