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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).
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