enzymes display either grooves at their surface or symmetrical
toroids that allow partial or complete substrate enclosure.18
MtNAS is original as it uses a rigid frame harboring an
internal asymmetrical cavity that precisely controls substrate
and reaction intermediate processing and translocation.
This work is supported by the ANR (project HEMOLI;
ANR-07-BLAN-0115-01) and the Commissariat a l’Energie
Atomique (CEA). We thank J. Varlin and J. Lavergne for
discussion. The coordinates for the MtNAS-E81Q–Reaction
intermediate complex have been deposited in the Protein Data
Bank (accession code 3O31).
Notes and references
1 C. Curie, G. Cassin, D. Couch, F. Divol, K. Higuchi, M. Le Jean,
J. Misson, A. Schikora, P. Czernic and S. Mari, Ann. Bot., 2009,
103, 1–11.
2 (a) I. Benes, H. Ripperger and A. Kircheiss, Experientia, 1983,
39, 261–262; (b) R. Rellan-Alvarez, J. Abadia and A. Alvarez-
Fernandez, Rapid Commun. Mass Spectrom., 2008, 22, 1553–1562;
(c) S. M. Reichman and D. R. Parker, Plant Physiol., 2002, 129,
1435–1438; (d) N. von Wiren, S. Klair, S. Bansal, J. F. Briat,
H. Khodr, T. Shioiri, R. A. Leigh and R. C. Hider, Plant Physiol.,
1999, 119, 1107–1114.
Fig. 3 Position of L110 and the secondary amine filter. Hydrogen
bonds between the reaction intermediate (AP2–Glu1) and the residues
in the cavity of MtNAS are depicted as dashed lines. Secondary
amines are selected at the position of Glu1 because primary amines
would have a hydrogen atom pointing toward L110 that could not be
stabilized.
compared with the bonds involved in stabilizing the glutamate
in its initial position. However, this does not really explain
why glutamate does not bind straightaway in S3. We believe
that the reason could be the very different affinities of the S3
subsite for secondary and primary amines. Indeed, details
of the interaction between the protein and the reaction
intermediate show that the nitrogen atom of Glu1 is involved
in two hydrogen bonds (one with Oe1 of Asn256 and one
intramolecular; Fig. 3). If one glutamate molecule were free to
translocate to subsite S3, the position of the third hydrogen of
its amine group would point toward the strictly conserved Leu
110. Because the side chain of this residue cannot engage any
hydrogen-bond, primary amines are clearly destabilized at this
position. Therefore, Leu110 plays a key role in the catalysis:
by rejecting primary amines from the bottom of the cavity and
admitting the secondary amine formed after the first catalytic
step, it ensures an ordered substrate binding and controls the
translocation process.
3 G. Weber, N. von Wiren and H. Hayen, BioMetals, 2008, 21,
503–513.
4 M. Takahashi, Y. Terada, I. Nakai, H. Nakanishi, E. Yoshimura,
S. Mori and N. K. Nishizawa, Plant Cell, 2003, 15, 1263–1280.
5 S. Lee, U. S. Jeon, S. J. Lee, Y. K. Kim, D. P. Persson, S. Husted,
J. K. Schjorring, Y. Kakei, H. Masuda, N. K. Nishizawa and
G. An, Proc. Natl. Acad. Sci. U. S. A., 2009, 106, 22014–22019.
6 L. Zheng, Z. Cheng, C. Ai, X. Jiang, X. Bei, Y. Zheng,
R. P. Glahn, R. M. Welch, D. D. Miller, X. G. Lei and
H. Shou, PLoS One, 2010, 5, e10190.
7 C. Dreyfus, D. Lemaire, S. Mari, D. Pignol and P. Arnoux, Proc.
Natl. Acad. Sci. U. S. A., 2009, 106, 16180–16184.
8 C. Dreyfus, D. Pignol and P. Arnoux, Acta Crystallogr., Sect. F:
Struct. Biol. Cryst. Commun., 2008, 64, 933–935.
9 M. Bouazaoui, J. Martinez and F. Cavelier, Eur. J. Org. Chem.,
2009, 2729–2732.
10 M. Bouazaoui, M. Larrouy, J. Martinez and F. Cavelier, Eur. J.
Org. Chem., 2010, 6609–6617.
11 R. P. McGeary, Tetrahedron Lett., 1998, 39, 3319–3322.
12 R. F. W. Jackson, R. J. Moore, C. S. Dexter, J. Elliot and
C. E. Mowbray, J. Org. Chem., 1998, 63, 7875–7884.
13 (a) J. Green, O. M. Ogunjobi, R. Ramage, A. S. J. Stewart,
S. Mccurdy and R. Noble, Tetrahedron Lett., 1988, 29,
4341–4344; (b) R. Ramage and J. Green, Tetrahedron Lett.,
1987, 28, 2287–2290.
14 (a) K. Wisniewski and A. S. Kolodziejczyk, Tetrahedron Lett.,
1997, 38, 483–486; (b) A. S. Kolodziejczyk, E. Sugajska,
B. Falkiewicz and K. Wisniewski, Synlett, 1999, 1606–1608.
15 J. A. Dunkle and J. H. Cate, Annu. Rev. Biophys. Biomol. Struct.,
2010, 39, 227–244.
16 A. Koglin and C. T. Walsh, Nat. Prod. Rep., 2009, 26, 987–1000.
17 P. H. Von Hippel, F. R. Fairfield and M. K. Dolejsi, Ann. N. Y.
Acad. Sci., 1994, 726, 118–131.
18 W. A. Breyer and B. W. Matthews, Protein Sci., 2001, 10,
1699–1711.
We now have snapshots of almost all the steps that lead
to the formation of tNA by MtNAS. A realistic molecular
movie can be generated that is based on experimental three-
dimensional structures (ESIw, Movie 1). Although not comparable
to much more complex enzymes such as the ribosome15 or
the non-ribosomal-peptide-synthase,16 the case of MtNAS is
interesting with regard to its processivity. Indeed, processive
enzymes are defined as proteins that remain attached to their
substrate and perform multiple catalytic cycles before disso-
ciating.17 In terms of the three-dimensional structure, processive
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 5825–5827 5827