Structure of the E. coli agmatinase, SPEB

Abstract

Agmatine amidinohydrolase, or agmatinase, catalyzes the conversion of agmatine to putrescine and urea. This enzyme is found broadly across kingdoms of life and plays a critical role in polyamine biosynthesis and the regulation of agmatine concentrations. Here we describe the high-resolution X-ray crystal structure of the E. coli agmatinase, SPEB. The data showed a relatively high degree of pseudomerohedral twinning, was ultimately indexed in the P31 space group and led to a final model with eighteen chains, corresponding to three full hexamers in the asymmetric unit. There was a solvent content of 38.5% and refined R/Rfree values of 0.166/0.216. The protein has the conserved fold characteristic of the agmatine ureohydrolase family and displayed a high degree of structural similarity among individual protomers. Two distinct peaks of electron density were observed in the active site of most of the eighteen chains of SPEB. As the activity of this protein is known to be dependent upon manganese and the fold is similar to other dinuclear metallohydrolases, these peaks were modeled as manganese ions. The orientation of the conserved active site residues, in particular those amino acids that participate in binding the metal ions and a pair of acidic residues (D153 and E274 in SPEB) that play a role in catalysis, are similar to other agmatinase and arginase enzymes and is consistent with a hydrolytic mechanism that proceeds via a metal-activated hydroxide ion.

Fig 1. Biosynthesis of polyamines from agmatine.

Putrescine can be generated from agmatine directly by an agmatinase enzyme (SPEB in E. coli), via two steps by the sequential action of agmatine deiminase and N-carbamoylputrescine amidohydrolase, or from ornithine by ornithine decarboxylase (ODC). Note that, although charges are not shown here for clarity, these metabolites are all expected to carry a positive charge at physiological pH.

Fig 2. Overall structure of SPEB.

The protomer of SPEB (A) shows the conserved α/β/α sandwich that is characteristic of the agmatine ureohydrolase family. The presumed biologically relevant unit, a hexamer with D3 symmetry (B), was observed in the crystal structure and was consistent with SEC data (S3 Fig in S1 File) and the prediction by PDBePISA (47).

Fig 3. Comparison of SPEB to other arginase family proteins.

The protomer of SPEB (A, green) superimposes well with the putative agmatinase from B. thailandensis (4DZ4, orange; RMSD 0.555 Å), the guanidinopropionase from P. aeruginosa (3NIP, blue; RMSD 0.793 Å), and the agmatinase from D. radiodurans (1WOG, gray; RMSD 1.13 Å). While the overall orientation of the protomers in the hexamer is similar, the structural similarity is much lower for the hexamers with an RMSD of 37.7 Å between SPEB and 4DZ4 (B, SPEB in yellow, red and blue helices for top 3 chains, bottom 3 chains shown in cyan, green and purple surface representation; 4DZ4 in green helices, see S6 Fig in S1 File for superposition with 3NIP and 1WOG).

Fig 4. Active site of SPEB.

The conserved active site residues of SPEB (A, chain A) are depicted in ball and stick representation with carbon atoms colored green, oxygen atoms colored red, and nitrogen atoms colored blue. Mn²⁺ ions are shown as purple spheres, while two active site water molecules are included as red spheres. A putative model of agmatine binding to SPEB (B, depicted with carbon atoms colored orange and nitrogen atoms colored blue) was generated by superposition to the hexane-1,6-diamine ligand in 1WOG (B, cyan line drawing, see structure analysis in Materials and Methods). The position of agmatine illustrates that protein-ligand binding is likely governed predominantly by polar contacts with the guanidinium group. Refer to Fig 5B and 5C for details about hydrogen bonding pattern and distances in the active site.

Fig 5. Metal binding in the SPEB active site.

Two clear peaks of electron density were observed in the structure and were modeled as Mn²⁺ ions (A, represented as purple spheres). Also shown are well-defined water molecules that may play a role in catalysis (represented as red spheres). A schematic (B) shows the coordination of the metal ions by active site residues, and the corresponding bond distances. The relative positions, and distances, of the water molecules (red spheres) is similarly depicted in a schematic (C) of the same site that was rotated 90°, relative to B, as shown.