X-ray structure of the amidase domain of AtzF, the allophanate hydrolase from the cyanuric acid-mineralizing multienzyme complex

Abstract

The activity of the allophanate hydrolase from Pseudomonas sp. strain ADP, AtzF, provides the final hydrolytic step for the mineralization of s-triazines, such as atrazine and cyanuric acid. Indeed, the action of AtzF provides metabolic access to two of the three nitrogens in each triazine ring. The X-ray structure of the N-terminal amidase domain of AtzF reveals that it is highly homologous to allophanate hydrolases involved in a different catabolic process in other organisms (i.e., the mineralization of urea). The smaller C-terminal domain does not appear to have a physiologically relevant catalytic function, as reported for the allophanate hydrolase of Kluyveromyces lactis, when purified enzyme was tested in vitro. However, the C-terminal domain does have a function in coordinating the quaternary structure of AtzF. Interestingly, we also show that AtzF forms a large, ca. 660-kDa, multienzyme complex with AtzD and AtzE that is capable of mineralizing cyanuric acid. The function of this complex may be to channel substrates from one active site to the next, effectively protecting unstable metabolites, such as allophanate, from solvent-mediated decarboxylation to a dead-end metabolic product.

FIG 1

Mineralization of atrazine in Pseudomonas sp. strain ADP. Atrazine (a) is hydrolyzed by AtzA, releasing chloride. The product (b) is further hydrolyzed by AtzB, releasing ethylamine (c), and subsequent hydrolysis of the resulting product (d) releases isopropylamine (e), generating cyanuric acid (f). AtzD hydrolyzes cyanuric acid to produce the unstable 1-carboxybiuret (g), which decomposes to biuret (h). Biuret is deaminated by AtzE to yield allophanate (i), which can spontaneously decompose to urea (j) or can be deaminated by AtzF to produce the unstable N-carboxycarbamate (k), which spontaneously decomposes first to carbamate (l) and then to ammonia. spon., spontaneous.

FIG 2

Alignment of the allophanate hydrolases from Pseudomonas sp. strain ADP (AtzF), Kluyveromyces lactis (AH_Kl), and Granulibacter bethesdensis (AH_Gb). Single underline, amidase domain; double underline, C-terminal domain; diamonds, active-site residues of the amidase domain; triangle, proposed catalytic histidine residue in the C-terminal domain; arrow, position where AtzF was truncated to produce AtzF468; asterisks, identical residues; colons, highly similar residues; periods, similar.

FIG 3

The AtzF structure and comparison to related structures. (A) A cartoon diagram of the dimer of the AtzF structure with the secondary structure colored by magenta for the beta sheet, cyan for alpha helices, and orange for loop structures. AtzF has two main domains: the catalytic domain and a second all-alpha-helical domain that forms the dimer interface. This has been highlighted by coloring these helices in green. (B) The AtzF structure superposed with structures with PDB accession numbers 4ISS and 4GYS. The structure with PDB accession number 4ISS is colored in cyan and has an extra domain which extends away from the rest of the molecule; AtzF is colored green, and the structure with PDB accession number 4GYS is colored magenta. The structures superpose well, despite limited sequence identity, with RMSD values of 1.3 to 1.6 Å. (C) Comparison of the AtzF dimer and the dimers with PDB accession numbers 4ISS and 4GYS. The figure shows how the dimers are similar and how the dimer with the extra domain (PDB accession number 4ISS) helps the dimer formation for this protein. (D) The catalytic site of these proteins. In two cases we see substrate mimetics bound into the catalytic site: in the case of AtzF we see clear density for malonate, whereas for the structure with PDB accession number 4ISS, there is tartrate.

FIG 4

Analysis of the multimerization of AtzF and Atzf₄₆₇ by SAXS. (A) High-resolution model of an AtzF tetramer (green and blue) superimposed on a dummy atom model derived from the SAXS data (gray density). (B) High-resolution model of dimeric AtzF₄₆₇ (green and blue) superimposed on a dummy atom model derived from the SAXS data (gray density). (C) Fit between the SAXS data and the AtzF tetramer model shown in panel A calculated using CRYSOL. (D) Fit between the measured AtzF₄₆₇ protein and a model of tetrameric AtzF₄₆₇ based on the AtzF tetramer model calculated using CRYSOL. (E) Fit between dimeric AtzF and the measured AtzF SAXS data calculated using CRYSOL. (F) Fit between the dimeric AtzF₄₆₇ model shown in panel B and the measured AtzF₄₆₇ SAXS data.

FIG 5

Activity of AtzF (and fragments) at different pHs and temperatures. k_cat values at different pH levels obtained at 28°C (A) and 4°C (B) for AtzF, AtzF₄₆₇, and AtzF H488A were compared. All the data points plotted had standard errors of less than 5.3%, and standard errors are presented in Table S2 in the supplemental material.

FIG 6

Effect of pH on residual activity of AtzF and its variants. Differences in the residual activity of AtzF, AtzF₄₆₇, and AtzF H488A at pH 7 (A) and pH 9 (B) were compared. Enzymes were incubated in pH 7 or pH 9 buffers for 5 min at temperatures ranging from 30 to 70°C before testing for residual activity.