Crystal structure of putative N-Acetyl-γ-glutamyl-phosphate reductase (AK071544) from rice (Oryza sativa)

Authors


Introduction.

Arginine is synthesized from glutamate via eight steps.1N-Acetyl-γ-glutamyl-phosphate reductase [AGPR; Enzyme Commission (EC): 1.2.1.38] catalyzes the third step of the arginine biosynthesis, in which N-acetyl-γ-glutamyl phosphate is converted to N-acetylglutamate-γ-semialdehyde by nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reductive dephosphorylation. This reaction is reversible. Therefore, AGPR also catalyzes oxidative phosphorylation of N-acetylglutamate-γ-semialdehyde to produce N-acetyl-γ-glutamyl phosphate as N-acetylglutamate-γ-semialdehyde dehydrogenase (NAGSA dehydrogenase). Aspartate-β-semialdehyde dehydrogenase (ASADH) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) are the enzymes that perform reductive dephosphorylation of β-aspartyl phosphate and oxidative phosphorylation of glyceraldehyde-3-phosphate, respectively.2 ASADH and GAPDH catalyze reactions similar to those catalyzed by AGPR, using NADP+ and NAD+ as coenzymes, respectively.2, 3 AGPR has some sequence similarity to ASADH and GAPDH, and is likely to have an overall folding similar to that of these enzymes.4 The crystal structures of ASADH and GAPDH revealed that the overall subunit structures of these two enzymes are similar.5, 6 A catalytic cysteine residue that functions as a nucleophile is conserved in both enzymes.

Recently, the crystal structures of putative AGPRs from Thermotoga maritima [Gene ID: Tm1782; Protein Data Bank (PDB) ID: 1vkn, hereafter TmAGPR] and from Arabidopsis thaliana (Gene ID: At2G19940; PDB ID: 1xyg, hereafter AtAGPR) have been deposited in the PDB. However, the details of these structures have not yet been reported.

In a systematic yeast-two hybrid screening to identify rice proteins interacting with key-regulators for the phytochrome signal transduction, the AK071544 protein, which is annotated as AGPR of Oryza sativa (OsAGPR), has been isolated as a phytochrome A-interacting protein by using the C-terminal domain of rice phytochrome A as the bait (N. Inagaki et al., unpublished data). In this work, we have determined the crystal structure of the AK071544 protein of rice (OsAGPR) to reveal its catalytic mechanism and interface of the interaction with phytochrome A. This is the first report of the crystal structure of AGPR.

Materials and Methods.

The AK071544 gene of the O. sativa complementary DNA (cDNA) library, the putative AGPR gene, codes 415 amino acid residues. The N-terminal region truncated AK071544 gene (corresponding to the region of Ala50–Pro415, 366 residues, 39.9 kDa) was expressed and purified as described elsewhere.7 The protein solution was dialyzed against 10 mM Tris-HCl buffer solution (pH 7.5) and concentrated to 10 mg/mL by centrifugal ultrafiltration.

Crystals of OsAGPR were obtained by the sitting-drop vapor diffusion method at 293 K. The protein solutions contained approximately 10 mg/mL OsAGPR and 10 mM Tris-HCl (pH 7.5), whereas the reservoir solutions contained 0.1 M Tris-HCl (pH 8.5), 0.72 M sodium formate, and 22.5% polyethylene glycol monomethyl ether 2000. The mixtures of protein solution and an equivalent volume of reservoir solution were equilibrated against the reservoir solutions. The crystal grew in about 10 days to an approximate size of 0.5 mm × 0.3 mm × 0.1 mm.

The X-ray diffraction data set of the crystal was collected at the SPring-8 BL44B2 at 90 K at the wavelength of 1.0080 Å and processed by the HKL2000 suite.8 The crystal-to-detector distance was set to 240 mm and diffraction images were recorded using an Area Detector Systems Corporation (ADSC) Quantum 210 charge-coupled device (CCD) detector with 0.3° oscillation and 3 s exposure per frame. The structure of OsAGPR was phased by the molecular replacement method using the structure of AGPR from T. maritima (TmAGPR, PDB ID: 1vkn) by the program MOLREP9 from CCP410 and refined by the programs of O and CNS.11, 12

Results and Discussion.

Overall structure.

The crystal diffracts X-rays beyond 1.8 Å resolution, but diffraction data at only less than 2.20 Å resolution were collected and processed due to the large length of the c axis. The crystal structure of OsAGPR has been determined and refined to a crystallographic R-factor of 17.1% (Rfree-factor of 21.3%) at 2.20 Å resolution. A summary of the data collection and refinement is shown in Table I. Root-mean-square-deviations (RMSDs) from ideal bond distances and angles were 0.005 Å and 1.3°, respectively.13 In a Ramachandran plot (Table I), 91.2% of the non-glycine residues were in the most favored regions of a ϕ-ψ plot.14 Four OsAGPR monomer molecules were found in the asymmetric unit, and these made a tetramer. We could trace 348 residues (Lys68–Pro415) of three monomers (chain ID: A–C) and 345 residues (Glu71–Pro415) of the other monomer (chain ID: D). The overall structure of OsAGPR is shown in Figure 1. The main-chain structure of OsAGPR is almost identical to those of TmAGPR (PDB ID, 1vkn) and AtAGPR (PDB ID, 1xyg). Moreover, the folding of OsAGPR is similar to that of ASADHs [e.g., with root-mean-square distances of 2.14 Å for the main-chain Cα atoms of the matched 155 amino acid residues against Haemophilus influenzae ASADH (PDB ID: 1pqu18)], which catalyze a similar reaction and usually exist as a dimer, with the exception of the inserted region of ASADHs [Fig. 2(A)]. Therefore, we discuss the catalytic cleft and the catalytic mechanism by comparing these two structures of the enzymes.

Table I. Summary of Data Collection and Refinement of OsAGPR
  • a

    Values in parentheses are for the outermost resolution shell.

  • b

    Rmerge = Σ|Ii − 〈Ii〉 |/Σ 〈Ii〉; where Ii is the observed intensity and 〈Ii〉 is the average intensity over symmetry equivalent measurements.

  • c

    R = Σ‖Fobs| − |Fcalc‖/Σ|Fobs|. Rfree is the same as R, but for a 5% subset of all reflections that were never used in crystallographic refinement.

  • d

    Percentages (%) of non-glycine and non-proline residues are in parentheses.

Data collection 
 Resolution range (Å)a50–2.20 (2.28–2.20)
 Space groupP61
 Cell constant: a (Å)86.11
 c (Å)316.3
 Wavelength (Å)1.0080
 Temperature (K)100
 Rmerge (%)a,b4.2 (6.4)
 Completeness (> 1σ, %)a97.2 (88.8)
 No. of observed reflections (> 1σ)667,362
 No. of unique reflections (> 1σ)65,608
 Redundancy (> 1σ, overall)10.2
I/σ(I)a50.8 (23.7)
Refinement 
 Resolution range (Å)20–2.20
 R-factor (%)c17.1
 Rfree-factor (%)c21.3
Ramachandran Plot 
 Region (residues)d: Most favored1092 (91.2)
  Allowed96 (8.0)
  Generously allowed3 (0.3)
  Disallowed7 (0.6)
Figure 1.

Ribbon model of the overall structure of the OsAGPR tetramer. This figure was drawn using MOLSCRIPT and Raster3D.15–17

Figure 2.

(A) Stereo view of the comparison of the Cα models of OsAGPR (red) and HiASADH complexed with NADP19 (blue; PDB ID: 1pqu) by stereo drawings of Cα-trace models. The aspartate-β-semialdehyde (green) covalently bonded to catalytic Cys136 and the phosphate ion (orange) interacted with Arg172 taken from the complex structure20 (PDB ID: 1nx6) are also superimposed. (B) Stereo view of the transparent superimposition of NADP (blue; PDB ID: 1pqu), covalently bonded aspartate-β-semialdehyde (ASA, green; PDB ID: 1nx6), and a phosphate ion (Pi; PDB ID: 1nx6) from the HiASADH complexes onto the putative active site of OsAGPR (pale red). The catalytic site and mechanism are thought to be similar between AGPRs and ASADHs. The flexible side-chain of Arg279 has variable conformations between the chain A–D of OsAGPR. This figure was drawn using MOLSCRIPT and Raster3D.15–17

Putative catalytic residues.

Gly79, Gly82, Tyr83, Arg172, Pro217, Gly218, Cys219, Tyr273, His278, and His280 in OsAGPR are completely conserved among known amino acid sequences and three-dimensional (3D) structures of AGPRs. These residues are located around the putative catalytic cleft. Gly79, Gly82, and Tyr83, located in the GXXGXXG motif, are thought to form a part of the NADP-binding region. Cys219 has been thought to be the catalytic nucleophile residue, because its 3D arrangement corresponds to that of Cys136 in H. influenzae ASADH (HiASADH), whose catalytic mechanism has been well investigated,18–20 and other ASADHs5, 21–25 [Fig. 2(B)]. The Sγ atom of Cys219 might have the role of attacking the carbonyl carbon Cδ atom of N-acetyl-γ-glutamyl-phosphate (AGP). Arg172 of OsAGPR corresponds to Arg103 of HiAGPR and has been thought to interact to a phosphate group of AGP. His277 in HiASADH, which is thought to accept a proton from the catalytic Cys136, is not conserved in AGPRs. His280 in OsAGPR is conserved in other AGPRs and adjacent to the thiol group of the catalytic Cys219. The distance between Nϵ of His280 and Sγ of Cys219 is approximately 4.2 Å. This is too far for the two to interact, but this histidine is completely conserved among AGPRs and likely works as a proton acceptor. Tyr273, His278, and/or Arg279 are also conserved residues and are thought to be related to the substrate recognition, for example, by hydrogen bonds to a carboxyl group of AGP. Tyr220, Ser248, Gly252, and Asn383 form a hydrophobic pocket and probably accept an acetyl group of AGP.

Coordinates.

The coordinates have been deposited in the PDB (ID: 2cvo).

Acknowledgements

Our thanks to Drs. Hisashi Naitow and Taiji Matsu (SPring-8 BL44B2) for their help during the data collection.

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