PDBsum entry 2buf

Transferase

PDB id: 2buf

Contents

Protein chains

(+ 5 more) 292 a.a. *

274 a.a. *

Ligands

NLG ×10

ADP ×10

Metals

_CL ×6

_MG ×8

* Residue conservation analysis

PDB id:

2buf

Name:

Transferase

Title:

Arginine feed-back inhibitable acetylglutamate kinase

Structure:

Acetylglutamate kinase. Chain: a, b, c, d, e, f, g, h, i, j, k, l. Synonym: NAG kinase, agk, n-acetyl-l-glutamate 5-phosphotransferase. Engineered: yes

Source:

Pseudomonas aeruginosa. Organism_taxid: 287. Expressed in: escherichia coli. Expression_system_taxid: 469008.

Biol. unit:

Hexamer (from PDB file)

Resolution:

2.95Å R-factor: 0.249 R-free: 0.267

Authors:

S.Ramon-Maiques,M.L.Fernandez-Murga,A.Vagin,I.Fita,V.Rubio

Key ref:

S.Ramón-Maiques et al. (2006). Structural Bases of Feed-back Control of Arginine Biosynthesis, Revealed by the Structures of Two Hexameric N-Acetylglutamate Kinases, from Thermotoga maritima and Pseudomonas aeruginosa. J Mol Biol, 356, 695-713. PubMed id: 16376937 DOI: 10.1016/j.jmb.2005.11.079

Date:

12-Jun-05 Release date: 13-Dec-05

Supersedes:

1uvd

Protein chains

Q9HTN2  (ARGB_PSEAE) -  Acetylglutamate kinase from Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1)

Seq: 301 a.a.

Struc: 292 a.a.

Protein chain

Q9HTN2  (ARGB_PSEAE) -  Acetylglutamate kinase from Pseudomonas aeruginosa (strain ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1)

Seq: 301 a.a.

Struc: 274 a.a.

Enzyme reactions

• Enzyme class: Chains A, B, C, D, E, F, G, H, I, J, K, L: E.C.2.7.2.8 - acetylglutamate kinase.
• Pathway: Ornithine Biosynthesis
• Reaction: N-acetyl-L-glutamate + ATP = N-acetyl-L-glutamyl 5-phosphate + ADP

N-acetyl-L-glutamate + ATP (Bound ligand (Het Group name = NLG) corresponds exactly) = N-acetyl-L-glutamyl 5-phosphate (Bound ligand (Het Group name = ADP) corresponds exactly) + ADP

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

reference

DOI no: 10.1016/j.jmb.2005.11.079

J Mol Biol 356:695-713 (2006)

PubMed id: 16376937

Structural Bases of Feed-back Control of Arginine Biosynthesis, Revealed by the Structures of Two Hexameric N-Acetylglutamate Kinases, from Thermotoga maritima and Pseudomonas aeruginosa.

S.Ramón-Maiques, M.L.Fernández-Murga, F.Gil-Ortiz, A.Vagin, I.Fita, V.Rubio.

ABSTRACT

N-Acetylglutamate kinase (NAGK) catalyses the second step in the route of arginine biosynthesis. In many organisms this enzyme is inhibited by the final product of the route, arginine, and thus plays a central regulatory role. In addition, in photosynthetic organisms NAGK is the target of the nitrogen-signalling protein P(II). The 3-D structure of homodimeric, arginine-insensitive, Escherichia coli NAGK, clarified substrate binding and catalysis but shed no light on arginine inhibition of NAGK. We now shed light on arginine inhibition by determining the crystal structures, at 2.75A and 2.95A resolution, of arginine-complexed Thermotoga maritima and arginine-free Pseudomonas aeruginosa NAGKs, respectively. Both enzymes are highly similar ring-like hexamers having a central orifice of approximately 30A diameter. They are formed by linking three E.coli NAGK-like homodimers through the interlacing of an N-terminal mobile kinked alpha-helix, which is absent from E.coli NAGK. Arginine is bound in each subunit of T.maritima NAGK, flanking the interdimeric junction, in a site formed between the N helix and the C lobe of the subunit. This site is also present, in variable conformations, in P.aeruginosa NAGK, but is missing from E.coli NAGK. Arginine, by gluing the C lobe of each subunit to the inter-dimeric junction, may stabilize an enlarged active centre conformation, hampering catalysis. Acetylglutamate counters arginine inhibition by promoting active centre closure. The hexameric architecture justifies the observed sigmoidal arginine inhibition kinetics with a high Hill coefficient (N approximately 4), and appears essential for arginine inhibition and for NAGK-P(II) complex formation, since this complex may involve binding of NAGK and P(II) with their 3-fold axes aligned. The NAGK structures allow identification of diagnostic sequence signatures for arginine inhibition. These signatures are found also in the homologous arginine-inhibited enzyme NAG synthase. The findings on NAGK shed light on the structure, function and arginine inhibition of this synthase, for which a hexameric model is constructed.

Selected figure(s)

Figure 2.
Figure 2. Architecture of arginine-sensitive NAGK. The TmNAGK ((a) and (b)) or PaNAGK ((e) and (f)) hexamers are viewed ((a) and (e)) along or ((b) and (f)) perpendicularly to the molecular 3-fold axis. Each homodimer is coloured differently. In (a) and (b) arginine, and in (e) and (f) MgADP and NAG are represented in space-filling representation and coloured. In (a) and (e) the arrowpoints indicate the interlaced N helices at one junction. (c) TmNAGK and (d) EcNAGK26 homodimers, viewed along their 2-fold axes. The N and C lobes are in blue and green, respectively, and the N helix is in red. In (c) arginine is shown in space-filling representation, and coloured. The ligands shown in (d) are in ball and stick representation, and are NAG and AMPPNP.

Figure 3.
Figure 3. Amino acid sequence and topology of secondary structure elements, and signature sequences of arginine-sensitive NAGK. (a) Sequence alignment of E. coli, P. aeruginosa and T. maritima NAGKs (Swissprot P0A6C8, Q9HTN2 and Q9X2A4, respectively), localizing the secondary structure elements as superimposed blue arrows (b-strands), and yellow (a-helices) or orange (N-terminal helix) rectangles. The residues conserved or conservatively replaced in all NAGKs are in red, those having decreased accessibility upon the binding of NAG, MgADP or arginine are indicated with dark green triangles, light green triangles and violet diamonds, respectively. Black and grey circles denote decreased accessibility upon homodimer and hexamer formation, respectively. Signature sequence traits associated with arginine inhibition are underlined. (b) Scheme of the topology of secondary structure elements found in NAGKs, where b-strands and a-helices are represented as triangles and circles, respectively, the strands of the central b-sheet are shadowed, and the colour code is red for the N helix (the only element missing in EcNAGK; represented as two circles because of the kink), and green and blue for the elements of the N and the C lobe, respectively. (c) Alignment (see Materials and Methods) of arginine-insensitive and arginine-sensitive NAGKs in the three regions (separated by vertical lines) where diagnostic signatures were identified. Residues found constantly and exclusively in arginine-sensitive NAGKs are highlighted in red. The K/R highlighted in blue is found constantly but not exclusively, in arginine-sensitive NAGKs. The residues highlighted in pink are exclusively (but not constantly) found in arginine-sensitive NAGKs. Yellow colouring highlights residues that are conserved or conservatively replaced in most NAGKs, irrespective of whether they are sensitive or insensitive to arginine. Rectangles and arrows above the alignment indicate, respectively, a-helices and b-strands, as they appear in PaNAGK. The horizontal line below the alignment marks the larger (see the text) sequence signature at the b15-aH-b16 region. A rectangle encloses the phenylalanine residues of yeast and Neurospora crassa NAGKs that when mutated resulted in hampered arginine inhibition.16 The abbreviations used and the Swissprot/Trembl (unless indicated otherwise) accession numbers (given between parentheses) are the following: ECOLI, E. coli (P0A6C8); SERMA, S. marcescens (encoded by nucleotides 4275578-4274805 of the S. marcescens genome, systematic_id=SMA4004, http://www.sanger.ac.uk/projects/s_marcescens/sma.art); BACSU, B. subtilis (P68729); BACST, Bacillus stearothermophilus (Q07905); PSEAE, P. aeruginosa (Q9HTN2); THEMA, T. maritima (Q9X2A4); CORGL, Corynebacterium glutamicum;13 SYNP7, S. elongatus, strain PCC7942 (Q6V1L5). The sequences of photosynthetic eukaryotes start after a predicted chloroplast signal targeting sequence that precedes the N-terminal extension: CREIN, Chlamydomonas reinhardtii (gene TC25068, http://www.tigr.org/tdb/tgi/chrgi); ORYSA, Oriza sativa (rice, Q949B4); ARATH: Arabidopsis thaliana (Q8LA25); the rice and A. thaliana NAGKs are assumed to be arginine-sensitive by similarity to the pea enzyme18 (for which no sequence is available) and also because both are known to interact with the nitrogen signalling protein P[II].19^ and 21 The fungal sequences start after the mitochondrial signal targeting sequence that precedes the N-terminal extension: YEAST, Saccharomyces cerevisiae (Q01217); NEUCR, N. crassa (P54898).

The above figures are reprinted by permission from Elsevier: J Mol Biol (2006, 356, 695-713) copyright 2006.

Figures were selected by an automated process.