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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