Key points are not available for this paper at this time.
The crystal structure of a microbial transglutaminase from Streptoverticillium mobaraense has been determined at 2.4 Å resolution. The protein folds into a plate-like shape, and has one deep cleft at the edge of the molecule. Its overall structure is completely different from that of the factor XIII-like transglutaminase, which possesses a cysteine protease-like catalytic triad. The catalytic residue, Cys64, exists at the bottom of the cleft. Asp255 resides at the position nearest to Cys64 and is also adjacent to His274. Interestingly, Cys64, Asp255, and His274 superimpose well on the catalytic triad “Cys-His-Asp” of the factor XIII-like transglutaminase, in this order. The secondary structure frameworks around these residues are also similar to each other. These results imply that both transglutaminases are related by convergent evolution; however, the microbial transglutaminase has developed a novel catalytic mechanism specialized for the cross-linking reaction. The structure accounts well for the catalytic mechanism, in which Asp255is considered to be enzymatically essential, as well as for the causes of the higher reaction rate, the broader substrate specificity, and the lower deamidation activity of this enzyme. The crystal structure of a microbial transglutaminase from Streptoverticillium mobaraense has been determined at 2.4 Å resolution. The protein folds into a plate-like shape, and has one deep cleft at the edge of the molecule. Its overall structure is completely different from that of the factor XIII-like transglutaminase, which possesses a cysteine protease-like catalytic triad. The catalytic residue, Cys64, exists at the bottom of the cleft. Asp255 resides at the position nearest to Cys64 and is also adjacent to His274. Interestingly, Cys64, Asp255, and His274 superimpose well on the catalytic triad “Cys-His-Asp” of the factor XIII-like transglutaminase, in this order. The secondary structure frameworks around these residues are also similar to each other. These results imply that both transglutaminases are related by convergent evolution; however, the microbial transglutaminase has developed a novel catalytic mechanism specialized for the cross-linking reaction. The structure accounts well for the catalytic mechanism, in which Asp255is considered to be enzymatically essential, as well as for the causes of the higher reaction rate, the broader substrate specificity, and the lower deamidation activity of this enzyme. Transglutaminase (TGase 1The abbreviations used are: TGase, transglutaminase; MTG, microbial TGase; FTG, fish-derived TGase; MIR, multiple isomorphous replacement 1The abbreviations used are: TGase, transglutaminase; MTG, microbial TGase; FTG, fish-derived TGase; MIR, multiple isomorphous replacement; protein-glutamine γ-glutamyltransferase, EC 2.3.2.13) catalyzes an acyl transfer reaction in which the γ-carboxyamide groups of peptide-bound glutamine residues act as the acyl donors. The most common acyl acceptors of TGase are the ε-amino groups of lysine residues within peptides or the primary amino groups of some naturally occurring polyamines (1Aeschlimann D. Paulsson M. Thromb. Haemostasis. 1994; 71: 402-415Crossref PubMed Scopus (492) Google Scholar, 2Folk J.E. Chung S.I. Methods Enzymol. 1985; 113: 358-375Crossref PubMed Scopus (250) Google Scholar). When lysine residues in proteins serve as acyl acceptors, intermolecular or intramolecular ε-(γ-glutamyl)lysine bonds are formed, resulting in the polymerization of proteins. TGases are widely distributed in various organisms, including vertebrates (3Chung S.I. Folk J.E. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 303-307Crossref PubMed Scopus (88) Google Scholar, 4Wong W.S. Batt C. Kinsella J.E. Int. J. Biochem. 1990; 22: 53-59Crossref PubMed Scopus (21) Google Scholar, 5Weraarchakul-Boonmark N. Jeong J.-M. Murthy S.N.P. Engel J.D. Lorrand L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9804-9808Crossref PubMed Scopus (33) Google Scholar, 6Grant F.J. Taylor D.A. Sheppard P.O. Mathewes S.L. Lint W. Vanaja E. Bishop P.D. O'Hara P.J. Biochem. Biophys. Res. Commun. 1994; 203: 1117-1123Crossref PubMed Scopus (47) Google Scholar, 7Yasueda H. Nakanishi K. Kumazawa Y. Nagase K. Motoki M. Matsui H. Eur. J. Biochem. 1995; 232: 411-419Crossref PubMed Scopus (54) Google Scholar), invertebrates (8Tokunaga F. Muta T. Iwanaga S. Ichinose A. Davie E.W. Kuma K. Mikata T. J. Biol. Chem. 1993; 268: 262-268Abstract Full Text PDF PubMed Google Scholar, 9Singh R.N. Metha K. Eur. J. Biochem. 1994; 225: 625-634Crossref PubMed Scopus (41) Google Scholar), mollusks (10Klein J.D. Guzman E. Kuehn G.D. J. Bacteriol. 1992; 174: 2599-2605Crossref PubMed Google Scholar), plants (11Margosiak S.A. Dharma A. Bruce-Carver M.R. Gonzales A.P. Louie D. Kuehn G.D. Plant Physiol. 1990; 92: 88-96Crossref PubMed Scopus (71) Google Scholar), and microorganisms (12Kanaji T. Ozaki H. Takao T. Kawajiri K. Ide H. Motoki M. Simonishi Y. J. Biol. Chem. 1993; 268: 11565-11572Abstract Full Text PDF PubMed Google Scholar). Among these TGases, the human blood coagulation factor XIII has been most characterized (13Ichinose A. Hendrickson L.E. Fujikawa K. Davie E.W. Biochemistry. 1986; 25: 6900-6906Crossref PubMed Scopus (153) Google Scholar, 14Lorand J.B. Pilkington T.R. Lorand L. Nature. 1966; 210: 1273-1274Crossref PubMed Scopus (44) Google Scholar, 15Gladner J.A. Nossal R. Thromb. Res. 1983; 30: 273-288Abstract Full Text PDF PubMed Scopus (44) Google Scholar, 16Hornyak T.J. Bishop P.D. Shafer J.A. Biochemistry. 1989; 28: 7326-7332Crossref PubMed Scopus (52) Google Scholar, 17Hornyak T.J. Shafer J.A. Biochemistry. 1991; 30: 6175-6182Crossref PubMed Scopus (56) Google Scholar, 18Bishop P.D. Teller D.C. Smith R.A. Lasser G.W. Gilbert T. Seale R.L. Biochemistry. 1990; 29: 1861-1869Crossref PubMed Scopus (85) Google Scholar). By catalyzing the cross-linking between fibrin molecules, factor XIII forms fibrin clots for hemostasis and heals a wound. The crystal structure of human factor XIII has been determined, revealing that it consists of four domains with a cysteine protease-like active site (19Yee V.C. Pedersen L.C., Le Trong I. Bishop P.D. Stenkamp R.E. Teller D.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7296-7300Crossref PubMed Scopus (321) Google Scholar, 20Yee V.C. Pedersen L.C. Bishop P.D. Stenkamp R.E. Teller D.C. Thromb. Res. 1995; 78: 389-397Abstract Full Text PDF PubMed Scopus (83) Google Scholar, 21Weiss M.S. Metzner H.J. Hilgenfeld R. FEBS Lett. 1998; 423: 291-296Crossref PubMed Scopus (136) Google Scholar, 22Pedersen L.C. Yee V.C. Bishop P.D., Le Trong I. Teller D.C. Stenkamp R.E. Protein Sci. 1994; 3: 1131-1135Crossref PubMed Scopus (138) Google Scholar). Many TGases are homologous to human factor XIII and share the common feature of Ca2+-dependent catalytic activity (3Chung S.I. Folk J.E. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 303-307Crossref PubMed Scopus (88) Google Scholar, 4Wong W.S. Batt C. Kinsella J.E. Int. J. Biochem. 1990; 22: 53-59Crossref PubMed Scopus (21) Google Scholar, 5Weraarchakul-Boonmark N. Jeong J.-M. Murthy S.N.P. Engel J.D. Lorrand L. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 9804-9808Crossref PubMed Scopus (33) Google Scholar, 6Grant F.J. Taylor D.A. Sheppard P.O. Mathewes S.L. Lint W. Vanaja E. Bishop P.D. O'Hara P.J. Biochem. Biophys. Res. Commun. 1994; 203: 1117-1123Crossref PubMed Scopus (47) Google Scholar, 7Yasueda H. Nakanishi K. Kumazawa Y. Nagase K. Motoki M. Matsui H. Eur. J. Biochem. 1995; 232: 411-419Crossref PubMed Scopus (54) Google Scholar, 8Tokunaga F. Muta T. Iwanaga S. Ichinose A. Davie E.W. Kuma K. Mikata T. J. Biol. Chem. 1993; 268: 262-268Abstract Full Text PDF PubMed Google Scholar). A tissue-type TGase from red sea bream liver (fish-derived TGase (FTG)) is an example of such factor XIII-like TGases and shows 33% sequence homology to human factor XIII (7Yasueda H. Nakanishi K. Kumazawa Y. Nagase K. Motoki M. Matsui H. Eur. J. Biochem. 1995; 232: 411-419Crossref PubMed Scopus (54) Google Scholar). The crystal structure of FTG has also been determined (23Noguchi K. Ishikawa K. Yokoyama K. Ohtsuka T. Nio N. Suzuki E. J. Biol. Chem. 2001; 276: 12055-12059Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The overall and active site structures of FTG are essentially similar to those of human factor XIII. A microbial TGase (MTG) has been isolated from the culture medium ofStreptoverticillium sp. S-8112 (24Ando H. Adachi M. Umeda K. Matsuura A. Nonaka M. Uchio R. Tanaka H. Motoki M. Agric. Biol. Chem. 1989; 53: 2613-2617Crossref Scopus (30) Google Scholar), which has been identified as a variant of Sv. mobaraense. This enzyme is the first TGase obtained from a nonmammalian source. Thus far, few TGases have been identified from microorganisms, particularly fromStreptoverticillium species (25Duran R. Junqua M. Schmitter J.M. Gancet C. Goulas P. Biochimie (Paris). 1998; 80: 313-319Crossref PubMed Scopus (54) Google Scholar). Although the physiological role of is this protein is from the as a and is by R. S. S. H. Eur. J. Biochem. 1998; PubMed Scopus Google Scholar). to TGases, the activity is (24Ando H. Adachi M. Umeda K. Matsuura A. Nonaka M. Uchio R. Tanaka H. Motoki M. Agric. Biol. Chem. 1989; 53: 2613-2617Crossref Scopus (30) Google Scholar). A sequence of by that the protein consists of amino with a of (12Kanaji T. Ozaki H. Takao T. Kawajiri K. Ide H. Motoki M. Simonishi Y. J. Biol. Chem. 1993; 268: 11565-11572Abstract Full Text PDF PubMed Google Scholar). The of is that of the factor XIII-like The amino sequence of homology to the factor XIII-like TGases or to in the protein sequence for the homologous TGases (25Duran R. Junqua M. Schmitter J.M. Gancet C. Goulas P. Biochimie (Paris). 1998; 80: 313-319Crossref PubMed Scopus (54) Google Scholar). to the factor XIII-like TGase, which possesses an active site of the sequence of a cysteine has been identified as the catalytic in the sequence of (12Kanaji T. Ozaki H. Takao T. Kawajiri K. Ide H. Motoki M. Simonishi Y. J. Biol. Chem. 1993; 268: 11565-11572Abstract Full Text PDF PubMed Google Scholar). A that the reaction and the substrate for the acyl of are higher and lower those of the factor XIII-like TGases such as liver TGase and FTG, N. Yokoyama K. Suzuki E. J. Agric. Chem. PubMed Scopus Google Scholar). the the deamidation activity of is that of FTG, that it is for the to the role of an acyl T. Y. Nio N. K. J. Sci. 2001; Scopus (47) Google Scholar). it is that has a novel structure and that catalytic mechanism is different from that of the factor XIII-like These including the higher reaction rate, the broader substrate for the acyl the lower activity for and the are for of is widely used to the and of such as and K. T. M. R. H. Agric. Biol. Chem. Scopus Google Scholar, M. Nio N. J. Sci. 1983; Scopus Google Scholar, L. P.J. J. Sci. Scopus Google Scholar, Y. A. J. J. 1995; Scopus Google Scholar). of the of the cross-linking of to are also the of and to for protein at various determined the structure of by the novel overall and active site structures of determined at 2.4 Å and the catalytic mechanism of this enzyme. of for the is an variant that has an at the of the amino sequence The is an variant in which the amino sequence the of the and has a as the and as K. N. K. K. Biochem. PubMed Scopus (56) Google Scholar). The has the amino sequence as the and be as or in the sequence to the sequence and the sequence by factor The which has the sequence of the The sequence to the sequence by K. N. K. K. Biochem. PubMed Scopus (56) Google Scholar). the with the site and the sequence of the the with The into the Its sequence and the as the and the in the by and by and by The the into the and the as E. as K. N. K. K. Biochem. PubMed Scopus (56) Google that by the of the at The from by and the obtained as K. N. K. K. Biochem. PubMed Scopus (56) Google Scholar). in and and for at by The to by and to a of of at the from to K. T. N. K. and D. in by the and the The and to a with of factor to of the of which for at to by and by to a with the with of the the with a of from to at a of and by to a with the with of the the with a of from to at a of that by and the of the by the of to a with The as to be as with the by The of and various from the and the for which obtained the The of at with the of the The in the a of and of and of and of the and the a few plate-like which to a for within the of the the crystal at a and The of the crystal are in I. The of the crystal a N. Methods Res. A. 1991; Scopus Google on at the of the for The to and the with to on the The the and W. Methods Enzymol. 276: Scopus Google Scholar). The crystal of to 2.4 Å resolution. to the with of a and The crystal four with a of of and The the and the of of of of the are in of the are in and of of the of for is the structure of a and are the structure and the of the and and are the and of the structure The the and the D. PubMed Scopus Google The of the are in and of of the for is the structure of a and are the structure and the of the and and are the and of the structure in a The of by the multiple isomorphous replacement The of the at with that with and The of the used for the are also in I. The and the the S. D. 1994; PubMed Scopus Google Scholar). The of the and the the on an The between the and four to the each site to the of The of the determined the by the of the The the in with of the The The of are in I. The Å of a and to The the in the on the of the Å The and a and this it that the crystal four and the the by the The determined by the of the and on the of and those of A. The the at Å resolution. The for the of and the The that the of of the be of the residues of the be to the The of to the of the A for and Scholar). The with the by the of this some of the protein the to to the and the for the within Å and The four molecules, each of amino and The in and are Å and A of the four in the the R.A. J.M. J. 1993; Google that of the residues are in the most of the residues are in of the residues are in and of the residues are in The overall structures of the four are The of the between the four from to the crystal A and and and are related by The between are the intermolecular in the the of these is is physiological The of each with adjacent residues of the related The 1The abbreviations used are: TGase, transglutaminase; MTG, microbial TGase; FTG, fish-derived TGase; MIR, multiple isomorphous replacement of A is with the and of the C. The of and the of are with the of the related and the of the related The 1The abbreviations used are: TGase, transglutaminase; MTG, microbial TGase; FTG, fish-derived TGase; MIR, multiple isomorphous replacement and of are with the and of the The of the in or the of 1The abbreviations used are: TGase, transglutaminase; MTG, microbial TGase; FTG, fish-derived TGase; MIR, multiple isomorphous replacement in the crystal This and The overall structure of is in The forms a with overall of a and has a deep cleft at the edge of the Cys64, the for the catalytic exists at the bottom of the cleft. this cleft as the active site cleft. The structure of to the and The and the are at the amino and of the These secondary structures are that a is by which are into The forms a this is between the and and is one between the of these and The first of exists on the of the of the and is of the and Cys64 resides on the between the and The the and and the the and on the and bottom of the of MTG, The structure of with the proteins in the Protein by the and the the with active site These the and of the proteins similar to be that has a novel The of are in the are in the active site and the of the is with are a of including 1The abbreviations used are: TGase, transglutaminase; MTG, microbial TGase; FTG, fish-derived TGase; MIR, multiple isomorphous Asp255, and in the active site cleft. the of the and residues of are a of including and on the around the active site cleft. These of the and residues the substrate of of The and are on the and residues and Cys64, and the residues are and This the The active site cleft exists at the of the of The of the cleft is The of the active site cleft are by of and are by a of Å between the of the and the of MTG, an 1The abbreviations used are: TGase, transglutaminase; MTG, microbial TGase; FTG, fish-derived TGase; MIR, multiple isomorphous and a between the and the of the cleft. These are with and and a structure with in The of resides in the of the active site cleft. is as a which has an of amino residues at the of the sequence R. S. S. H. Eur. J. Biochem. 1998; PubMed Scopus Google Scholar). The has The position of the of the it to that the the active site cleft and the from the active the it has been that the reaction of is higher that of N. Yokoyama K. Suzuki E. J. Agric. Chem. PubMed Scopus Google Scholar). The crystal structure of MTG, in which the and Cys64 are to each is with the that the of the catalytic A from to forms of the active site cleft. to the this a The of this to shows the in in the molecule. The bottom of the active site cleft is of the the and the between the and Cys64 exists on the of this The bottom of the active site cleft is in Although Cys64 is by such as Asp255, and the of Cys64 has the with and is to the of Thus far, the crystal structures of TGases, human factor XIII and red sea bream liver TGase have been determined (19Yee V.C. Pedersen L.C., Le Trong I. Bishop P.D. Stenkamp R.E. Teller D.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7296-7300Crossref PubMed Scopus (321) Google Scholar, K. Ishikawa K. Yokoyama K. Ohtsuka T. Nio N. Suzuki E. J. Biol. Chem. 2001; 276: 12055-12059Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The overall structures of FTG and human factor XIII each other. between and The overall structure of is completely different from that of FTG, a that be from the of sequence and the different of these to the structure of MTG, FTG as well as human factor XIII of four and by Yee (19Yee V.C. Pedersen L.C., Le Trong I. Bishop P.D. Stenkamp R.E. Teller D.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7296-7300Crossref PubMed Scopus (321) Google Scholar). The active site of FTG exists in the which amino Although the structure of the consists of and and to the the overall of and the of FTG are different the between the active site structures of and FTG is in the of the secondary structures around the active of and FTG are The active site Cys64 in and in FTG, both the of the in This of the position of the is also in cysteine and L.C. Yee V.C. Bishop P.D., Le Trong I. Teller D.C. Stenkamp R.E. Protein Sci. 1994; 3: 1131-1135Crossref PubMed Scopus (138) Google Scholar). this is by the and in in each The catalytic triad of FTG consists of and and on the of this XIII and some cysteine such as J. Biochemistry. PubMed Scopus Google and A. Scopus Google Scholar), also share a similar of and the catalytic triad. the in the active site of MTG, is such cysteine protease-like catalytic triad as This is the most and between and the factor XIII-like Interestingly, in the Asp255 and His274 the to those of and in FTG, The Cys64, Asp255, and of the superimpose well on the catalytic triad of FTG and with Å for the and the and in the the of the and to be to the Many share similar structures of active including a catalytic triad and an the of overall This first in the structures of and is as a of convergent J. Biol. 1972; PubMed Scopus Google Scholar). The the active site structures and the in the overall structures between and the factor XIII-like TGases imply that the between these is a of convergent such are Asp255 and His274 the role of and in the catalytic triad of the factor XIII-like TGase, have developed a catalytic mechanism specialized for the TGase reaction. The of be in a has a broader substrate for the acyl and a higher reaction the factor XIII-like TGases N. Yokoyama K. Suzuki E. J. Agric. Chem. PubMed Scopus Google Scholar). the structures of FTG and human factor the of the catalytic residues with the of residues in FTG and in factor and are to the (19Yee V.C. Pedersen L.C., Le Trong I. Bishop P.D. Stenkamp R.E. Teller D.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7296-7300Crossref PubMed Scopus (321) Google Scholar, K. Ishikawa K. Yokoyama K. Ohtsuka T. Nio N. Suzuki E. J. Biol. Chem. 2001; 276: 12055-12059Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The resides on the of the and the active site of the is that the of and the acyl causes the in which the is from the catalytic and the is to the of the active site and the of the factor XIII-like TGase, Cys64 of is to the and with the of the of the active site cleft the between the enzyme and These between and the factor XIII-like TGases be the for the in the substrate and the reaction to the in the and of the active site cleft of to the acyl and acyl of factor XIII-like TGases (19Yee V.C. Pedersen L.C., Le Trong I. Bishop P.D. Stenkamp R.E. Teller D.C. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7296-7300Crossref PubMed Scopus (321) Google Scholar, K. Ishikawa K. Yokoyama K. Ohtsuka T. Nio N. Suzuki E. J. Biol. Chem. 2001; 276: 12055-12059Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar). The of these are with the of these in of the of the active site cleft is with the of and which the of a acyl the as in the of and residues and and residues and are in the of the active site cleft. Interestingly, an the substrate for the acyl of that peptides amino residues and residues at the of are of MTG, catalytic with the peptides residues at the of T. M. Nio N. Motoki M. Biochem. PubMed Scopus Google Scholar). These results that the of within the acyl to the of the active site cleft of and that an acyl with and on the of to with the enzyme A cysteine protease-like catalytic mechanism of human factor XIII has been on the of the of the active between these L.C. Yee V.C. Bishop P.D., Le Trong I. Teller D.C. Stenkamp R.E. Protein Sci. 1994; 3: 1131-1135Crossref PubMed Scopus (138) Google Scholar). The catalytic triad of factor XIII consists of and the mechanism, and act in the acyl transfer reaction. which is with to the of the active site and to the of on the with the cysteine and are considered to the as the L.C. Yee V.C. Bishop P.D., Le Trong I. Teller D.C. Stenkamp R.E. Protein Sci. 1994; 3: 1131-1135Crossref PubMed Scopus (138) Google Scholar). in the the of the secondary structure of the active site between and FTG is the catalytic triad is in the active site of a cysteine protease-like catalytic mechanism for in which Asp255 the role of the in the factor XIII-like A catalytic mechanism of is in A of the of an acyl the of the and Asp255 a to the and an is an acyl such as the of the residue, the active and the of Asp255, which is a of the acyl and the is from the and the catalytic reaction is The of the to Asp255 is by the the amino residues with at the position nearest a protein in which Asp255 is by catalytic activity to a that Asp255 is for the reaction. M. K. T. K. D. and E. the in N. M. Yokoyama K. T. Ishikawa K. D. Suzuki E. FEBS Lett. PubMed Scopus Google Scholar). to this mechanism, Asp255 be at the far, is for the of the of be this is well in the protein with of the and and the of Asp255 in to be for the substrate of the acyl the amino groups are the species such as by the with the deamidation activity of is that of FTG, T. Y. Nio N. K. J. Sci. 2001; Scopus (47) Google Scholar). the the role of His274 in the catalytic reaction the and the between the Asp255 and His274 are and the of Asp255 and the of His274 a This a role in the of the active in this Asp255 and His274 as the and This is completely that of the factor XIII-like TGases and to the of Asp255 in reaction D. an in which His274 is by activity to the that His274 is for the The of the between Asp255 and that the for the and the for the catalytic be and the catalytic of His274. the of the factor XIII-like TGases, the for the residues that the are and the structure of MTG, exists the position to of J. Biochemistry. PubMed Scopus Google or A. Scopus Google Scholar), which is the the as well as Cys64, Asp255, and is in the amino sequence of the (25Duran R. Junqua M. Schmitter J.M. Gancet C. Goulas P. Biochimie (Paris). 1998; 80: 313-319Crossref PubMed Scopus (54) Google Scholar). in the of MTG, the this have determined the structure of a novel of TGase from a Although overall structure is completely different from those of the factor XIII-like TGases and the cysteine the secondary structure of the catalytic is similar to a between these on this around the active have that Asp255 of the role of the in the cysteine protease-like catalytic triad. have been to the mechanism M. R.E. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus (88) Google Scholar), and residues serve as the and the is is the first enzyme that a Although for the mechanism is including the of the structure of an or to this novel catalytic of
Kashiwagi et al. (Fri,) studied this question.
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