Identification and characterization of γ-glutamylamine cyclotransferase, an enzyme responsible for γ-glutamyl-ε-lysine catabolism

Article abstract of DOI:10.1074/jbc.M109.082099

γ-Glutamylamine cyclotransferase (GGACT) is an enzyme that converts γ-glutamylamines to free amines and 5-oxoproline. GGACT shows high activity toward γ-glutamyl-ε-lysine, derived from the breakdown of fibrin and other proteins cross-linked by transglutaminases. The enzyme adopts the newly identified cyclotransferase fold, observed in γ- glutamylcyclotransferase (GGCT), an enzyme with activity toward γ-glutamyl-α-amino acids (Oakley, A. J., Yamada, T., Liu, D., Coggan, M., Clark, A. G., and Board, P. G. (2008) J. Biol. Chem. 283, 22031-22042). Despite the absence of significant sequence identity, several residues are conserved in the active sites of GGCTand GGACT, including a putative catalytic acid/base residue (GGACT Glu⁸²). The structure of GGACT in complex with the reaction product 5-oxoproline provides evidence for a commoncatalytic mechanism in both enzymes. The proposed mechanism, combined with the three-dimensional structures, also explains the different substrate specificities of these enzymes. Despite significant sequence divergence, there are at least three subfamilies in prokaryotes and eukaryotes that have conserved the GGCT fold and GGCT enzymatic activity.

Full text of DOI:10.1074/jbc.M109.082099

Protein Structure and Folding:
Identification and Characterization of γ
-Glutamylamine Cyclotransferase, an
Enzyme Responsible for γ
-Glutamyl-?-lysine Catabolism
Aaron J. Oakley, Marjorie Coggan and Philip
G. Board
J. Biol. Chem. 2010, 285:9642-9648.
doi: 10.1074/jbc.M109.082099 originally published online January 28, 2010
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 285, NO. 13, pp. 9642–9648, March 26, 2010
© 2010 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.
Identification and Characterization of ␥-Glutamylamine
Cyclotransferase, an Enzyme Responsible for
␥-Glutamyl-⑀-lysine Catabolism*
Received for publication, November 4, 2009, and in revised form, December 23, 2009 Published, JBC Papers in Press, January 28, 2010, DOI 10.1074/jbc.M109.082099
Aaron J. Oakley‡, Marjorie Coggan§, and Philip G. Board§1
From the ‡Division of Molecular and Health Technologies, Commonwealth Scientific and Industrial Research Organization,
Parkville, Victoria 3052 and the §John Curtin School of Medical Research, Australian National University, Canberra,
Australian Capital Territory 2601, Australia
␥-Glutamylamine cyclotransferase (GGACT) is an enzyme
that converts ␥-glutamylamines to free amines and 5-oxopro-
line. GGACT shows high activity toward ␥-glutamyl-⑀-lysine,
derived from the breakdown of fibrin and other proteins cross-
linked by transglutaminases. The enzyme adopts the newly
identified cyclotransferase fold, observed in ␥-glutamylcyclo-
transferase (GGCT), an enzyme with activity toward ␥-glu-
tamyl-␣-amino acids (Oakley, A. J., Yamada, T., Liu, D., Coggan,
M., Clark, A. G., and Board, P. G. (2008) J. Biol. Chem. 283,
22031–22042). Despite the absence of significant sequence
identity, several residues are conserved in the active sites of
GGCT and GGACT, including a putative catalytic acid/base res-
idue (GGACT Glu82). The structure of GGACT in complex with
the reaction product 5-oxoproline provides evidence for a com-
mon catalytic mechanism in both enzymes. The proposed mech-
anism, combined with the three-dimensional structures, also
explains the different substrate specificities of these enzymes.
Despite significant sequence divergence, there are at least three
subfamilies in prokaryotes and eukaryotes that have conserved
the GGCT fold and GGCT enzymatic activity.
demonstrated to be active toward a range of L-␥-glutamyl con-
jugates with mono- and polyamines and amino acids. The
action of GGACT in all cases is to cyclize the ␥-glutamyl moi-
ety, producing 5-oxo-L-proline and the free alkylamine. No
activity was detected toward L-glutamine or L-␤-aspartyl-L-⑀-
lysine. Furthermore, derivatives of L-␥-glutamyl-L-⑀-lysine in
which the ␣-amino or ␣-carboxyl functional group of the glu-
tamyl moiety is blocked do not serve as substrates, nor do any of a
range of L-␥-glutamyl-L-␣-amino acids (3). Based on these results,
it was proposed that GGACT functions in the latter stages of the
catabolism of the products of transglutaminases (3).
We reported recently the identification, cloning, and three-
dimensional structure of an enzyme with related catalytic activ-
ity to GGACT but with distinct specificity: ␥-glutamylcyclo-
transferase (GGCT) (4). Unlike GGACT, this enzyme is active
toward a range of L-␥-glutamyl-␣-amino acids (5, 6). GGCT
catalyzes the penultimate step in glutathione catabolism,
whereby L-␥-glutamyl-L-␣-cysteine and other L-␥-glutamyl-L-
␣-amino acid dipeptides formed by the ␥-glutamyl cycle are
catabolized to 5-oxo-L-proline and a free amino acid. (L-Glu-
tamic acid is subsequently formed from 5-oxo-L-proline by the
action of 5-oxoprolinase.) We demonstrated that GGCT is a
homodimer of 20,994-Da subunits and has a distinctive mixed
␣/␤-topology with six ␤-strands, five ␣-helices, and four short
310 helices, with strands ␤1–5 forming a barrel structure (4).
This fold has also been observed in several proteins of unknown
function from animals, plants, bacteria, and archaea. One such
structure, solved by the Joint Center for Structural Genomics
Consortium, is a mouse protein (AIG2-like domain 1; Protein
Data Bank code 1VKB) of unknown function (7). The gene
encoding this protein is distinct from the mouse gene encoding
GGCT. Furthermore, a homolog of mouse AIG2-like domain 1
known as A2LD1 was predicted to exist in the human genome.
The structure of the mouse protein possesses a cleft similar to
the proposed active site of human GGCT. Tantalizingly, the
proposed catalytic residue (Glu98 in human GGCT) and other
active-site residues are structurally conserved in the mouse
protein and in the amino acid sequence of its human homolog.
These observations led us to the hypothesis that this mouse
protein and its human homolog are also cyclotransferases, pos-
sibly the long sought after GGACT. Here, we describe the clon-
ing, expression, purification, catalytic activity, and three-di-
mensional structure of human A2LD1 and positively identify it
as GGACT. A common mechanism is proposed to act in both
Proteins can be cross-linked via the side chains of glutamine
and lysine by transglutaminases. This reaction results in the
formation of ammonia and an L-␥-glutamyl-L-⑀-lysine isopep-
tide bond linking the two polypeptide chains. The existence of
such ␥-glutamyl-⑀-lysine links was unambiguously demon-
strated in factor XIIIa-cross-linked fibrin: extensive proteolytic
degradation of cross-linked fibrin resulted in the formation of
L-␥-glutamyl-L-⑀-lysine (1). It was concluded that L-␥-glu-
tamyl-L-⑀-lysine is not broken down by conventional proteoly-
sis. The breakdown of the isodipeptide is instead catalyzed by
␥-glutamylamine cyclotransferase (GGACT),2 first purified
from rabbit kidney (2, 3). The partially purified enzyme was
* This work was supported by Australian Research Council Discovery Grant
DP0880027 and National Health and Medical Research Council Project
Grant 525458.
The atomic coordinates and structure factors (codes 3JUB, 3JUC, and 3JUD) have
been deposited in the Protein Data Bank, Research Collaboratory for Structural
Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
1 To whom correspondence should be addressed: John Curtin School of Med-
ical Research, Australian National University, P. O. Box 334, Canberra, ACT
2601, Australia. Tel.: 61-2-6125-4714; Fax: 61-2-6125-4712; E-mail: Philip.
Board@anu.edu.au.
2 The abbreviations used are: GGACT, ␥-glutamylamine cyclotransferase;
GGCT, ␥-glutamylcyclotransferase; QC, glutaminyl cyclase.
9642 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 285•NUMBER 13•MARCH 26, 2010

Structure of ␥-Glutamylamine Cyclotransferase
GGCT and GGACT. The proposed mechanism, combined volumes of human GGACT (6 mg/ml in 20 mM Tris-HCl (pH
with the three-dimensional structural data, also explains the 8.0) and 0.1 mM dithiothreitol) were mixed with the precipi-
different substrate specificities of these enzymes.
tants (total volume of 0.3 ␮l). The trays were incubated in and
drops imaged by Rigaku Minstrel systems at 8 or 20 °C. After
the initial conditions were identified, crystals for x-ray analysis
EXPERIMENTAL PROCEDURES
Cloning—A human expressed sequence tag clone (GenBankTM were grown by the vapor diffusion hanging drop method using
accession number BU156875) from a melanoma cell line was Greiner௡ 24-well plates at 4 °C. 2 ␮l of protein was mixed with
identified by sequence alignment with a mouse protein with a an equal volume of reservoir solution on a siliconized coverslip
GGCT-like fold (Protein Data Bank code 1VKB). The clone was (Hampton Research) prior to suspension over reservoirs con-
obtained from the I.M.A.G.E. Consortium, and the coding taining 1 ml of precipitant. A complex of wild-type GGACT
region of the cDNA was amplified with primers GGACT- with 5-oxo-L-proline was obtained by soaking crystals with arti-
F
(5Ј-CTCCGCGGTGGAATGGCCCTAGTCTTCGTG-3Ј) ficial mother liquor containing this compound at 100 mM. Crys-
and GGACT-R (5Ј-CTAAGCTTATCATCTGTTCTCCC- tals of mutant E82Q (5.9 mg/ml in 20 mM Tris-HCl (pH 8.0) and
GGGG-3Ј) and cloned into pGEM-T (Promega). The pGEM-T 0.1 mM dithiothreitol) were grown under identical conditions.
clone was sequenced by the Australian Cancer Research Foun-
X-ray data were collected at the Australian Synchrotron
dation Biomedical Resource Facility of the John Curtin School using Blu-Ice (11). Crystals were transferred to cryoprotectant
of Medical Research to confirm the sequence. The cloned priortoflashcoolingusinganOxfordCryojetsamplecoolerat100
cDNA was excised from the pGEM-T vector with KspI and K. Data were collected using an ADSC area detector. Data were
HindIII and then ligated into the same sites in the pHUE processed with MOSFLM (12) and scaled with SCALA (13).
vector (8). The resulting clone, pHUE-GGACT, was trans-
Structure Solution and Refinement—Automated molecular
fected into Escherichia coli BL21(DE3) for protein expres- replacement in MOLREP (14) was used for the determination
sion. Production of E82Q and E82A mutants was undertaken of initial structure factor phase angles. Model building and
using a QuikChange site-directed mutagenesis kit (Strat- refinement were conducted using REFMAC5 (15) and COOT
agene). The mutations were confirmed by DNA sequencing. (16). Ramachandran plot statistics were derived from PRO-
Expression and Purification—Expression of recombinant CHECK (17). Statistics for the final models are given in Table 1.
enzymes in pHUE allows the rapid purification of the expressed
Structure Analysis—The DALI server was used to find repre-
protein by nickel-agarose chromatography and the subsequent sentatives of the cyclotransferase fold in the Protein Data Bank.
removal of all additional N-terminal residues by cleavage with STAMP (18) was used to superimpose the structures onto
the catalytic domain of mouse ubiquitin-specific protease 2 as human GGACT and to create an initial structure-based
described in detail previously (8). The recombinant enzyme was sequence alignment, which was edited for clarity.
dialyzed in 20 mM Tris-HCl (pH 8.0) and 0.1 mM dithiothreitol
RESULTS
and concentrated to ϳ10 mg/ml. The purified enzyme was
stored frozen until required.
Gene, Protein, and Enzymatic Characterization—Examina-
Enzymatic Analysis—Activity with L-␥-glutamyl-L-␣-alanine tion of NCBI Human Genome Database Entrez Gene entries
as a substrate was determined by a previously published spec- showed that the GGACT gene (A2LD1, Gene ID87769) is quite
trophotometric method that links the release of alanine to the small (2.187 kb) and is located on chromosome 13q32.3. It is
oxidation of NADH (9, 10). Activity with L-␥-glutamyl-L-⑀-ly- interesting to note that the gene contains only a single intron
sine was determined by quantifying the release of 5-oxo-L-pro- that falls within the 5Ј-noncoding region of the gene transcript.
line by a modification of a method described by Orlowski et al. The human GGACT gene (A2LD1) encodes a protein of 153
(5). The reaction mixture contained 20 ␮l of 1 M Tris-HCl (pH amino acids with a predicted molecular mass of 17,327 Da. The
8.0), 20 ␮l of 0.15 M L-␥-glutamyl-L-⑀-lysine (Bachem), 5 ␮l of human A2LD1 cDNA was expressed in E. coli as a 17,300-Da
enzyme, and 155 ␮l of water. The reaction was incubated at protein. SDS-PAGE of the purified recombinant enzyme under
37 °C for 15 min and stopped by heating at 90 °C for 5 min. The reducing conditions showed a protein that migrated within the
sample was then chilled on ice, and the inactivated protein was expected size range. Gel filtration of the native protein revealed
removed by centrifugation in a microcentrifuge. An aliquot of a molecular mass of 18 kDa, which is compatible with a mono-
100 ␮l of the supernatant was added to a 1-ml column of Dowex meric structure (data not shown). Analysis of the potential
50 equilibrated in water. The sample was then washed through ␥-glutamylcyclotransferase activity of the recombinant enzyme
the column with 0.9 ml of water, followed by a second wash with revealed that it is inactive with L-␥-glutamyl-␣-amino acid sub-
1 ml of water. The unreacted L-␥-glutamyl-L-⑀-lysine bound to strates such as L-␥-glutamyl-L-␣-cysteine and L-␥-glutamyl-L-
the Dowex 50, and the 5-oxoproline was in the unbound fraction. ␣-alanine but has distinct activity with L-␥-glutamyl-L-⑀-lysine
The entire flow-through fraction was collected, and the concen- (2.7 Ϯ 0.06 ␮mol/min/mg). In contrast, recombinant human
tration of 5-L-oxoproline was determined spectrophotometrically GGCT is active with L-␥-glutamyl-L-␣-alanine (50.3 Ϯ 1.22
at 205 nm with an extinction coefficient of 2600 (5).
␮mol/min/mg) but inactive with L-␥-glutamyl-L-⑀-lysine. As a
Crystallization and Data Collection—All crystals were result of this clear specificity, we will refer to the product of the
grown at the Collaborative Crystallization Center using the so-called A2LD1 gene as ␥-glutamylamine cyclotransferase.
vapor diffusion method. Initial screens were conducted using
Crystal Structure—Crystals of human GGACT appeared in
the PACT and JCSG screens. 50 ␮l of screening solution was several polyethylene glycol-based conditions in the JCSG and
dispensed into the reservoirs of Innovadyne plates, and equal PACT screens. The best crystals grew at 8 °C with JCSG condi-
MARCH 26, 2010•VOLUME 285•NUMBER 13
JOURNAL OF BIOLOGICAL CHEMISTRY 9643

Structure of ␥-Glutamylamine Cyclotransferase
TABLE 1
Crystallographic data
PDB, Protein Data Bank; r.m.s.d., root mean square deviation.
Structure
GGACT/5-oxoproline
3JUC
GGACT
3JUB
GGACT E82Q mutant
PDB code
3JUD
X-ray data
Beamline
PX-1
P212121
PX-1
P212121
PX-2
P212121
Space group
Unit cell parameters
a ϭ 36.0, b ϭ 41.9, c ϭ 84.0 Å;
␣ ϭ ␤ ϭ ␥ ϭ 90°
30–1.2 (1.26–1.20)a
277,499 (38,387)
40,593 (5782)
22.2 (5.2)
a ϭ 36.2, b ϭ 42.5, c ϭ 84.8 Å;
␣ ϭ ␤ ϭ ␥ ϭ 90°
43–1.2 (1.26–1.20)
369,388 (39,839)
40,928 (5805)
27.5 (10.7)
a ϭ 36.3, b ϭ 42.4, c ϭ 84.8 Å;
␣ ϭ ␤ ϭ ␥ ϭ 90°
38–0.98 (1.03–0.98)
494,078 (72,260)
74,200 (10,958)
10.4 (2.5)
Resolution range (Å)
Total no. of observations
No. of unique reflections
b
͗I/␴I͘
R
merge (%)c
5.8 (32.3)
5.5 (15.4)
10.4 (68.8)
Completeness
99.9 (99.7)
98.5 (97.0)
97.7 (100.0)
Multiplicity
6.8 (6.6)
9.0 (6.9)
6.7 (6.8)
͗B͘ from Wilson plot (Å2)
8.7
8.7
5.0
Refinement statistics
Resolution range (Å)
42–1.20 (1.23–1.20)
38,497 (2917)
2032
42–1.20 (1.23–1.20)
38,832 (2906)
2053
42–0.98 (1.01–0.98)
70,357 (5455)
3736
No. of reflections used in refinement
No. of reflections (Rfree set)
R
R
work (%)d
12.3 (14.9)
13.7 (17.0)
1433
11.7 (14.8)
9.5 (12.8)
1514
15.3 (23.9)
18.0 (26.0)
1450
free (%)d
No. of atoms
͗B͘ of structure (Å2)
r.m.s.d. from ideal geometry
Bond lengths (Å)
Bond angles
9.0
9.8
8.4
0.023
1.910°
0.132
0.012
0.022
1.922°
0.11
0.029
2.313°
0.153
0.013
Chiral centers (Å3)
General planes (Å)
0.011
a Numbers in parentheses refer to the highest resolution bin.
b Angle brackets refer to mean values.
c
R
merge ϭ ⌺h⌺i͉Ihi Ϫ ͗Ih͉͘/⌺h⌺iIh.
d Rfactor ϭ ⌺hʈFo͉ Ϫ ͉Fcʈ/⌺͉Fo͉, where Fo and Fc are the observed and calculated structure factors, respectively. Rfree was calculated from 5% of the diffraction data not used in
refinement.
tion C3 (20% (w/v) polyethylene glycol 3350 and 0.2 M ammo- amine group of oxoproline is 3.2 Å from the Glu82 carboxylate
nium nitrate) as the precipitant. Crystals grew as rods with group. The loop containing residues 7–10 has a backbone con-
dimensions of 220 ϫ 100 ϫ 80 ␮m. Crystals were cryoprotected formation that causes the main chain amino groups to be ori-
by transfer to artificial mother liquor with the polyethylene gly- ented into the cavity. The oxoproline carboxylic acid moiety
col 3350 concentration increased to 25 or 30% (w/v). X-ray data accepts hydrogen bonds from the main chain amino groups of
collection statistics are given in Table 1. The structure was Tyr7 and Gly8. The carbonyl oxygen of oxoproline accepts a
solved using the mouse GGACT homolog AIG2-like domain 1 hydrogen bond from the backbone amino and side chain O-␥
(Protein Data Bank code 1VKB) as a search model. Several moieties of Thr9. The aromatic face of the side chain of Tyr7
cycles of model building and refinement followed, during which forms the bottom of the cavity. The ligand 5-oxo-L-proline sits
the mouse sequence in the search model was mutated to match lies ϳ3.6 Å from this residue (Fig. 3). Using this structure as a
Ϫ
the human sequence. Water molecules and four NO3 groups, template, the substrate L-␥-glutamyl-L-⑀-lysine has been mod-
one of which binds in the active site, were also built into the eled into the active site (Fig. 3B). Similarly, substrate or oxopro-
model. The final model has excellent electron density (Fig. 1A) line has been modeled in the active site of GGCT (Fig. 3, C and
except for the surface loop linking strands ␤4 and ␤5 (residues D). Patterns of hydrogen bonding are conserved in these
99–113), which appears disordered. The equivalent loop is also structures.
absent in the model of the mouse homolog. The final structure
Site-directed Mutagenesis—We demonstrated previously
and its topology are illustrated in Fig. 2. The topology of that Glu98 is crucial for catalytic activity in GGCT (4). To deter-
GGACT is similar to that of GGCT, i.e. a five-strand ␤-barrel mine the importance of the equivalent residue in GGACT
decorated with helices and connecting loops. In both struc- (Glu82), we mutated this residue to glutamine or alanine. The
tures, the active site is found in a cavity located on the side of the E82Q and E82A mutants had no catalytic activity, and the E82A
barrel adjacent to strands ␤1 and ␤5. The active site of GGACT enzyme was unstable and readily precipitated. We crystallized
is unambiguously identified by the binding of the reaction the E82Q mutant under conditions identical to those used for
product 5-oxo-L-proline (Figs. 1B and 3A). In GGACT, strand the wild-type enzyme (Fig. 1C). The E82Q mutant and wild-
␤2a crosses over strand ␤3b so as to reverse topological order type structures superimpose with a root mean square deviation
with respect to strands ␤2b and ␤3a (Fig. 2). This distinctive of 0.352 Å over 144 C-␣ atoms. The only difference in the active
“crossover” feature is conserved in all structures with this fold. site is a small rotation of Gln82 about the C-␥–C-␦ bond with
The cavity is formed by Tyr7, Gly8, and Thr9 in the loop follow- respect to Glu82. The binding patterns of the water molecule
Ϫ
ing strand ␤1 (a conserved motif in the cyclotransferase family; and an NO3 group binding in the active site are identical in the
see below), Leu10, Glu82, Tyr88, and Tyr119. The secondary mutant and wild-type structures.
9644 JOURNAL OF BIOLOGICAL CHEMISTRY
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Structure of ␥-Glutamylamine Cyclotransferase
FIGURE 3. A, structure of the active site of GGACT in complex with 5-oxo-L-
proline; B, model of GGACT in complex with L-␥-glutamyl-L-⑀-lysine; C, model
of GGCT in complex with 5-oxo-L-proline; D, model of GGCT in complex with
L-␥-glutamyl-L-␣-cysteine. In all cases, carbon atoms of the protein and ligand
are yellow and green, respectively. Potential hydrogen bonds are shown as
thin black lines.
Comparison of GGACT with Related Structures—A DALI
search for structural homologs revealed a limited number of
proteins with this fold. These were from both prokaryotic and
eukaryotic species and included Pyrococcus horikoshii (Protein
Data Bank code 1V30), E. coli (code 1XHS), Arabidopsis thali-
ana (code 2G0Q), and Bacillus subtilis (code 2QIK), as well as
the previously identified human GGCT and mouse GGACT
homologs (codes 2PN7 and 1VKB, respectively). The super-
posed structures and structure-based sequence alignment (Fig.
4) show several conserved features: ␤-barrel topology with two
strands “crossing over” as described above, a highly conserved
helix (in GGACT numbering, helix ␣1), a binding cavity formed
on one side of the ␤-barrel, a loop following strand ␤1 contain-
ing a conserved (V/A)YG(S/T) motif, a conserved tyrosine in
strand ␤4, and an aromatic residue in strand ␤5.
FIGURE 1. Models of GGACT in the vicinity of the active site shown in stick
form with 2mFo ؊ DFc electron density, contoured at 1␴ shown in
chicken wire representation. A and B, GGACT and GGACT, respectively, in
complex with 5-oxo-L-proline; C, GGACT E82Q mutant.
Phylogenetic Relationships—Despite the conservation of the
overall fold and the active-site architecture, there is consider-
able sequence divergence between the different proteins exhib-
iting the GGCT fold. A direct alignment of the sequence of
human GGACT with that of human GGCT showed Ͻ10%
amino acid identity (Fig. 4). Similarly, pairwise alignment of the
GGCT and GGACT sequences with the prokaryotic members
of this structural family revealed a similar low level of sequence
identity. To gain some insight into the evolution of this fold and
the diversity within this broad protein family, we undertook
BLAST searches to identify homologous proteins in different
species. Multiple sequence alignments using the COBALT
alignment tool (NCBI) (19) were then used to build a phyloge-
netic tree. Although there are many sequences available for this
FIGURE 2. A, schematic representation of GGACT, with bound nitrate groups
represented in stick form. The N and C termini are labeled. B, topology dia-
gramofGGACT. Interactionsbetween␤-strandsareindicatedbyblackrectan-
gles. The broken rectangles adjacent to strands ␤2a and ␤4 indicate the wrap-
ping of the ␤-sheet to form a barrel.
MARCH 26, 2010•VOLUME 285•NUMBER 13
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Structure of ␥-Glutamylamine Cyclotransferase
related proteins from P. horikoshii
(Protein Data Bank code 1V30) and
E. coli (code 1XHS) confirm the
association between this fold and
␥-glutamylcyclotransferase
enzy-
matic activity in prokaryotes. Al-
though we did not find evidence for
GGCT fold proteins in yeast or fun-
gal species, it is possible that they
have diverged beyond the level of
sequence identity that can be
identified by sequence similarity
searches, and these await identifica-
tion via structural studies.
DISCUSSION
The enzymatic activity known as
␥-glutamylamine cyclotransferase
has been unambiguously assigned
to the human gene known as
A2LD1. The human GGACT pro-
tein is clearly similar to the mouse
protein AIG2-like domain 1, and
the human and mouse genes are on
syntenic chromosomes. The two
proteins can be superimposed with
a root mean square deviation of 0.68
Å over 138 C-␣ atoms, and the puta-
tive active-site residues are con-
served. It is therefore likely that the
mouse protein has GGACT activity.
The structure of GGACT bound to
the reaction product 5-oxo-L-pro-
line (Figs. 1B and 3A) unambigu-
ously identifies the active site. The
GGACT residues in proximity to
this entity, Tyr7, Gly8, Thr9, Glu82,
FIGURE 4. Sequence alignment of Protein Data Bank structures adopting the cyclotransferase fold.
Sequences are labeled by Protein Data Bank codes, except for human GGACT (hGGACT) and GGCT (hGGCT).
Protein Data Bank code 2QIK contains two cyclotransferase domains that have been split into 2QIK-1 and
2QIK-2. Secondary structure elements of human GGACT are denoted above the sequences. Where significant
structural homology exists, the residues are shown in uppercase letters. Residues lining the active-site cavity are
indicated with green triangles. Possible catalytic glutamate residues are highlighted with a red star. Completely
conserved (black) and highly conserved (gray) residues are highlighted.
type of analysis, the sequences used here were selected to pro- and Tyr88, are structurally equivalent to Tyr22, Gly23, Ser24,
vide a broad evolutionary perspective. A neighbor joining tree Glu98, and Tyr105 in GGCT, respectively, strongly suggesting
(Fig. 5) indicated that there are three main families of pro- that this compound binds in an analogous fashion in both
teins with the ␥-glutamylcyclotransferase fold (GGCT-like, enzymes. A model of the 5-oxo-L-proline-GGCT complex was
GGACT-like, and BtrG-like), and there is evidence that each therefore produced (Fig. 3C). We propose that these residues
group has retained ␥-glutamylcyclotransferase activity. It is evi- form the binding site for the L-␥-glutamyl portion of the sub-
dent from the lack of sequence identity that these families have strates of both enzymes (Fig. 3, B and D). In both models, the ␣-
evolved separately for a considerable period. The GGCT-like and ␥-amino groups of the glutaminyl moieties of the sub-
family appears to be restricted to animals with sequences iden- strates are in close proximity to Glu82 (GGACT) and Glu98
tified in a range of species from Caenorhabditis elegans to (GGCT). We propose that Glu82 (GGACT) and Glu98 (GGCT)
humans. Our previous studies showed that the human GGCT share the same function, i.e. a general acid/base, and pro-
enzyme (Protein Data Bank code 2PN7) has ␥-glutamylcyclo- pose the following reaction mechanism: the ␣-amino group of
transferase activity (4). The GGACT-like family is also the L-␥-glutamyl moiety is deprotonated by Glu82/Glu98, with
restricted to eukaryotes but clearly includes plants, as con- the concomitant nucleophilic attack of this amine onto the side
firmed by the structure from A. thaliana (Protein Data Bank chain amide carbon atom. The resulting oxyanion intermediate
code 2G0Q). In this study, we have shown that members of the collapses to form 5-oxo-L-proline, with the now protonated
GGACT-like family have ␥-glutamylcyclotransferase activity. Glu82/Glu98 residue donating a hydrogen ion to the amine of
In contrast, the BtrG-like family appears to be restricted to the ␣- or ⑀-linked amino portion of the substrate (Fig. 6). We
prokaryotes. The BtrG protein from Bacillus circulans has been have demonstrated by site-directed mutagenesis and kinetics
shown to have ␥-glutamylcyclotransferase activity in an antibi- experiments that Glu82 (GGACT) and Glu98 (GGCT) (4) are
otic synthesis pathway (20), and the structures of the sequence- essential for activity. The structural integrity of E82Q indicates
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VOLUME 285•NUMBER 13•MARCH 26, 2010

Structure of ␥-Glutamylamine Cyclotransferase
ence of GGACT for substrates with
extended aliphatic amines (lysine,
spermidine, etc.) conjugated to the
␥-glutamyl group.
The B. subtilis homolog (named
YkqA, of unknown function; Pro-
tein Data Bank code 2QIK) has two
cyclotransferase domains contained
within one polypeptide chain. Only
the second has a glutamyl residue
equivalent to Glu82 in GGACT. This
protein possibly has cyclotrans-
ferase-like activity in its C-terminal
domain, with a regulatory function
in the N-terminal domain. The
E. coli homolog (named YtfP, of
unknown function; Protein Data
Bank code 1XHS) has an arginine
residue in place of GGACT residue
Glu82, suggesting that this protein is
not a cyclotransferase but might
bind
a similar substrate. The
A. thaliana protein (known as
At5g39720.1, of unknown function;
Protein Data Bank code 2G0Q) is
clearly a member of the GGCT
structural family and has a con-
served active-site glutamyl residue,
suggesting that it may have cyclo-
transferase activity. This is consis-
tent with the previous detection of
␥-glutamylcyclotransferase activity
in tobacco suspension cultures (21).
At this juncture, the lack of struc-
tural similarity of the GGCT family
of cyclotransferases and the glu-
taminyl cyclases (QCs) (EC 2.3.2.5)
should be noted. The QCs catalyze
the formation of N-terminal 5-oxo-
proline from its glutaminyl (or glu-
tamyl) precursor on peptides. This
modification is required by certain
proteins for biological function in
either mediating interaction with
FIGURE 5. Neighbor joining tree depicting the probable phylogenetic relationships between selected
proteins with the ␥-glutamylcyclotransferase fold. Representative structures from each subfamily are
shown.
FIGURE 6. Proposed reaction mechanism for GGACT and GGCT. Glu represents Glu82 (GGACT) or Glu98
(GGCT).
that a glutamyl residue at this position is not essential for fold- receptors or stabilizing proteins against N-terminal degrada-
ing. Our model of substrate binding in GGACT and GGCT can tion. QCs are classified as belonging to two groups: mammalian
further explain the substrate specificities of both enzymes. In QCs and a second group containing enzymes from bacteria,
our models, the groups to which the glutamyl moieties are plants, and parasites. The structure of human QC, representa-
linked are orientated out of the active site and toward the sur- tive of the first group, adopts an ␣/␤-open sandwich fold similar
face, and the residues of GGACT and GGCT share little struc- to the two-zinc exopeptidases (22) and requires zinc as a cofac-
tural similarity in this vicinity. In our GGCT model with sub- tor. Although human QC is unrelated to the GGCT family in
strate, Arg30 is involved in a salt bridge with the ␣-carboxylic either sequence or three-dimensional structure, it does appear
acid of the cysteine residue, compatible with the known prefer- to contain a catalytic acid/base residue (Glu201) analogous in
ence of GGCT for L-␥-glutamyl-L-␣-amino acid substrates. function to Glu82 in GGACT. In human QC, a Zn2ϩ ion is
GGACT lacks an arginine residue at the equivalent location and proposed to stabilize an oxyanion intermediate during cataly-
does not act on L-␥-glutamyl-L-␣-amino acids. The active site is sis. In GGACT, the Thr9 amino and side chain hydroxyl groups
narrower in GGACT compared with GGCT, partly due to the appear to perform this function. As with GGCT and GGACT,
presence of Phe81 (Fig. 7). This is compatible with the prefer- mutation of the proposed acid/base residue in human QC
MARCH 26, 2010•VOLUME 285•NUMBER 13
JOURNAL OF BIOLOGICAL CHEMISTRY 9647

Structure of ␥-Glutamylamine Cyclotransferase
␥-glutamylcyclotransferase activity, as the BtrG protein from
B. circulans catalyzes the formation of 5-oxoproline from an
intermediate in the synthesis of the antibiotic butirosin (20).
Thus, although the primary amino acid sequences of this struc-
tural family are incredibly diverse, the conserved fold is very
strongly associated with conserved ␥-glutamylcyclotransferase
activity. Because there is such great sequence diversity between
the three groups within this structural family, there is a strong
possibility that additional subfamilies with sequence diversity
beyond the reach of BLAST alignments may be identified by
structural studies in the future.
Acknowledgments—We thank the technical staff of the Bio21-C3 Cen-
ter for help with crystallization. This work was undertaken on the
PX-1 and PX-2 beamlines at the Australian Synchrotron (Victoria,
Australia).
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