Crystal structure of the guanylyl cyclase Cya2.
Cyclic GMP (cGMP) is an important second messenger in eukaryotes. It is formed by guanylyl cyclases (GCs), members of the nucleotidyl cyclases class III, which also comprises adenylyl cyclases (ACs) from most organisms. To date, no structures of eukaryotic GCs are available, and all bacterial class III proteins were found to be ACs. Here we describe the biochemical and structural characterization of the class III cyclase Cya2 from cyanobacterium Synechocystis PCC6803. Cya2 shows high specificity for GTP versus ATP, revealing it to be the first bacterial GC, and sequence similarity searches indicate that GCs are also present in other bacteria. The crystal structure of Cya2 provides first structural insights into the universal GC family. Structure and mutagenesis studies show that a conserved glutamate, assisted by an interacting lysine, dominates substrate selection by forming hydrogen bonds to the substrate base. We find, however, that a second residue involved in substrate selection has an unexpected sterical role in GCs, different from its hydrogen bonding function in the related ACs. The structure identifies a tyrosine that lines the guanine binding pocket as additional residue contributing to substrate specificity. Furthermore, we find that substrate specificity stems from faster turnover of GTP, rather than different affinities for GTP and ATP, implying that the specificity-determining interactions are established after the binding step.
Rauch
Annika
A
Department of Physiological Chemistry, Ruhr-University Bochum, Universitaetsstrasse 150, 44801 Bochum, Germany.
Leipelt
Martina
M
Russwurm
Michael
M
Steegborn
Clemens
C
D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry*; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D000458:Cyanobacteria; D018698:Glutamic Acid; D006160:Guanosine Triphosphate / Q000378:metabolism; D006162:Guanylate Cyclase / Q000737:chemistry*; D006860:Hydrogen Bonding; D011485:Protein Binding; D013379:Substrate Specificity; D046939:Synechocystis / Q000737:chemistry*
D001426:Bacterial Proteins; D018698:Glutamic Acid; D006160:Guanosine Triphosphate; D000255:Adenosine Triphosphate; D006162:Guanylate Cyclase