PDBsum entry: 2w01

Lyase

PDB id: 2w01

Contents

Protein chains

(+ 0 more) 201 a.a. *

Waters ×468

* Residue conservation analysis

PDB id:

2w01

Name:

Lyase

Title:

Crystal structure of the guanylyl cyclase cya2

Structure:

Adenylate cyclase. Chain: a, b, c, d, e, f. Fragment: catalytic domain, residues 434-635. Synonym: guanylyl cyclase cya2. Engineered: yes

Source:

Synechocystis sp.. Organism_taxid: 1148. Strain: pcc 6803. Expressed in: escherichia coli. Expression_system_taxid: 469008.

Resolution:

2.31Å R-factor: 0.198 R-free: 0.282

Authors:

A.Rauch,M.Leipelt,M.Russwurm,C.Steegborn

Key ref:

A.Rauch et al. (2008). Crystal structure of the guanylyl cyclase Cya2. Proc Natl Acad Sci U S A, 105, 15720-15725. PubMed id: 18840690 DOI: 10.1073/pnas.0808473105

Date:

08-Aug-08 Release date: 30-Sep-08

Protein chains

P72951  (P72951_SYNY3) -  Adenylate cyclase

Seq: 756 a.a.

Struc: 201 a.a.

 Gene Ontology (GO) functional annotation 

• Biological process: intracellular signal transduction (2 terms)
• Biochemical function: phosphorus-oxygen lyase activity (1 term)

DOI no: 10.1073/pnas.0808473105

Proc Natl Acad Sci U S A 105:15720-15725 (2008)

PubMed id: 18840690

Crystal structure of the guanylyl cyclase Cya2.

A.Rauch, M.Leipelt, M.Russwurm, C.Steegborn.

ABSTRACT

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.

Selected figure(s)

Figure 1.
Cyclase activities of the recombinant Cya2 catalytic domain. (A) Cyclase activity of Cya2 with 3 mM of either GTP or ATP as substrate nucleotide. (B) Substrate saturation curve for Cya2. GC activities assayed at various GTP concentrations were fitted assuming Michaelis–Menten kinetics, yielding a K[m] value of 0.11 mM and a V[max] of 1.8 nmol/(mg × min). (C) Inhibition of Cya2 activity, at a fixed GTP concentration of 3 mM, by increasing concentrations of ATP/Mg^2+. (D) Substrate saturation curve for Cya2 with ATP as substrate determined by using a RIA. The Michaelis–Menten fit yielded a K[m] of 0.10 mM for this substrate.

Figure 3.
Crystal structure of Cya2 catalytic domain. (A) Overall structure of the Cya2 homodimer. The two monomers are colored green and blue, and the secondary structure elements are labeled in one monomer. (B) Overlay of the Cya2 catalytic domain (green) with the AC enzyme CyaC (red). Secondary structure elements displaying differences between the two cyclases are labeled. (C) Active site of the Cya2 dimer. Residues mentioned in the text are labeled; residues supplied by the second monomer of the dimer are marked with an asterisk. (D) Cya2 active site with a modeled GTPαS ligand. The nucleotide was positioned in Cya2 after superposition with the structure of a CyaC/ATPαS complex; the nucleotide base was then modified manually to guanosine.

Figures were selected by the author.