N-Acetyl-L-glutamate kinase from Escherichia coli: cloning of the gene, purification and crystallization of the
recombinant enzyme, and preliminary X-ray analysis of the free and ligand-bound forms
NOEMI BUENO LOPEZ
Introduction
• N-acetyl-L-glutamate kinase (NAGK) phosphorylates the acetylglutamate (NAG) in the second step of microbial arginine biosynthesis.
• In many microorganisms NAGK is a crucial control point, being feed-back inhibited by the final product arginine.
• The P. aeruginosa enzyme is a homodimer of a 29-kDa subunit, that form higher oligomers.
• Bacterial and chloroplast NAGK polypeptides are composed of 258-304 amino acid residues, exhibiting considerable sequence identity.
• Substantial sequence similarity (~20% identity) is found with carbamate kinase (CK), the final enzyme of the arginine deiminase pathway of arginine catabolism.
• CK is a homodimer of 30 kDa and catalyzes the transfer of the -phosphoryl group of ATP to a COO- group, whose 3D structure we have recently determined.
• These similarities suggest that CK and NAGK are structurally and functionally similar.
• Since CK presents a new / fold, NAGK and CK might be members of a new structural family.
• This family could also include: -glutamyl kinase and long chain fatty acyl CoA synthetase..
Experimental
• The NAGK gene was PCR-amplified from E. coli DNA.
• The BspHI and BlpI digested amplified fragment was ligated using T4 ligase into the NcoI and BlpI sites of plasmid pET-15b.
• Plasmid pNAGK24 was isolated. Restricition analysis and automated DNA sequencing confirmed the presence of the full NAGK gene.
• E. coli BL21(DE3) cells were transformed with pNAGK24.
• Cells were grown to A600=0.6 (37°C) in 1.5 l LB broth with 75 g/ml ampicillin and induced for 3h with 1 mM IPTG. Harvesting by centrifugation.
• Ammonium sulphate precipitation of the supernatant (40-60 % saturation) (at 0 ºC).
• Anion exchange chromatography (Q-Sepharose Fast Flow) equilibrated with buffer A.
• Affinity chromatography (Affigel Blue column) in buffer 20 mM Tris-HCl pH 7.5, 20 mM MgCl2, 0.2 mM DTT.
• Pure NAGK was eluted with buffer supplemented with 2.5 mM ATP and 20 mM NAG,
• Precipitation with ammonium sulphate (70% saturation). Stored at 4° C.
• Crystallization using the hanging drop vapor diffusion method.
• Conditions: 10 mg/ml NAGK in 10 mM phosphate Na/K pH 7.0, 1 mM DTT, alone or supplemented with 24 mM NAG, 30 mM MgCl2 and 6 mM AMPPNP ("substrates").
• The best crystals were produced in about a week at 22º C.
• Without substrates, 0.1 M Na citrate pH 5.6, 26-32 % PEG 4K and 0.1-0.3 M ammonium acetate.
• With substrates, crystals were obtained with with 0.1 M Na-acetate pH 4.6, 27-32 % PEG-MME 2K (from Hampton) and 0.1-0.3 M ammonium sulphate. Crystals (0.3 mm) were examined with an Image Plate (MAR RESEARCH) area detector mounted on a Rigaku rotating copper anode
• Data were collected at 100 K temperature with crystals flash cooled.
Results and discussion • The product generated by PCR-amplification of E. coli DNA exactly matched
in size and DNA sequence with the NAGK gene.
• Extracts of E. coli BL21(DE3) transformed with an expression plasmid (pNAGK24) that contains as an insert the amplified DNA.
• The enzyme exhibited similar activity (63.4 U/mg protein) to the purest bacterial preparation reported.
36
24
66
45
29
20.1
kDa 1 2 3• Automated Edman
degradation yielded the expected N-terminal sequence (MNPLIIK).
Lane 1. Cells transformed with plasmids pET-15b.
Lane 2. Transformed with pNAGK24 plasmid.
Lane 3. Purified enzyme.
36
24
97
66
45
29
20.1
14.2
116
kDa - DMS - DMS
NAGK Aldolase
• Cross-linking with dimethylsuberimidate indicates that E. coli NAGK forms dimers.
• NAGK crystals in the absence of substrates diffracted at least 2 Å resolution - Data set at 2.95 Å resolution (93.4 % completeness; Rmerge
12.1). - Space group P6122 or P6522.- Cell parameters a= b=78.6 Å and c= 278.0 Å. - 2 monomers in the asymmetric unit.
• NAGK crystals in the presence of substrates diffracted to at least 1.8 Å.
– Data set collected at 2.3 Å (90.4 % completeness, Rmerge 11.5).– Space group is C2221.– Cell parameters a=60.0 Å, b= 71.9 Å and c= 107.4 Å.– One monomer in the asymmetric unit.
Crystals without substrates Crystals with substrates
• Molecular replacement using CK yielded suggestive but not conclusive solutions.
References.
Gil-Ortiz, F., Ramon-Maiques, S., Marina A., Fita, I. and Rubio V. (1999) N-Acetyl-L-glutamate kinase from Escherichia coli: cloning of the gene, purification and crystallization of the recombinant enzyme, and preliminary X-ray analysis of the free and ligand-bound forms. Acta Crystallog. D 55: 1350-1352.
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