PDBsum entry: 2fb2

Ligand binding protein

PDB id: 2fb2

Contents

Protein chains

327 a.a. *

Ligands

SO4 ×2

SAM-SF4 ×2

SF4 ×2

Waters ×177

* Residue conservation analysis

PDB id:

2fb2

Name:

Ligand binding protein

Title:

Structure of the moaa arg17/266/268/ala triple mutant

Structure:

Molybdenum cofactor biosynthesis protein a. Chain: a, b. Synonym: moaa. Engineered: yes. Mutation: yes

Source:

Staphylococcus aureus. Organism_taxid: 1280. Gene: moaa. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Dimer (from PQS)

Resolution:

2.25Å R-factor: 0.196 R-free: 0.237

Authors:

P.Haenzelmann,H.Schindelin

Key ref:

P.Hänzelmann and H.Schindelin (2006). Binding of 5'-GTP to the C-terminal FeS cluster of the radical S-adenosylmethionine enzyme MoaA provides insights into its mechanism. Proc Natl Acad Sci U S A, 103, 6829-6834. PubMed id: 16632608 DOI: 10.1073/pnas.0510711103

Date:

08-Dec-05 Release date: 09-May-06

Protein chains

P69848  (MOAA_STAA8) -  Molybdenum cofactor biosynthesis protein A

Seq: 340 a.a.

Struc: 327 a.a.*

* PDB and UniProt seqs differ at 3 residue positions (black crosses)

 Gene Ontology (GO) functional annotation 

• Cellular component: molybdopterin synthase complex (1 term)
• Biological process: metabolic process (2 terms)
• Biochemical function: catalytic activity (6 terms)

DOI no: 10.1073/pnas.0510711103

Proc Natl Acad Sci U S A 103:6829-6834 (2006)

PubMed id: 16632608

Binding of 5'-GTP to the C-terminal FeS cluster of the radical S-adenosylmethionine enzyme MoaA provides insights into its mechanism.

P.Hänzelmann, H.Schindelin.

ABSTRACT

The first step in molybdenum cofactor biosynthesis, the conversion of 5'-GTP to precursor Z, an oxygen-sensitive tetrahydropyranopterin is catalyzed by the S-adenosylmethionine (SAM)-dependent enzyme MoaA and the accessory protein MoaC. This reaction involves the radical-initiated intramolecular rearrangement of the guanine C8 atom. MoaA harbors an N-terminal [4Fe-4S] cluster, which is involved in the reductive cleavage of SAM and generates a 5'-deoxyadenosyl radical (5'-dA*), and a C-terminal [4Fe-4S] cluster presumably involved in substrate binding and/or activation. Biochemical studies identified residues involved in 5'-GTP binding and the determinants of nucleotide specificity. The crystal structure of MoaA in complex with 5'-GTP confirms the biochemical data and provides valuable insights into the subsequent radical reaction. MoaA binds 5'-GTP with high affinity and interacts through its C-terminal [4Fe-4S] cluster with the guanine N1 and N2 atoms, in a yet uncharacterized binding mode. The tightly anchored triphosphate moiety prevents the escape of radical intermediates. This structure also visualizes the L-Met and 5'-dA cleavage products of SAM. Rotation of the 5'-dA ribose and/or conformational changes of the guanosine are proposed to bring the 5'-deoxyadenosyl radical into close proximity of either the ribose C2' and C3' or the guanine C8 carbon atoms leading to hydrogen abstraction.

Selected figure(s)

Figure 2.
Fig. 2. Determination of MoaA substrate-binding by equilibrium dialysis. (A) Nucleotide specificity. MoaA (75 µM) was equilibrated with the listed nucleotides (150 µM); n.d., not detected. (B) 5'-GTP/5'-ATP competition. WT MoaA (wt) and variants (75 µM) were dialyzed against equimolar concentrations (150 µM) of 5'-GTP (black bar) and 5'-ATP (white bar). (C) 5'-GTP binding. WT MoaA and variants (75 µM) were dialyzed against 5'-GTP (150 µM). Error bars denote the SD from the mean of at least four independent experiments. CN A, triple Cys Ala variant of the N-terminal FeS cluster; black bars, WT MoaA and residues not involved in 5'-GTP binding; light gray bars, residues important for 5'-GTP binding; dark gray bars, residues less important in 5'-GTP binding; white bar, R17/266/268A triple variant.

Figure 3.
Fig. 3. Structure of MoaA in complex with 5'-GTP. (A) 2F[o]-F[c] map of the C-terminal [4Fe-4S] cluster with bound 5'-GTP contoured at one times the rms deviation. (B) Electrostatic potential (electropositive in blue, electronegative in red contoured at ±10 kT) surrounding the hydrophilic channel. [4Fe-4S] clusters, L-Met, 5'-dA, 5'-GTP, and residues important for 5'-GTP binding are displayed. (C) Stereoview of 5'-GTP interactions with surrounding active site residues (dashed lines). Carbon atoms of residues in hydrogen-bonding distance are in white, and carbon atoms of residues within a distance of 4 Å are in green. (D) Superposition of MoaA in complex with 5'-GTP (gray), without 5'-GTP (PDB ID code 1TV8; green) and the R17/266/268A variant (yellow).

Figures were selected by an automated process.