The gene cluster for agmatine catabolism of Enterococcus faecalis: study of recombinant putrescine transcarbamylase and agmatine deiminase and a snapshot of agmatine deiminase catalyzing its reaction

Abstract

Enterococcus faecalis makes ATP from agmatine in three steps catalyzed by agmatine deiminase (AgDI), putrescine transcarbamylase (PTC), and carbamate kinase (CK). An antiporter exchanges putrescine for agmatine. We have cloned the E. faecalis ef0732 and ef0734 genes of the reported gene cluster for agmatine catabolism, overexpressed them in Escherichia coli, purified the products, characterized them functionally as PTC and AgDI, and crystallized and X-ray diffracted them. The 1.65-Angstroms-resolution structure of AgDI forming a covalent adduct with an agmatine-derived amidine reactional intermediate is described. We provide definitive identification of the gene cluster for agmatine catabolism and confirm that ornithine is a genuine but poor PTC substrate, suggesting that PTC (found here to be trimeric) evolved from ornithine transcarbamylase. N-(Phosphonoacetyl)-putrescine was prepared and shown to strongly (K(i) = 10 nM) and selectively inhibit PTC and to improve PTC crystallization. We find that E. faecalis AgDI, which is committed to ATP generation, closely resembles the AgDIs involved in making polyamines, suggesting the recruitment of a polyamine-synthesizing AgDI into the AgDI pathway. The arginine deiminase (ADI) pathway of arginine catabolism probably supplied the genes for PTC and CK but not those for the agmatine/putrescine antiporter, and thus the AgDI and ADI pathways are not related by a single "en bloc" duplication event. The AgDI crystal structure reveals a tetramer with a five-blade propeller subunit fold, proves that AgDI closely resembles ADI despite a lack of sequence identity, and explains substrate affinity, selectivity, and Cys357-mediated-covalent catalysis. A three-tongued agmatine-triggered gating opens or blocks access to the active center.

FIG. 1.

Agmatine catabolism gene cluster and purification and crystallization of PTC and AgDI. (A) Gene organization of the two strands of the sequenced region, showing the number of amino acid (aa) residues expected for each gene product and the length (in base pairs) of the intergenic regions. Genes are given the identifiers of the TIGR database, together with the agc designations given to them here. The positions of the three predicted stem-loops are indicated by the open circles, and a putative cre box preceding agcB is indicated with a gray rectangle. The gene in the opposite strand corresponds to a luxR regulator. (B and C) SDS-PAGE and Coomassie blue staining analyses of the various steps of the purifications of PTC and AgDI. The crude extracts are the postsonication supernatants. Panel B also includes a blank extract of E. coli BL21 cells transformed with the parental pET-22 plasmid carrying no gene insert, to highlight the differences from the extracts of cells transformed with the plasmids carrying the gene for PTC or for AgDI. Molecular mass marker proteins were from Sigma (Dalton Mark VII-L). Results of enzyme activity assays for PTC and AgDI are shown below the purification steps at which the activities were assayed. The activities obtained when putrescine was replaced by 10 mM ornithine (for OTC) or when agmatine was replaced by 5 mM arginine (for ADI) are also shown. Values of <0.1 or <0.2 are below the detection limits for assays giving no activity. (D) Crystals of both enzymes that were obtained and used for diffraction studies. Bars, 0.1 mm.

FIG. 2.

Investigation of the oligomeric state of E. faecalis PTC and AgDI, using gel filtration. A semilogarithmic plot of molecular mass versus elution volume (expressed as K_d [see Materials and Methods]) from the Superdex 200HR column is shown. The circles correspond to the following protein standards: cytochrome c (12.3 kDa), lactalbumin (14.2 kDa), carbonic anhydrase (29.0 kDa), ovalbumin (42.7 kDa), bovine serum albumin (66.4 kDa), the dimer of bovine serum albumin (132.9 kDa), Pyrococcus furiosus carbamate kinase (68.8 kDa), intact (97.1 kDa) and truncated (31.9 kDa) aspartokinase III of E. coli, alcohol dehydrogenase (146.8 kDa), aldolase (156.8 kDa), Thermotoga maritima N-acetyl-l-glutamate kinase (182.0 kDa), amylase (223.8 kDa), catalase (230.3 kDa), ferritin (440 kDa), and thyroglobulin (669 kDa). The triangle and square denote, respectively, the positions of elution of the peaks of E. faecalis PTC and AgDI, assuming that PTC is a trimer (sequence-deduced mass, 120,273 Da) and AgDI is a tetramer (deduced mass, 165,412 Da).

FIG. 3.

Effects of PAPU on E. faecalis PTC activity. (A) Inhibition and lack of inhibition of E. faecalis PTC and OTC, respectively, by PAPU. Activities are given as fractions of the activity in the absence of PAPU. (B and C) Influence of PAPU on the kinetic parameters of PTC for putrescine (Put) and on the K_m for carbamoyl phosphate (Carb-P_i), respectively.

FIG.4.

Structure of AgDI. (A and B) Ribbon representations of the monomer of E. faecalis AgDI (A) and of the catalytic domain of Mycoplasma arginini ADI (PDB entry 1S9R without residues 75 to 148, which are not a part of the catalytic domain) (B), both containing the covalently bound substrate in space-filling representation. α helices, β sheets, and loops are colored red, yellow, and green, respectively. (C) Stereo view of the active site of AgDI, showing the density map of 2F_obs − F_calc, contoured at the 0.9 σ level, around the covalent adduct. The substrate is colored yellow, and the surrounding protein residues are colored gray. O, N, and S atoms are colored red, blue, and green, respectively. (D) Interatomic distances (in angstroms) between the catalytic protein residues and the substrate around the reactive carbon center. The interactions with a fixed water molecule (W1) believed to be important in the mechanism are also represented. The density map of 2F_obs − F_calc, contoured at 0.75 σ for the covalent amidino complex, is shown. (E) Correspondence between amino acid sequence and secondary structure. Bars, arrows, and lines above the structure denote, respectively, α helices, β strands, and loops (only long loops are depicted), numbered in ascending order from N to C terminus and, when belonging to a repeat, in parentheses and having a subscript that denotes the repeat number. Open triangles under the sequence denote residues having decreased accessibility upon the binding of agmatine. Circles denote decreased accessibility upon dimer (open) and tetramer (shaded) formation. The gray sequence backgrounds highlight residues that are invariant in the AgDIs of E. faecalis, Streptococcus mutans, Pseudomonas aeruginosa, and Arabidopsis thaliana (SwissProt accession numbers Q837U5, Q8DW17, Q9I6J9, and Q8GWW7, respectively). (F) Ribbon diagram of AgDI dimer viewed perpendicularly to the molecular twofold axis. Coloring and substrate representation are as in panel A. (G) Ribbon representation of the AgDI tetramer viewed along one of the three twofold molecular axes. The two subunits of the two dimers (as defined in the text) are shown in different shades of red or blue. The covalently bound substrate is shown in space-filling representation.

FIG. 5.

Proposed five-step mechanism for the AgDI reaction. Step 1 leads to the formation of the first tetrahedral carbon center intermediate as a consequence of the attack by the activated thiol of Cys357. Asp96 and the nonprotonated primary N of the guanidinium group may induce deprotonation of the thiol group. A proton is extracted by His218, which forms a charge relay system with Glu157. In step 2 the tetrahedral intermediate collapses to the triagonal amidino intermediate, with liberation of ammonia. Asp96, Asp220, and His218 help stabilize the leaving ammonia and the positive charge development in the amidino group. In step 3 ammonia is replaced by water positioned for attack on the carbon center, interacting with the same groups as the ammonia. The intermediate revealed here by X-ray crystallography corresponds to one of the two complexes (either the ammonia or the water complex) with the amidino intermediate. Step 4 is the formation of the second tetrahedral carbon intermediate. His218 helps this step by abstracting one proton from water. The final step is the collapse of the tetrahedral intermediate to carbamoylputrescine and the regenerated thiol group.