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(Received for publication, July 24,
1995; and in revised form, September 25, 1995) From the
Crystallographic analysis and site-directed mutagenesis have
been used to identify the catalytic and oligosaccharide recognition
residues of
peptide-N
Peptide-N Two crystal structures of PNGase F, in
different crystal forms, were recently
published(6, 7) . The molecular structure, shown in Fig. 1, consists of two eight-stranded antiparallel
Figure 1:
Stereo ribbon diagram showing the
In this
paper we describe the results of site-directed mutagenesis studies,
resulting in the identification of the active site area, and of the
crystallographic analysis of the complex of the enzyme with the product
disaccharide N,N`-diacetylchitobiose. These studies
lead to the unambiguous identification of the catalytic residues and
the detailed analysis of the oligosaccharide recognition site.
The D60N, E206Q, and
E118Q mutants were overexpressed in E. coli, purified in large
quantities, and crystallized. All three crystallized in the same space
group under nearly identical conditions as the wild-type
enzyme(16) . The crystallographic analysis showed that their
structures are essentially identical to that of the wild-type enzyme,
confirming that the reduced activities are caused by diminished
catalytic functionality or impaired substrate binding, not by altered
protein conformation. (
The
crystals of the complex were grown under identical conditions and
belong to the same space group as those of the uncomplexed protein. The
structure of the complex of the wild-type enzyme has been fully refined
to an R value of 0.197 at 2.0-Å resolution. Fig. 2shows the electron density of the disaccharide in the
refined structure. The protein conformation is essentially unaffected
by the binding of the ligand. The least-squares fit of the main chain
atoms of all but the four N-terminal residues has a root mean square
deviation of 0.33 Å and a maximum deviation of 1.30 Å. All
of the largest deviations are in loops that are far removed from the
binding cleft. The main difference in the cleft is a slight change in
the orientation of the side chain of Trp-191, with a shift in the
position of N
Figure 2:
Stereodiagram showing the electron density
for the N,N`-diacetylchitobiose in the final
2F
In solution, N,N`-diacetylchitobiose exists in an equilibrium
state containing a mixture of Fig. 3shows the
location and orientation of the N,N`-diacetylchitobiose molecule in the cleft of the
enzyme. The molecule is inserted edgewise into the cleft and spans the
distance between the two groups of acidic residues in the cleft, with
O
Figure 3:
Detailed image of the interactions of N,N`-diacetylchitobiose with PNGase F showing the
hydrogen bonding interactions between the disaccharide, water
molecules, and the protein. Aromatic residues also make important
contacts with the substrate. Trp-191 is positioned nearly perpendicular
to the disaccharide and forms a hydrogen bonding contact rather than a
hydrophobic interaction. Trp-120 forms a hydrogen bond with O
Figure 4:
Schematic diagram showing the
intermolecular hydrogen bonding contacts between PNGase F, N,N`-diacetylchitobiose and water molecules. Protein
residues are indicated with single-letter amino acid code and sequence
number in rectangular boxes, water molecules are indicated by a number,
corresponding to their number in the file deposited with the Protein
Data Bank. The reducing end GlcNAc residue is on the left. Hydrogen
bonding distances, in Å, are shown in italics. Note that
Wat
Figure 5:
GRASP-image (23) showing the
molecular envelope of PNGase F, cut-off through the oligosaccharide
binding cleft to show the penetration of the N-acetyl group of
the first GlcNAc residue into the cavity in the core of the molecule.
The carbonyl oxygen makes hydrogen bonds with two water molecules in
the cavity, while the methyl group is in close proximity to a
hydrophobic area, primarily formed by the Ile-82 side chain. The
protein surface is colored according to its electrostatic potential.
Potentials greater than 9 T are blue, those less than
-22 T are red, and neutral ones are white.
The solvent-accessible surface of the protein is reduced by
less than 200 Å Site-directed mutagenesis experiments were used to determine
that the active site of PNGase F is located in the cleft at the
interface between the two domains of the molecule and showed that three
acidic residues, Asp-60, Glu-118, and Glu-206, are essential for
activity. The combination of this information with the crystal
structure analysis of the complex with N,N`-diacetylchitobiose clearly reveals the distinct
roles of these residues in the mechanism of action of the enzyme.
Mutagenesis of Asp-60 to the corresponding asparagine results in total
loss of activity. This residue is in direct hydrogen bonding contact
with O The geometry of the complex structure in the area of Glu-118 clearly
illustrates why this residue is important for substrate binding and why
the E118Q mutation impairs substrate binding. O These studies have unambiguously identified
the active site residues and the oligosaccharide binding mechanism of
PNGase F. The exact nature of the mechanism of action of this enzyme is
at this time still unclear. However, it is obvious from the lack of
serine or threonine residues in the active site area that, contrary to
what was initially expected, the mechanism is not related to those of L-asparaginase or glycosylasparaginase. These studies will
form the basis for further analysis, which will be focused on the
analysis of the interaction of the enzyme with the peptide component of
the substrate.
The atomic
coordinates (code 1PNF) have been deposited in the Protein Data Bank,
Brookhaven National Laboratory, Upton, NY.
Volume 270,
Number 49,
Issue of December 8, 1995 pp. 29493-29497
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
-(N-acetyl-
-
D
-glucosaminyl)asparagine
Amidase F (*)
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
-(N-acetyl-
-D-glucosaminyl)asparagine
amidase F (PNGase F), an amidohydrolase that removes intact
asparagine-linked oligosaccharide chains from glycoproteins and
glycopeptides. Mutagenesis has shown that three acidic residues,
Asp-60, Glu-206, and Glu-118, that are located in a cleft at the
interface between the two domains of the protein are essential for
activity. The D60N mutant has no detectable activity, while E206Q and
E118Q have less than 0.01 and 0.1% of the wild-type activity,
respectively. Crystallographic analysis, at 2.0-Å resolution, of
the complex of the wild-type enzyme with the product, N,N`-diacetylchitobiose, shows that Asp-60 is in
direct contact with the substrate at the cleavage site, while Glu-206
makes contact through a bridging water molecule. This indicates that
Asp-60 is the primary catalytic residue, while Glu-206 probably is
important for stabilization of reaction intermediates. Glu-118 forms a
hydrogen bond with O
of the second N-acetylglucosamine residue of the substrate and the low
activity of the E118Q mutant results from its reduced ability to bind
the oligosaccharide. This analysis also suggests that the mechanism of
action of PNGase F differs from those of L-asparaginase and
glycosylasparaginase, which involve a threonine residue as the
nucleophile.
-(N-acetyl--D-glucosaminyl)asparagine
amidase F (PNGase F, (
)peptide N-glycanase) is a
34.8-kDa amidohydrolase secreted by Flavobacterium
meningosepticum(1) . The enzyme cleaves the
-aspartylglucosamine bond of asparagine-linked oligosaccharides,
converting the asparagine residue to an aspartic acid(2) . The
1-amino-oligosaccharide product spontaneously hydrolyzes to ammonia and
the intact oligosaccharide chain(3) . The enzyme has a broad
substrate specificity, but both the amino and carboxyl groups of the
asparagine residue have to be in peptide linkage, while the
oligosaccharide must consist at least of the N,N`-diacetylchitobiose core,
GlcNAc14GlcNAc. The enzyme is highly sensitive to
modifications of this core; an
-13-fucose substituent on the
asparagine-proximal, or reducing end, GlcNAc completely blocks PNGase F
activity(4) , but an
-16-fucose substituent has no
effect. The enzyme is extensively used as a biochemical tool for the
study and analysis of glycoproteins(5) , including the analysis
of oligosaccharides as well as the deglycosylation of glycoproteins for
structural analysis.
-sandwiches that lie side-by-side so that the interface runs the
full length of the -sheets. Very little information regarding the
mechanism of action of the enzyme was available when the structures
were determined, and the assignment of a possible active site was based
primarily on information obtained from the studies of other
asparaginases: L-asparaginase (8) and
glycosylasparaginases(9, 10) . The active site of L-asparaginase includes a triad of hydrogen-bonded Asp-Lys-Thr
residues, where the threonine is thought to be the nucleophile in the
reaction mechanism. Glycosylasparaginases also have an essential
threonine residue. This information, combined with observation of
surface features of the molecule, led to the identification of three
possible substrate binding sites(6) : a bowl on one face of the
molecule that contains a group of residues that somewhat resemble the
active site of L-asparaginase, a shallow S-shaped cleft on the
opposite face of the molecule that contains a number of acidic residues
and threonine residues, and a deep cleft at the interface between the
two domains at one end of the molecule. This cleft, formed by the loops
connecting -strands between the -sheets contains several
acidic residues and serines, as well as many aromatic residues. Most
remarkable is the presence of five tryptophan residues.
-carbon tracing of the three-dimensional structure of PNGase F
with N,N`-diacetylchitobiose in the substrate binding
cleft. The molecule consists of two nearly identical eight-stranded
antiparallel -barrel domains with jelly roll motifs. Side chains
of representative residues in the three areas initially identified as
possible active sites are shown: Thr-101, lower back; Thr-72, lower
front; Glu-118, top front; and, Asp-60 and Glu-206, top rear. The
figure was prepared with program
Molscript(21) .
Site-directed Mutagenesis
The coding sequence of
PNGase F was cloned into pLITMUS 29 (New England Biolabs).
Site-directed mutagenesis was performed following the Kunkel
protocol(11) , using 39-base pair oligonucleotides bearing the
site of mutation at positions 22-24. All substitutions were
confirmed by DNA sequencing at the mutation site in the pLITMUS 29
vector and again by sequencing of the full PNGase F gene in the pMAL p2
construct. Single colonies were picked until at least two independent
clones could be derived that harbored the same mutation. The New
England Biolabs pMAL p2 fusion kit and protocol were used for
expression and purification. Cells were grown at 37 °C and
expression was induced after 3 h by addition of 1 mM
isopropyl-1-thio--D-galactopyranoside. Incubation was
continued for 8 h at 30 °C followed by 12 h at room temperature.
After purification over an amylose column, the fusion protein was
cleaved using a 12-h room temperature incubation with 3% Factor Xa,
resulting in an 85% yield of the cleavage product. A Q-Sepharose
(Pharmacia Biotech Inc.) column was used for the final purification to
homogeneity. Enzymatic activity of the fusion proteins was measured by
enzyme concentration-dependent gel-shift assays of the deglycosylation
of denatured RNase B. An HPLC assay(12) , using a
didansylfetuin glycopeptide, Leu-Ala-Asn(CHO)-AeCys-Ser as the
substrate, was used to compare the activities of the most interesting
mutants with that of the wild-type enzyme and for the competitive
inhibition experiments.X-ray Crystallography
Solutions containing a 30:1
molar ratio of N,N`-diacetylchitobiose (Fine grade,
Seikagaku America) to PNGase F were prepared by adding lyophilized
disaccharide to 100-µl volumes of protein at concentrations of
about 10 mg/ml. These solutions were incubated at 37 °C for 1 h
prior to set-up of crystallization experiments. X-ray diffraction data
for the N,N`-diacetylchitobiose complex of the
wild-type enzyme were measured using a Rigaku R-Axis image plate area
detector equipped with focussing mirrors. The crystal was flash-cooled
to approximately -140 °C immediately after addition of an
equal volume of 50% glycerol to the crystallization drop. A total of
104,022 reflections were measured to 2.0-Å resolution. These were
averaged to unique 26,902 data with an R (
I -
<I>
/
<I>) of 0.044. A F > 2
(F) cut-off was applied to the data, which
are 94.1% complete. The structure was refined with the program X-PLOR (13) , using the protein coordinates of the room temperature
wild-type structure as the starting point. After the initial simulated
annealing refinement of the protein molecule, electron density for the N,N`-diacetylchitobiose was observed in
(2F
- F
) and (F - F) maps displayed
with the program CHAIN(14) . Further refinement using
alternating X-PLOR molecular dynamics refinement and model building led
to an R value of 0.197 for the data between 10.0 and 2.0
Å. The validity of the N,N`-diacetylchitobiose
modeling was checked intermittently with omit maps that were calculated
after simulated annealing refinement in which atoms within an 8-Å
sphere of the binding cleft area were omitted. The final model consists
of 2458 protein atoms, 29 N,N`-diacetylchitobiose
atoms, one sulfate anion, and 266 water molecules. The root mean square
deviations from ideality of the final model are: bonds 0.010 Å,
angles 1.67°, dihedrals 27°. Only Trp-86 falls outside the
allowed regions of the Ramachandran plot, while 90.4% of the
non-glycine residues are in the most favored region as defined in the
program PROCHECK(15) . The average thermal parameters are 18.4
Å
for the main chain atoms, 19.7 Å for the side chain atoms, and 16.6 Å for the
chitobiose. Details of the structure analysis of the mutant enzymes and
their complexes will be published elsewhere.
Site-directed Mutagenesis
Table 1lists
the mutants prepared as part of the search for catalytic residues.
Initially, selected residues in the three possible substrate binding
sites were mutated: acidic residues to the corresponding amides and
serine or threonine residues to alanine. Two of the mutants of residues
in the bowl area, T101A and D102N, have somewhat reduced activity,
while the third one, D99N, is fully active. The latter residue was
considered to be the most likely candidate for the acidic residue in
the L-asparaginase-like active site. Likewise, the two mutants
of residues in the S-shaped cleft, T71A and T72A, are fully active.
Therefore, these two areas were eliminated from further consideration.
The cleft at the interface between the two domains at the top end of
the molecule has two areas of interest that are about 12 Å apart
at opposite ends of the cleft. Both areas include acidic residues as
well as other polar residues that could be involved in catalysis or
substrate recognition. One group includes Glu-118, Ser-154, Ser-155,
and Asp-157. The E118Q mutant has greatly reduced activity, but the
S154A, S155A, and D157N mutants are fully active, indicating that only
the glutamic acid directly interacts with the substrate. At the other
end of the cleft, Asp-60 and Glu-206 are in hydrogen bonding contact
with each other via a very tightly bound water molecule. This water
molecule also makes additional contacts with Tyr-85 and Arg-248. When
the acidic residues were mutated, the D60N mutant was found to have
absolutely no activity, while the activity of E206Q was extremely low.
To further evaluate this site, three additional mutants were prepared:
D60E, E206D, and Y85F. The very low activity levels of the acidic
residue mutants confirm their role in catalysis while suggesting strict
geometric requirements. The hydroxyl group of Tyr-85, however, is not
of great importance in view of the relatively high activity of the Y85F
mutant. No other hydroxyl groups are present in the active site cleft.
Therefore, the mechanism of action of PNGase F, unlike those of L-asparaginase and glycosylasparaginase, does not appear to
involve an hydroxyl group as the nucleophile.
)X-ray Crystallographic Analysis of the
N,N`-Diacetylchitobiose Complex
PNGase F hydrolyzes
glycopeptides containing the minimal oligosaccharide moiety N,N`-diacetylchitobiose, GlcNAc14GlcNAc.
The initial oligosaccharide product of the PNGase F hydrolysis contains
the 1-amino-N,N`-diacetylchitobiose core, which
spontaneously degrades to N,N`-diacetylchitobiose.
This compound was tested as a potential inhibitor against the standard
PNGase F substrate, dansyl-Leu-Ala-Asn(CHO)-dansyl-AeCys-Ser using an
HPLC assay(12) . N,N`-Diacetylchitobiose
inhibited substrate hydrolysis by 36% at a 12.5-fold molar excess over
substrate but by only 50% at a 100-fold excess. Even though the
inhibitory activity of the disaccharide was relatively poor,
co-crystallization experiments were conducted, using a 30:1 molar ratio
of N,N`-diacetylchitobiose to PNGase F.
by 0.95 Å. The average thermal
parameters for the protein molecule are significantly lower in the
complex: 18.4 Å
and 19.5 Å for the
main chain and side chain atoms in the complex, compared with 23.2 and
25.4 Å in the uncomplexed structure. Interestingly,
the change in the thermal parameters is uniformly distributed over the
molecule and does not result from large changes in the binding cleft
area. The N,N`-diacetylchitobiose molecule has an
average thermal parameter of 16.6 Å with a range from
11.0 to 26.0 Å. The N-acetyl group of the
first GlcNAc is most deeply buried into the protein molecule and
correspondingly has the lowest thermal parameters.
- F
map, contoured at 1.5
of the
map.
- and -configurations of the
O-hydroxyl group at the reducing end. In the wild-type
PNGase F complex structure, only the -conformation is observed,
despite the fact that the oligosaccharide moiety attached to the
N of the asparagine is in the
-configuration. The
glycosidic link between the GlcNAc residues is in an extended
conformation with torsion angles,
(O
-C-O-C
)
-85° and
(C
-O-C
-C
)
-123°. These angles are well within the range of those
observed for other
-14-linked disaccharides in complexes
with lectins(17, 18) but are most similar to those
seen in complexes of lysozyme with GlcNAc
multimers(19, 20) .
of the reducing end GlcNAc residue forming hydrogen bonds
with Asp-60 and the water molecule, Wat, that connects
Asp-60 and Glu-206. O
of the second GlcNAc forms a hydrogen
bond with Glu-118. Fig. 4shows a schematic diagram of all
important intermolecular contacts. The first GlcNAc residue makes
extensive contacts with the protein, although several are mediated by
tightly bound water molecules. One interesting feature, shown in Fig. 5, is the location of the N-acetyl group, which
extends into a deep hole in the interface between the two domains of
the protein. The contacts of the second GlcNAc are much weaker and only
O and O
are involved in direct hydrogen bonds
with the protein. On the reducing end, Asp-60 forms hydrogen bonds with
O of the N,N`-diacetylchitobiose on one
side and with Wat on the other side. Glu-206 does not
make any direct contacts with the disaccharide. The position and
orientation of the disaccharide in the cleft easily explains the
inability of the enzyme to process substrates with an
-13-fucose on the first GlcNAc residue (5) and the
lack of interference of O
substitutions; O
is
buried and inaccessible while O is fully exposed to the
solvent.
but also appears to be positioned correctly to be able to make a
hydrophobic contact with the next residue, the first mannose, in the
intact substrate. The figure was prepared with the program
SECTOR(22) .
(349) is present twice, once in contact with
O
and once with Arg-61.
in the complex: 12,395 Å for the protein in the complex versus 12,587
Å in the uncomplexed structure. This is explained in
part by the location of the molecule toward one side of the cleft,
making close contacts with many residues of the N-terminal domain but
allowing for a layer of water molecules to form bridging contacts with
the residues of C-terminal domain. Many of these water molecules,
including Wat, Wat
, and Wat
,
are in nearly identical positions in the uncomplexed and complex
structures. Of all the water molecules involved in contacts with the
protein and the disaccharide, only Wat
and Wat
have no water molecule within 0.5 Å in the uncomplexed
structure. However, all water molecules that only make contacts with
the N,N`-diacetylchitobiose molecule are disordered
in the uncomplexed structure.
of the reducing-end GlcNAc residue. This atom
replaces the N of the asparagine in the substrate.
Therefore, this residue must be of primary importance in the catalytic
mechanism. The fact that the enzyme selects for the
-configuration
of O of the N,N`-diacetylchitobiose while
the N of the substrate is in the
-configuration
may prove to be important for further studies of the mechanism of
action. Glu-206, whose amide mutant has very low residual activity, is
not in direct contact with the product and is more likely to play a
secondary role, such as stabilization of a reaction intermediate or
interaction with the O of the substrate asparagine.
of the
second GlcNAc residue accepts a hydrogen atom in its hydrogen bond with
N of Trp-120. Therefore, it must donate a hydrogen
atom in its hydrogen bond to O
of Glu-118.
O
of the Glu-118 side chain is also involved in a
hydrogen bond with another hydrogen donor, Ser-155. The glutamine side
chain of the E118Q mutant cannot accept two hydrogen atoms, thus
destabilizing this interaction and decreasing the ability to bind the
substrate. Co-crystallization experiments of the mutants with the N,N`-diacetylchitobiose confirm this interpretation.
While the D60N and E206Q mutants also crystallize as complexes with the N,N`-diacetylchitobiose bound in the active site
cleft, no electron density corresponding to the N,N`-diacetylchitobiose molecule could be located in
the electron density map of crystals of the E118Q mutant grown under
identical conditions.
)-(N-acetyl--D-glucosaminyl)asparagine
amidase; HPLC, high performance liquid chromatography.)
We thank Yiqiu Zhang for assistance with the
crystallization of the complex.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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