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§1§ The Crystal Structures of Apo and Complexed Saccharomyces cerevisiae
GNA1 Shed Light on the Catalytic Mechanism of an Amino-sugar
N-Acetyltransferase[15]* §1§
1. [16]Caroline Peneff[17]‡[18]§,
2. [19]Dominique Mengin-Lecreulx[20]¶ and
3. [21]Yves Bourne[22]‡[23]‖
1.
From the ^‡UMR 6098 CNRS, 31 chemin Joseph Aiguier, 13402 Marseille
Cedex 20, France and ^¶UMR 8619 CNRS, Université Paris-Sud, Bâtiment
430, 91405 Orsay Cedex, France
[24]Next Section
§2§ Abstract §2§
The yeast enzymes involved in UDP-GlcNAc biosynthesis are potential
targets for antifungal agents. GNA1, a novel member of the Gcn5-related
N-acetyltransferase (GNAT) superfamily, participates in UDP-GlcNAc
biosynthesis by catalyzing the formation of GlcNAc6P from AcCoA and
GlcN6P. We have solved three crystal structures corresponding to the
apo Saccharomyces cerevisiae GNA1, the GNA1-AcCoA, and the
GNA1-CoA-GlcNAc6P complexes and have refined them to 2.4, 1.3, and 1.8
Å resolution, respectively. These structures not only reveal a stable,
β-intertwined, dimeric assembly with the GlcNAc6P binding site located
at the dimer interface but also shed light on the catalytic machinery
of GNA1 at an atomic level. Hence, they broaden our understanding of
structural features required for GNAT activity, provide structural
details for related aminoglycosideN-acetyltransferases, and highlight
the adaptability of the GNAT superfamily members to acquire various
specificities.
The Gcn5-related N-acetyltransferases (GNATs)^1 represent a large
superfamily of functionally diverse enzymes that catalyze the transfer
of an acetyl group from AcCoA to the primary amine of a wide range of
acceptor substrates (for a review, see Ref. [25]1). Recently,
three-dimensional structural information has become available with the
structures of two aminoglycoside N-acetyltransferases fromSerratia
marcescens (SmAAT) ([26]2) and Enterococcus faecium (EfAAT) ([27]3);
five histone acetyltransferases (HATs): PCAF ([28]4), HAT1 ([29]5),
Tetrahymena GCN5 (tGCN5) ([30]6, [31]7), yeast GCN5 (yGCN5) ([32]8),
and Hpa2 ([33]9); and one arylalkylamineN-acetyltransferase, the
serotoninN-acetyltransferase (AANAT) ([34]10). These structures have
revealed a structurally conserved GNAT core that is largely involved in
AcCoA binding and incorporates elements from the four conserved
sequence motifs (A to D) initially identified by sequence analysis
([35]11,[36]12).
Although the GNAT AcCoA binding site is well documented, the binding
site of the acceptor substrate has been characterized in only two
cases: the histone binding site in the tGCN5-CoA-H3 peptide complex
structure ([37]7) and the serotonin binding site in the
AANAT-bisubstrate analog complex structure ([38]10). These two
structures also provide the first glances into the catalytic machinery
of GNAT. Yet, a better understanding of the diverse modes of acceptor
substrate recognition and the catalytic mechanism of the GNATs as well
as further insights into the evolution of this superfamily still await
additional structural studies of GNAT-substrate complexes.
Glucosamine-6-phosphate N-acetyltransferase 1 (GNA1) is a novel
amino-sugar N-acetyltransferase member of the GNAT superfamily. GNA1
holds a key position in the pathway toward de novo synthesis of the
essential metabolite UDP-GlcNAc and is present in various eukaryotic
organisms (Fig. [39]1 A). The GNA1 murine homologue, EMeg32, which
possesses an extra 31-residue NH[2]-terminal region compared with the
yeast homologues, has recently been characterized ([40]13). EMeg32 is
essential for embryonic development, and its inactivation in mouse
embryonic fibroblasts generates resistance to apoptotic stimuli and
defects in cell proliferation ([41]14). GNA1 has also been
characterized in yeast ([42]15) and shown to be essential to the
survival of Saccharomyces cerevisiae in which it controls multiple cell
cycle steps ([43]16). In addition, Candida albicans GNA1 null mutants
exhibit reduced virulence when injected into mice ([44]17), making GNA1
a potential target for the development of new antifungal agents.
[45]Figure 1
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Figure 1
Sequence conservation and quality of the GNA1 structure. A, sequence
alignment of GNA1 homologues. Conserved and similar residues are
highlighted withblack and gray backgrounds, respectively. GNA1
secondary structure elements forming the structurally conserved GNAT
core are shown in black. The sequence alignment for SmAAT and Hpa2 is
based on a structural comparison with GNA1. Subunit 1 residues involved
in the GlcNAc6P and AcCoA binding sites are identified by filled
circles and filled triangles, respectively, and are shown in black for
the residues making GNAT conserved interactions. The unfilled circles
indicate subunit 2 residues that complete the GlcNAc6P binding site of
subunit 1. The four GNAT sequence motifs areboxed. B, stereoview of the
1.3 Å resolution experimental, solvent-flattened, averaged electron
density map contoured at 1.25 ς around an AcCoA molecule.
We present here three high resolution crystal structures of GNA1. The
apo GNA1 and the binary GNA1-AcCoA complex structures were solved
independently using MAD techniques and refined to 2.4 and 1.3 Å
resolution, respectively (Table [49]I). The structure of the
GNA1-CoA-GlcNAc6P ternary complex was solved by molecular replacement
and refined to 1.8 Å resolution (Table[50]I). These three structures
reveal GNA1 catalytic features and provide the first complete picture
of an amino-sugar GNAT active site as a first step toward the
development of specific inhibitors.
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Table I
Structural statistics
[53]Previous Section[54]Next Section
§2§ EXPERIMENTAL PROCEDURES §2§
§5§ Expression and Purification of the Native and Selenomethionyl
Proteins §5§
Full-length GNA1 was amplified from genomic S. cerevisiae DNA, cloned
into the pQE30 expression vector, and transformed into M15pREP4
(Qiagen) Escherichia coli cells. Protein expression was induced with
0.1 mmisopropyl-1-thio-β-d-galactopyranoside for 20 h at 37 °C.
Selenomethionyl GNA1 was produced as previously published ([55]18). The
recombinant His-tagged native and selenomethionyl enzymes were purified
via nickel affinity and anion exchange chromatographies, dialyzed
against 10 mm Tris-HCl, pH 8, 150 mm NaCl, 1 mm dithiothreitol, and
concentrated to 10 mg/ml. The activity of these enzymes was verified.
§5§ Crystallization §5§
Crystals of the native and selenomethionyl protein were grown at 20 °C
using the hanging-drop vapor diffusion method by mixing equal volumes
of the protein solution and the reservoir, which contained 17–22%
polyethylene glycol 600 and 0.2/0.4m imidazole/malate, pH 5.1. Crystals
obtained from the apo protein belong to the orthorhombic space group
P2[1]2[1]2[1] and contain 6 molecules/asymmetric unit. Those obtained
from GNA1 preincubated overnight with 1 mm AcCoA or CoA belong to the
monoclinic space group C2 and contain 4 molecules/asymmetric unit. For
data collection, the crystals were transferred to a reservoir solution
containing 32% polyethylene glycol 600 and flash-frozen at 100 K in
gaseous nitrogen.
§5§ Data Collection §5§
A three-wavelength MAD experiment was performed at the ESRF beamline
ID14-4 (European Synchroton Radiation Facility, Grenoble, France) on
both the AcCoA-complexed and apo selenomethionyl protein crystals at
2.8 and 3.2 Å resolution, respectively. High resolution data sets were
collected at 2.4 and 1.3 Å resolution on an apo GNA1 crystal and a
GNA1-AcCoA complex crystal, respectively, on beamline ID14-2 (ESRF,
Grenoble). A 1.8 Å resolution data set was collected at EMBL-X31 (DESY,
Hamburg) from a GNA1-CoA complex crystal soaked in the reservoir
solution supplemented with 1 mm GlcNAc6P for 6 days.
§5§ Structure Solution, Model Building, and Refinement §5§
All data were processed and reduced using DENZO ([56]19) and the CCP4
program suite ([57]20). The apo and AcCoA-complexed GNA1 structures
were solved independently using SOLVE ([58]21). The experimental MAD
electron density maps were improved by solvent flattening,
non-crystallographic symmetry averaging, and phase extension with the
program DM ([59]20). The apo GNA1 model was built manually using
TURBO-FRODO ([60]22). The GNA1-AcCoA complex model was built
automatically using ARP/wARP ([61]23). These two models were refined
against their respective high resolution data set using the program CNS
([62]24), including bulk solvent and anisotropic B-factor corrections.
NCS restraints were used only for the apo GNA1 model. High temperature
factors and weak electron density maps are associated with residues
Gln-52 to Lys-57 in the two models. The 3 intertwined dimers of the apo
GNA1 model have an average root mean square deviation of 0.7 Å for all
Cα atoms. In the GNA1-AcCoA complex model, the root mean square
deviation value between the 2 dimers is 0.4 Å for all Cα atoms. The
GNA1-CoA-GlcNAc6P complex structure was obtained from a rigid body
refinement using the GNA1-AcCoA complex as a starting model. Fourier
difference maps clearly revealed the location of the bound CoA and
GlcNAc6P in two of the four molecules. The structure of the
GNA1-CoA-GlcN6P complex was also solved at 2.5 Å; superimposition of
the two ternary complexes (GNA1-CoA-GlcNAc6P and GNA1-CoA-GlcN6P)
revealed that GlcN6P (the substrate) and GlcNAc6P (the reaction
product) were positioned similarly. Because the GNA1-CoA-GlcNAc6P
structure was obtained at a higher resolution than that of
GNA1-CoA-GlcN6P, we only considered in the analysis the
GNA1-CoA-GlcNAc6P complex structure. The stereochemistry of the refined
models was analyzed by PROCHECK ([63]25); no residue was found in the
disallowed regions of the Ramachandran plot. The coordinates of apo,
AcCoA-, and CoA-GlcNAc6P-complexed GNA1 have been deposited in the
Protein Data Bank (accession codes [64]1I21, [65]1I12, and [66]1I1D).
Figs. [67]1, 2, 4, and 5 were generated with SPOCK ([68]26) and
Raster3D ([69]27) except for Fig. [70]1 A, which was computed with
Alscript ([71]28).
[72]Previous Section[73]Next Section
§2§ RESULTS AND DISCUSSION §2§
§5§ Overall Structure §5§
The three-dimensional structures of GNA1 in its apo state and complexed
forms with AcCoA or with CoA and GlcNAc6P have been solved and refined
at 2.4, 1.3, and 1.8 Å resolution, respectively. Overall, the electron
density is well defined for these structures (Fig. [74]1 B) except for
a surface loop comprising residues Gln-52 to Lys-57 (cf.“Experimental
Procedures”). As predicted from sequence analysis, GNA1 shares
structural similarities with other GNAT superfamily members ([75]1,
[76]11). The GNA1 fold consists of a central core, composed of a mixed
5-stranded β-sheet flanked by 4 α-helices, and a COOH-terminal strand
β6, which is projected away from the central core (Fig.[77]2 A).
[78]Figure 2
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Figure 2
Structure of GNA1 and AcCoA, GlcNAc6P binding sites. A, ribbon
representations of the GNA1 fold (left) and the intertwined GNA1 dimer
(right). In subunit 1, the GNA1 secondary structure elements forming
the structurally conserved GNAT core are shown ingreen, the exchanged
β-strand in yellow, and the remaining structural elements in cyan.
Subunit 2 is shown in magenta with its exchanged strand β6 inred. The
molecular surface of AcCoA-(B) and CoA-GlcNAc6P-(C) complexed GNA1,
oriented as in Fig.[82]1 B (left view) and color-coded (B) as in Fig.
[83]1 B, with the regions undergoing small structural rearrangements
upon AcCoA binding displayed under a transparent surface (the cyan and
yellow bonds refer to the apo and AcCoA-complexed GNA1 models,
respectively). AcCoA is shown with carbon (white), nitrogen (blue),
sulfur (green), oxygen (red), and phosphorous (purple) atoms. C, the
color code is according to the electrostatic potential with positive
and negative charges shown inblue and red, respectively. The essential
catalytic Tyr-143 is displayed through a transparent surface. CoA and
GlcNAc6P are shown with yellow carbon atoms. D, stereoview of the
GlcNAc6P binding site with residues from subunit 1 and 2 shown in cyan
and magenta, respectively. The dotted lines indicate hydrogen bonds.
Residues within the GNAT conserved β-bulge are displayed in green.
The GNA1 structure is dimeric in the crystal as well as in solution, as
attested from gel filtration data (not shown). The crystalline dimer is
made of two intertwined GNA1 monomers in which strand β6 of one subunit
exchanges with the identical strand from the other subunit (Fig. [84]2
A). A β-strand exchange between subunits in a dimer is an unusual
feature among GNATs and has been observed only in the HAT Hpa2
structure ([85]9). In all other structurally characterized GNAT, except
Hat1 that lacks a β6 strand ([86]5), the hinge loop preceding strand β6
folds back onto its own subunit. This difference is reminiscent of
three-dimensional domain-swapped proteins in which the loop that
precedes the exchanged domain can switch from a closed to an opened
conformation thereby leading to either a monomeric or a dimeric form
([87]29). In the case of GNA1 or Hpa2, the α4–β6 loop is too small to
undergo such a conformational switch, and the dimeric assembly is
further stabilized by a hydrophobic interface, two features that make
three-dimensional domain swapping unlikely. Nonetheless, the monomeric
GNATs and the intertwined dimers of GNA1 and Hpa2 are most probably
related by divergent evolution from a common ancestor, and the
evolutionary mechanisms that have led to dimer formation may have
included three-dimensional domain swapping.
§5§ The Cofactor Binding Site §5§
In each subunit of the GNA1 dimer, AcCoA is positioned in a large
hydrophobic cleft located at the site where the two parallel strands,
β4 and β5, diverge because of a β-bulge in strand β4 that positions the
side chains of Glu-98 and Asp-99 on the same face of the β-sheet. The
presence of this β-bulge is remarkably well conserved among GNATs,
which suggests a critical role for this structural element in the
formation of the AcCoA binding site.
AcCoA adopts a conformation similar to that described in other
AcCoA-complexed GNAT structures ([88]1). The acetyl group of AcCoA,
which marks the active site, is located between strand β5 and the
β-bulge and is largely stabilized by contacts with the protein; the two
carbon atoms contract hydrophobic interactions with residues Ile-100,
Leu-133, and Tyr-143, and the carbonyl oxygen inserts into an oxyanion
hole formed by the backbone amides of residues Asp-99 and Ile-100. Such
an oxyanion hole has been observed in the structurally
relatedN-myristoyltransferase ([89]30) but is a unique feature within
the structurally characterized GNATs.
Superimposition of the apo and AcCoA-complexed GNA1 structures shows
that AcCoA binding induces subtle structural rearrangements that are
confined to the edges of the cleft and result in a slightly narrower
cleft. Residues 102–109 in the α3–β5 loop and 134–143 in the β5–α4 loop
plus the N-cap of α4 move by ∼1.3 and 1.1 Å, respectively, toward the
center of the cleft (Fig. [90]2 A). Whether these conformational
changes, induced upon cofactor binding, are a prerequisite for acceptor
substrate binding as shown for other GNATs ([91]31-33) needs to be
ascertained by kinetic studies. A detailed comparison with other GNATs
reveals that these rearrangements differ from those reported for (i)
tGCN5, in which the cofactor-binding cleft opens slightly upon AcCoA
binding to accommodate the histone tail ([92]7); and (ii) AANAT, in
which a major rearrangement of the α1-loop-α2 region occurs upon AcCoA
binding to complete the serotonin binding site ([93]10). Therefore,
although the binding of AcCoA is similar among GNATs, it induces
different conformational changes that can contribute to the specific
binding of the acceptor substrate.
§5§ The Acceptor Substrate Binding Site §5§
The GNA1 amino-sugar binding site exhibits an atypical architecture, as
it is built at the dimer interface and involves residues from the
exchanged β-strand, two features found only in a few intertwined
oligomeric structures such as that of bovine seminal ribonuclease
([94]34). GlcNAc6P binds at the base of the AcCoA cleft within a small
pocket that is lined mostly with electronegative residues except for a
patch of positively charged residues that specifically accommodate the
6-phosphate group (Fig.[95]2 B). Remarkably, the GlcNAc6P acetyl group
is positioned similarly to the cofactor acetyl group in the GNA1-AcCoA
complex structure (Fig. [96]2 B). The sugar-6-phosphate establishes
numerous hydrogen bonds, mainly via side chain atoms, together with a
few hydrophobic contacts such as that found between Leu-27 and the
β-face of the sugar ring (Fig. [97]2 C). Superimposition of the
structures of the cofactor-complexed SmAAT ([98]2) or EfAAT ([99]3)
(the two structurally characterized aminoglycoside GNATs) on that of
the GNA1-CoA-GlcNAc6P complex shows that SmAAT Phe-51, EfAAT Trp-25,
and GNA1 Leu-27 are identically positioned, an observation that
supports a common functional role for these residues, thereby
identifying an aminoglycoside recognition feature of GNATs.
§5§ Catalytic Mechanism §5§
Several reports on GlcN6PN-acetyltransferases suggest that catalysis
requires sulfhydryl group-containing residues, such as a cysteine that
could act as a nucleophile in a two-step mechanism involving the
formation of a covalent acetyl-cysteine enzyme intermediate ([100]35,
[101]36). In contrast, structural and kinetic data available for GNATs
support a mechanism proceeding through a direct nucleophilic reaction
of acceptor substrate on AcCoA ([102]1, [103]32, [104]33).
In the GNA1-AcCoA complex structure, the cysteine residue closest to
the AcCoA acetyl group lies 6.5 Å apart, too far to play a role in
acetyl transfer. Furthermore, no other appropriate nucleophile residue
is found in the proximity of the acetyl group, which makes the
formation of an acetyl-enzyme intermediate very unlikely and supports
the hypothesis of a single-step mechanism as suggested for GNATs.
Consistent with this hypothesis is the position of the amino group of
the product GlcNAc6P (similar to that of the substrate GlcN6P;cf.
“Experimental Procedures”), which is ideal to allow a direct
nucleophilic attack at the AcCoA carbonyl (Fig.[105]2 B). In addition,
the nucleophilic character of the amine is enhanced by the hydrogen
bond it establishes with the backbone carbonyl of Asp-134 (Fig. [106]2
C). The AcCoA carbonyl is polarized via hydrogen bonds to the backbone
amides of Asp-99 and Ile-100, located in the oxyanion hole, a feature
that facilitates the nucleophilic attack and stabilizes the negative
charge building up on the oxygen atom of the tetrahedral reaction
intermediate (Fig.[107]3). Finally, the Tyr-143 hydroxyl group, which
lies within hydrogen bond distance of the AcCoA sulfur atom (Fig.[108]2
B), could serve to stabilize the thiolate anion of the departing CoA
molecule. Tyr-143 also establishes hydrophobic contacts with the acetyl
group, probably playing a role in correctly positioning the acetyl
group for the reaction. A critical role of Tyr-143 in catalysis is
supported by mutagenesis data ([109]15). A close inspection of the
active site of GNA1 also pinpoints two significant structural and
functional differences with others members of the GNAT superfamily.
[110]Figure 3
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Figure 3
Schematic representation of the proposed GNA1 catalytic mechanism.
The first striking difference resides within the GNA1 β-bulge which,
when compared with the β-bulges in other GNAT structures, shows a
markedly different hydrogen bonding pattern. The GNAT β-bulge is an
irregularity of the antiparallel β structure in which two residues on
one strand are facing a single residue on the other strand ([114]37).
Such a β-bulge is formed when two consecutive residues in a β-strand
direct their backbone carbonyl or amide toward the same side of the
strand, thereby breaking the typical alternated pattern of a
β-structure. In GNA1, the backbone amides of Asp-99 and Ile-100 are
projected toward the active site and form the oxyanion hole, whereas
the carbonyls of Glu-98 and Asp-99 point toward β3. In all other
members of the GNAT superfamily (except EfAAT in which a proline
perturbs the bulge conformation ([115]3)), the situation is reversed;
the oxyanion hole is absent because the two consecutive backbone amides
are now directed toward strand β3 (establishing an hydrogen bond with
the backbone carbonyl of the facing residue), whereas two consecutive
main chain carbonyls are found pointing into the active site
(Fig.[116]4). These two carbonyls have been suggested to play a role in
acceptor substrate binding in tGCN5 ([117]7) and in the stabilization
of catalytic water molecules in AANAT ([118]10); this indicates that,
in addition to a common role in structuring the cofactor binding site,
the β-bulge could also fulfill other nonconserved catalytic functions.
[119]Figure 4
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Figure 4
The β-bulge structure in GNA1 and comparison with another GNAT
superfamily member. The structure of GNA1-CoA-GlcNAc6P (thick sticks
withlight gray carbons) is superimposed with that of AANAT (thin sticks
with dark gray carbons) in the vicinity of the β-bulge. Nitrogen and
oxygen atoms from the protein main chain or the acetylated amine of
GlcNAc6P are shown inblack. Light and dark gray dotted lines indicate
hydrogen bonds in the structures of GNA1 and AANAT, respectively.
The second important difference concerns the deprotonation of the
acceptor substrate amino group prior to the reaction. In the case of
tGCN5 and AANAT, a chain of well ordered water molecules, or “proton
wire,” connecting the acceptor substrate amino group to the proposed
catalytic bases tGCN5 Glu-122 or AANAT His-120 was suggested to be
involved in this proton removal ([123]7, [124]10). In the
GNA1-CoA-GlcNAc6P complex, a similar proton wire is observed, leading
to Glu-98 in which the side chain occupies a similar position as that
of tGCN5 Glu-122 and AANAT His-120. However, the E98A mutation does not
abolish the GNA1 activity ([125]15), suggesting that Glu-98 might not
function as the general base. Nevertheless, deprotonation prior to the
reaction might not be necessary in the case of GNA1, because the pK [a]
of GlcN6P (∼7.75) is lower than that of other GNAT acceptor substrates
such as lysine (8.95) or serotonin (∼10). This hypothesis is also
supported by the fact that the optimum pH of purified mammalian GlcN6P
N-acetyltransferases lies in the alkaline range ([126]35, [127]36) and
by the lower K [m] value ofS. cerevisiae GNA1 for GlcN6P at pH 8 than
at pH 7.5 ([128]13), suggesting that GNA1 may preferentially bind the
basic/deprotonated form of GlcN6P.
§5§ Substrate Specificity among GNATs §5§
Although the GNAT enzymes share structural similarities, they have
distinct acceptor specificities, consistent with their implication in
various biological processes. A comparative analysis of the complexes
of GNA1-CoA-GlcNAc6P, tGCN5-CoA-H3 peptide ([129]7), and
AANAT-bisubstrate analog ([130]7, [131]10) highlights the structural
determinants responsible for the substrate specificities among GNATs.
Importantly, this knowledge is essential for the design of specific
inhibitors for medical applications.
The structural comparison of these three complexes reveals that both
the NH[2]- and COOH-terminal regions diverge between the different GNAT
structures and are important for substrate specificity. The
NH[2]-terminal structural differences concern the α1-loop-α2 region and
are relatively minor, whereas more dramatic changes occur in the
COOH-terminal end. In AANAT, the α4–β6 loop orients toward the active
site as it folds back on its own subunit. This loop, along with the
α1–α2 loop, almost covers the active site, thus facilitating the
binding of a hydrophobic substrate (Fig.[132]5 A). In tGCN5, the
20-residue segment inserted between α4 and β6 contributes to one side
of the substrate binding canal, providing specific binding residues for
the histone tail (Fig. [133]5 B).
[134]Figure 5
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Figure 5
Structural comparison of substrate binding sites of AANAT , tGCN5, and
GNA1. The molecular surfaces around the active site of the
AANAT-bisubstrate analog (A), the tGCN5-CoA-H3peptide (B), and the
GNA1-CoA-GlcNAc6P complex structures (C) are viewed with a similar
orientation. From top to bottom, the serotonin-like moiety and the
acetyl group are shown in red, the H3 peptide backbone in yellow, with
its reactive Lys-14 side chain in orange, and GlcNAc6P in purple.
Structural divergences within the NH[2]- and COOH-terminal regions are
highlighted in dark blue andblue, respectively. The β3–β4 insertion
loop unique to AANAT is shown in brown.
In GNA1, the shorter α4–β6 loop does not participate directly in
acceptor substrate binding, but it forces strand β6 to extend and
exchange with the identical strand of the other subunit in the dimer.
Instead of using this loop, GNA1 exploits its intertwined oligomeric
state to achieve specific binding, because an important part of the
GNA1 active site consists of residues from the other subunit in the
dimer (Fig. [138]5 C). In contrast, this region is partly replaced in
monomeric AANAT by a long loop inserted between strands β3 and β4,
which contributes largely to one wall of the serotonin binding site
(Fig. [139]5 A). For the monomeric tGCN5, the short β3–β4 turn
contributes to the canal-shaped active site designed to accommodate a
long peptidic chain (Fig. [140]5 B).
§5§ Role in the Cell Cycle §5§
GNA1 was shown to control multiple cell cycle steps in S. cerevisiae
([141]16). It is still unclear whether this role is related to the
N-acetyltransferase activity of GNA1 in UDP-GlcNAc biosynthesis (which
implies a physiological link between UDP-GlcNAc and cell cycle
progression) or if it is the consequence of an additional function of
GNA1.
The hypothesis of an additional HAT activity for GNA1 was addressed,
but no HAT activity could be detected in vitro([142]15). Interestingly,
a comparison of GNA1 with the related GNAT structures reveals that the
closest structural homologue of GNA1 is the HAT Hpa2, which also adopts
an intertwined dimeric structure ([143]9). Superimposition of the two
structures reveals differences in the relative arrangement of the two
subunits, resulting in different acceptor substrate binding sites. A
narrow open-ended channel, in which the histone tail could insert, is
found in the Hpa2 structure instead of the rounded pocket of GNA1,
which seems unlikely to accommodate an extended and bulky histone tail.
Could GNA1 fulfill an additional function via the noncovalent
association with a particular cell compartment or with a protein
partner? An association with the cytoplasmic face of organelle
membranes has been described for EMeg32 (the GNA1 murine homologue)
which also co-purifies with the cdc48 homologue protein (p97/VCP)
([144]13). Double-hybrid systematic experiments performed in S.
cerevisiae revealed interactions between GNA1 and a priori unrelated or
unknown proteins ([145]38). Further biochemical experiments are needed
to determine the biological relevance of these protein/membrane
interactions.
UDP-GlcNAc is a key precursor of chitin (a component of the yeast and
fungal cell wall) as well as of the glycosylphosphatidylinositol anchor
of membrane-bound proteins and is essential to N-linked glycosylation
and O-GlcNAc modification of proteins. Glycosyltransferases involved in
N-glycosylation, such as the yeast GPT/alg7, which uses UDP-GlcNAc as a
substrate, have been suggested to play a role in the cell cycle
([146]39). In addition, a recent report shows that EMeg32-dependent
UDP-GlcNAc levels influence cell cycle progression and apoptosis
signaling ([147]14). Hence, the role of GNA1 in cell cycle progression
appears to be linked to its key GlcN6P N-acetyltransferase activity in
de novo UDP-GlcNAc biosynthesis. The structural data presented here
have allowed us to propose a catalytic mechanism for GNA1, as well as
providing a structural template for GNA1 homologues and related
aminoglycosides GNATs. Finally, these results further exemplify the
remarkable diversity of the GNAT superfamily and represent a critical
step toward the development of specific inhibitors.
[148]Previous Section[149]Next Section
§2§ ACKNOWLEDGEMENTS §2§
We thank Véronique Charrier, Bernard Henrissat, Pascale Marchot,
Florence Fassy, and Gerlind Sulzenbacher for helpful discussions,
Frédérique Pompéo for enzymatic assays, and the ESRF staff for
technical support in data collection.
[150]Previous Section[151]Next Section
§2§ Footnotes §2§
* [152]↵* This work was supported by grants from the CNRS to UMR 6098
(Marseille, Y. B.) and to UMR 8619 (Orsay, D. M.-L.).The costs of
publication of this article were defrayed in part by the payment of
page charges. The article must therefore be hereby marked
“advertisement” in accordance with 18 U.S.C. Section 1734 solely to
indicate this fact.
The atomic coordinates and the structure factors (code , , and )
have been deposited in the Protein Data Bank, Research
Collaboratory for Structural Bioinformatics, Rutgers University,
New Brunswick, NJ ([153]http://www.rcsb.org/).
* [154]↵§ Holder of a CNRS Ph.D. fellowship.
* [155]↵‖ To whom correspondence should be addressed. Tel.:
+33-4-91-16-45-08; Fax: +33-4-91-16-45-36; E-mail:
yves@afmb.cnrs-mrs.fr.
* Published, JBC Papers in Press, February 9, 2001, DOI
10.1074/jbc.M009988200
* Abbreviations:
GNAT
Gcn5-relatedN-acetyltransferase
HAT
histone acetyltransferase
AANAT
serotonin N-acetyltransferase
MAD
multi-wavelength anomalous dispersion
*
+ Received November 2, 2000.
+ Revision received February 9, 2001.
* The American Society for Biochemistry and Molecular Biology, Inc.
[156]Previous Section
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