Mutational Analysis of the Catalytic Domain of O-Linked N-Acetylglucosaminyl Transferase
2005; Elsevier BV; Volume: 280; Issue: 42 Linguagem: Inglês
10.1074/jbc.m504948200
ISSN1083-351X
AutoresBrooke D. Lazarus, Mark D. Roos, John A. Hanover,
Tópico(s)Proteoglycans and glycosaminoglycans research
ResumoO-Linked N-acetylglucosaminyltransferase (OGT) catalyzes the transfer of O-linked GlcNAc to serine/threonine residues of a variety of target proteins, many of which have been implicated in such diseases as diabetes and neurodegeneration. The addition of O-GlcNAc to proteins occurs in response to fluctuations in cellular concentrations of UDP-GlcNAc, which result from nutrients entering the hexosamine biosynthetic pathway. However, the molecular mechanisms involved in sugar nucleotide recognition and transfer to protein are poorly understood. We employed site-directed mutagenesis to target potentially important amino acid residues within the two conserved catalytic domains of OGT (CD I and CD II), followed by an in vitro glycosylation assay to evaluate N-acetylglucosaminyltransferase activity after bacterial expression. Although many of the amino acid substitutions caused inactivation of the enzyme, we identified three amino acid residues (two in CD I and one in CD II) that produced viable enzymes when mutated. Structure-based homology modeling revealed that these permissive mutants may be either in or near the sugar nucleotide-binding site. Our findings suggest a model in which the two conserved regions of the catalytic domain, CD I and CD II, contribute to the formation of a UDP-GlcNAc-binding pocket that catalyzes the transfer of O-GlcNAc to substrate proteins. Identification of viable OGT mutants may facilitate examination of its role in nutrient sensing and signal transduction cascades. O-Linked N-acetylglucosaminyltransferase (OGT) catalyzes the transfer of O-linked GlcNAc to serine/threonine residues of a variety of target proteins, many of which have been implicated in such diseases as diabetes and neurodegeneration. The addition of O-GlcNAc to proteins occurs in response to fluctuations in cellular concentrations of UDP-GlcNAc, which result from nutrients entering the hexosamine biosynthetic pathway. However, the molecular mechanisms involved in sugar nucleotide recognition and transfer to protein are poorly understood. We employed site-directed mutagenesis to target potentially important amino acid residues within the two conserved catalytic domains of OGT (CD I and CD II), followed by an in vitro glycosylation assay to evaluate N-acetylglucosaminyltransferase activity after bacterial expression. Although many of the amino acid substitutions caused inactivation of the enzyme, we identified three amino acid residues (two in CD I and one in CD II) that produced viable enzymes when mutated. Structure-based homology modeling revealed that these permissive mutants may be either in or near the sugar nucleotide-binding site. Our findings suggest a model in which the two conserved regions of the catalytic domain, CD I and CD II, contribute to the formation of a UDP-GlcNAc-binding pocket that catalyzes the transfer of O-GlcNAc to substrate proteins. Identification of viable OGT mutants may facilitate examination of its role in nutrient sensing and signal transduction cascades. O-Linked N-acetylglucosaminyltransferase (OGT) 3The abbreviations used are: OGTO-linked N-acetylglucosaminyltransferaseHBPhexosamine biosynthetic pathwayTPRtetratricopeptide repeatGTGPFglycogen phosphorylase/glycosyltransferase familymOGTmitochondrial OGT. is a nucleocytoplasmic enzyme that catalyzes the addition of a single GlcNAc residue, in an O-glycosidic linkage to serine or threonine residues of target proteins (1Lubas W.A. Frank D.W. Krause M. Hanover J.A. J. Biol. Chem. 1997; 272: 9316-9324Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar, 2Kreppel L.K. Blomberg M.A. Hart G.W. J. Biol. 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The O-GlcNAc modification occurs on a wide variety of proteins in macromolecular complexes including nuclear pore proteins (7Hanover J.A. Cohen C.K. Willingham M.C. Park M.K. J. Biol. Chem. 1987; 262: 9887-9894Abstract Full Text PDF PubMed Google Scholar, 8Holt G.D. Snow C.M. Senior A. Haltiwanger R.S. Gerace L. Hart G.W. J. Cell Biol. 1987; 104: 1157-1164Crossref PubMed Scopus (316) Google Scholar, 9Davis L.I. Blobel G. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 7552-7556Crossref PubMed Scopus (252) Google Scholar), RNA polymerase II (10Kelly W.G. Dahmus M.E. Hart G.W. J. Biol. Chem. 1993; 268: 10416-10424Abstract Full Text PDF PubMed Google Scholar, 11Cervoni L. Turano C. Ferraro A. Ciavatta P. Marmocchi F. Eufemi M. FEBS Lett. 1997; 417: 227-230Crossref PubMed Scopus (4) Google Scholar) and associated transcription factors (12Jackson S.P. Tjian R. Cell. 1988; 55: 125-133Abstract Full Text PDF PubMed Scopus (650) Google Scholar, 13Yang X. Su K. Roos M.D. Chang Q. Paterson A.J. 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Cell Biol. 1997; 17: 2550-2558Crossref PubMed Scopus (377) Google Scholar), synapsins (20Cole R.N. Hart G.W. J. Neurochem. 1999; 73: 418-428Crossref PubMed Scopus (95) Google Scholar, 21Luthi T. Haltiwanger R.S. Greengard P. Bahler M. J. Neurochem. 1991; 56: 1493-1498Crossref PubMed Scopus (38) Google Scholar), oncogene products (22Chou T.Y. Dang C.V. Hart G.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 4417-4421Crossref PubMed Scopus (185) Google Scholar, 23Medina L. Grove K. Haltiwanger R.S. Glycobiology. 1998; 8: 383-391Crossref PubMed Scopus (64) Google Scholar), and tumor suppressor proteins (24Shaw P. Freeman J. Bovey R. Iggo R. Oncogene. 1996; 12: 921-930PubMed Google Scholar). These findings are in accord with a broad physiological role for O-GlcNAc modification in such varied cellular processes as transport, transcription, cell shape, cell signaling, and apoptosis (25Hanover J.A. FASEB J. 2001; 15: 1865-1876Crossref PubMed Scopus (253) Google Scholar). There is also evidence suggesting that OGT plays a role in sensing cellular levels of UDP-GlcNAc synthesized by the hexosamine biosynthetic pathway (HBP) (26McClain D.A. Lubas W.A. Cooksey R.C. Hazel M. Parker G.J. Love D.C. Hanover J.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 10695-10699Crossref PubMed Scopus (276) Google Scholar, 27Marshall S. Bacote V. Traxinger R.R. J. Biol. Chem. 1991; 266: 4706-4712Abstract Full Text PDF PubMed Google Scholar). Increased intracellular glucose concentrations result in increased flow through the HBP, leading to an elevation of UDP-GlcNAc and the O-GlcNAcylation of many proteins (28Buse M.G. Robinson K.A. Marshall B.A. Hresko R.C. Mueckler M.M. Am. J. Physiol. 2002; 283: E241-E250Crossref PubMed Scopus (91) Google Scholar). Furthermore, elevated O-GlcNAcylation of proteins is linked to insulin resistance and the onset of diabetes (29Hazel M. Cooksey R.C. Jones D. Parker G. Neidigh J.L. Witherbee B. Gulve E.A. McClain D.A. Endocrinology. 2004; 145: 2118-2128Crossref PubMed Scopus (59) Google Scholar, 30Hawkins M. Barzilai N. Chen W. Angelov I. Hu M. Cohen P. Rossetti L. Diabetes. 1996; 45: 1734-1743Crossref PubMed Scopus (38) Google Scholar, 31Wells L. Vosseller K. Hart G.W. Cell Mol. Life Sci. 2003; 60: 222-228Crossref PubMed Scopus (178) Google Scholar). The determination of the function of OGT as a nutrient sensor and the mechanism underlying sugar nucleotide recognition and transfer are therefore of interest. O-linked N-acetylglucosaminyltransferase hexosamine biosynthetic pathway tetratricopeptide repeat glycogen phosphorylase/glycosyltransferase family mitochondrial OGT. The OGT enzyme is comprised of two distinct regions, an N-terminal domain containing a varying number of tetratricopeptide repeat (TPR) motifs and a C-terminal catalytic domain (1Lubas W.A. Frank D.W. Krause M. Hanover J.A. J. Biol. Chem. 1997; 272: 9316-9324Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar, 2Kreppel L.K. Blomberg M.A. Hart G.W. J. Biol. Chem. 1997; 272: 9308-9315Abstract Full Text Full Text PDF PubMed Scopus (612) Google Scholar) (Fig. 1). The TPR motif mediates protein-protein interactions (32Goebl M. Yanagida M. Trends Biochem. Sci. 1991; 16: 173-177Abstract Full Text PDF PubMed Scopus (377) Google Scholar, 33Liu F.H. Wu S.J. Hu S.M. Hsiao C.D. Wang C. J. Biol. Chem. 1999; 274: 34425-34432Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar), and a minimum of three TPRs are required for substrate binding (34Das A.K. Cohen P.W. Barford D. EMBO J. 1998; 17: 1192-1199Crossref PubMed Scopus (715) Google Scholar). We have recently solved the three-dimensional structure of the TPR domain of OGT (35Jinek M. Rehwinkel J. Lazarus B.D. Izaurralde E. Hanover J.A. Conti E. Nat. Struct. Mol. Biol. 2004; 11: 1001-1007Crossref PubMed Scopus (228) Google Scholar), which was shown to have a similar binding motif to importin-α; importin-α uses a string of conserved asparagines residues to bind a number of different, structurally unrelated substrates. The crystal structure also showed that the nucleocytoplasmic form of OGT exists as a dimer and that residues involved in formation of the dimer interface are present in the TPR domain. The three reported isoforms of OGT, nucleoplasmic OGT (ncOGT), mitochondrial OGT (mOGT), and short OGT (sOGT), have 12.5, 9.5, and 2.5 TPRs, respectively (36Hanover J.A. Yu S. Lubas W.B. Shin S.H. Ragano-Caracciola M. Kochran J. Love D.C. Arch. Biochem. Biophys. 2003; 409: 287-297Crossref PubMed Scopus (181) Google Scholar). The C-terminal catalytic region of OGT contains two conserved domains known as CD I and CD II (37Roos M.D. Hanover J.A. Biochem. Biophys. Res. Commun. 2000; 271: 275-280Crossref PubMed Scopus (51) Google Scholar), originally identified by their organizational similarity with the catalytic domain and lectin domain of the GalNAc transferases (38Hagen F.K. Hazes B. Raffo R. deSa D. Tabak L.A. J. Biol. Chem. 1999; 274: 6797-6803Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 39Breton C. Imberty A. Curr. Opin. Struct. Biol. 1999; 9: 563-571Crossref PubMed Scopus (171) Google Scholar). The two conserved domains of OGT also show similarities to glycosyltransferases that have a single monosaccharide-binding site. Our aim was to perform a mutational analysis of residues in the C-terminal catalytic domain that may be important for UDP-GlcNAc sensing, thus providing information regarding the catalytic roles of CD I and CD II. To identify potentially important amino acid residues that could be targeted for mutagenesis, sequence comparisons were made with OGT homologues, as well as with other glycosyltransferase families for which a mechanism has been proposed for catalytic function (38Hagen F.K. Hazes B. Raffo R. deSa D. Tabak L.A. J. Biol. Chem. 1999; 274: 6797-6803Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar, 40Cid E. Gomis R.R. Geremia R.A. Guinovart J.J. Ferrer J.C. J. Biol. Chem. 2000; 275: 33614-33621Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar, 41Morera S. Imberty A. Aschke-Sonnenborn U. Ruger W. Freemont P.S. J. Mol. Biol. 1999; 292: 717-730Crossref PubMed Scopus (99) Google Scholar, 42Vrielink A. Ruger W. Driessen H.P. Freemont P.S. EMBO J. 1994; 13: 3413-3422Crossref PubMed Scopus (227) Google Scholar, 43Persson K. Ly H.D. Dieckelmann M. Wakarchuk W.W. Withers S.G. Strynadka N.C. Nat. Struct. Biol. 2001; 8: 166-175Crossref PubMed Scopus (315) Google Scholar, 44Tenno M. Saeki A. Kezdy F.J. Elhammer A.P. Kurosaka A. J. Biol. Chem. 2002; 277: 47088-47096Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar). The approach taken was to make single amino acid substitutions at conserved residues in both the CD I and CD II regions of human mOGT (1Lubas W.A. Frank D.W. Krause M. Hanover J.A. J. Biol. Chem. 1997; 272: 9316-9324Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar), followed by recombinant expression in Escherichia coli. Because E. coli does not possess a gene for OGT, any OGT activity recovered from these extracts is due to expression of the recombinant enzyme. E. coli extracts were used in an in vitro assay system, where wild type mOGT and mutant OGT enzymes were tested for their ability to glycosylate Nup62, a nuclear pore protein known to be an excellent OGT substrate. Here we show that many of the amino acid substitutions made in both the CD I and CD II domains of OGT are critical for function and may be involved in either maintaining structure or catalysis. Most of the residues mutated in OGT were inactive; however, two amino acids in CD I (Asp422 and Tyr434) and one amino acid in CD II (Phe752) retained similar levels of enzyme activity as wild type. Homology modeling of these residues to other glycosyltransferases in the glycogen phosphorylase superfamily suggests that they may be either in or close to the active site of the enzyme. These results provide important new insights into the structure/function of this enzyme and may help understand the mechanism by which OGT recognizes UDP-GlcNAc and catalyzes transfer to substrate proteins. Site-directed Mutagenesis—Thirty mOGT single point mutants were generated using the QuikChange site-directed mutagenesis kit from Stratagene (see TABLE ONE), with the pET-32 human mOGT expression vector (1Lubas W.A. Frank D.W. Krause M. Hanover J.A. J. Biol. Chem. 1997; 272: 9316-9324Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar) as a template. The sequence was confirmed in each case and was shown to produce an open reading frame with the appropriate amino acid change.TABLE ONEOGT mutant constructs for CD I and CD II Amino acid substitutions made to residues in the CD I and CD II domains of OGT are shown.CD ICD IIY387AF721AG402SW735AD407AW748AD422AF752AY434AF776AD438AL796AF439AW812AG453SC836SF460AC839SG472AW878AE482AD488AD505AW536AG538SY539AD549AD554AE556AE568A Open table in a new tab Production of Human mOGT and mOGT Mutants from an E. coli Protein Expression System—The pET-32 human mOGT expression vector and the pET-32 human mOGT mutant expression vectors were transformed into competent BL21(DE3) cells (Novagen). As previously described (45Lubas W.A. Hanover J.A. J. Biol. Chem. 2000; 275: 10983-10988Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar), the cells were grown overnight at room temperature and 220 rpm in LB medium (KD Medical) supplemented with 50 μg/ml Ampicillin (Sigma). The cells were centrifuged at 3000 rpm for 10 min in a Beckman GS-6R centrifuge, and the pellet was resuspended in 1/20 of the original volume of lysis buffer containing 20 mm Tris-HCl, pH 7.5, 2 mm EDTA, 1 mg/ml lysozyme, 0.1% Triton X-100, and complete mini EDTA-free protease inhibitor mixture tablet (Roche Applied Science). Lysozyme digestion was performed at room temperature for 5 min. The lysate was subjected to freeze/thaw cycles by sonication on ice (3 × 10 s) until DNA was sheared. The supernatant obtained after centrifugation at 14,000 × g for 10 min was frozen in aliquots and stored at -80 °C. The total protein concentrations were determined using the BCA protein assay protocol (Pierce). OGT levels were detected by immunoblotting using an anti-His tag antibody (AbCam). O-GlcNAc Transferase Assay—As described previously (45Lubas W.A. Hanover J.A. J. Biol. Chem. 2000; 275: 10983-10988Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar), bacterial extracts containing human recombinant mOGT and mOGT mutant proteins were added to a 40-μl reaction mixture containing 50 mm Tris-HCl, pH 7.5, 1 mm dithiothreitol, 12.5 mm MgCl2, 0.2 nCi of UDP-[14C]GlcNAc (American Radiolabeled Chemicals), and 1 μg of recombinant, purified Nup62. The reactions were incubated for 60 min at 37 °C with shaking and were stopped by adding 4× SDS-PAGE sample buffer (Invitrogen) and boiling for 3 min. SDS-PAGE was performed using precast 4–12% NuPage gels (Invitrogen), followed by staining with Simply Blue Safestain (Invitrogen) for 60 min and destaining with distilled water for 60 min. Glycosylation of Nup62 with [14C]-GlcNAc was visualized after treatment with En3Hance (PerkinElmer Life Sciences) for 60 min by fluorography using BIOMAX-MR film (Kodak) or the Fujifilm BAS-1500 phosphorus imager. Densitometry of x-ray film was performed with Image J software, whereas densitometry of phosphorimaging data was performed with Image Gauge 3.0 software. OGT enzyme activity was also measured using ScintiStrip wells (Wallac) precoated with Nup62 (1Lubas W.A. Frank D.W. Krause M. Hanover J.A. J. Biol. Chem. 1997; 272: 9316-9324Abstract Full Text Full Text PDF PubMed Scopus (421) Google Scholar, 46Lubas W.A. Smith M. Starr C.M. Hanover J.A. Biochemistry. 1995; 34: 1686-1694Crossref PubMed Scopus (59) Google Scholar). For kinetic assays, OGT concentrations were initially determined by Ponceau S staining of Western blots. Image J software was used to determine the level of OGT protein compared with total protein levels determined by BCA. More recent experiments used fast green staining of nitrocellulose, followed by quantitation by infrared imaging using an Odyssey Western blot scanner. Native Gel Electrophoresis—E. coli lysates containing the same amounts of recombinant mOGT and mOGT mutant proteins were prepared in 2× native sample buffer (Invitrogen) and loaded onto a precast 4–12% Tris-glycine Novex gel (Invitrogen). Following electrophoresis, the gels were immunoblotted with an anti-His tag antibody (AbCam) to detect recombinant protein. Aldolase (160 kDa) and catalase (240 kDa) were used as molecular mass standards (Serva). Site-directed Mutagenesis of CD I Amino Acid Residues—The catalytic region of OGT consists of two highly conserved domains, CD I and CD II. Because the CD I domain of OGT may encode part of the sugar nucleotide-binding domain (36Hanover J.A. Yu S. Lubas W.B. Shin S.H. Ragano-Caracciola M. Kochran J. Love D.C. Arch. Biochem. Biophys. 2003; 409: 287-297Crossref PubMed Scopus (181) Google Scholar), a number of acidic residues in this domain were mutated. Acidic residues in the galactosyltransferase families are known to be important for recognition and stabilization of the sugar nucleotide substrate (44Tenno M. Saeki A. Kezdy F.J. Elhammer A.P. Kurosaka A. J. Biol. Chem. 2002; 277: 47088-47096Abstract Full Text Full Text PDF PubMed Scopus (43) Google Scholar, 47Zhang Y. Malinovskii V.A. Fiedler T.J. Brew K. Glycobiology. 1999; 9: 815-822Crossref PubMed Scopus (19) Google Scholar). Seven Asp residues and three Glu residues were mutated to Ala. Gly538 was substituted for Ser to evaluate a similar mutation in Arabidopsis OGT that causes disruption to giberellin signaling (Spindly phenotype) (48Jacobsen S.E. Binkowski K.A. Olszewski N.E. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 9292-9296Crossref PubMed Scopus (278) Google Scholar). Three other Gly to Ser substitutions were used to determine whether other Gly residues within CD I would have an effect on the enzyme activity of mOGT. Three Tyr residues, two Phe residues, and a Trp residue were mutated to Ala to evaluate the importance of aromatic residues in CD I. A complete list of CD I mutations is presented in TABLE ONE. Enzyme activity for wild type mOGT and the CD I mutant constructs was determined by the O-GlcNAc transferase assay (see "Experimental Procedures"), and incorporation of [C14]GlcNAc into Nup62 was visualized by fluorography (Fig. 2B). The bands were then quantified, and the resulting values were normalized as percentages of mOGT activity (Fig. 2A). All of the data were normalized to the concentration of OGT in each sample (Fig. 2C). The following mutations were not included for enzymatic analysis, because we could not produce sufficient amounts of protein: Y387A, F439A, D505A, W536A, Y539A, D549A, D554A, and E556A. With the exception of Y434A and D422A, all of the CD I mutants analyzed had a significant inhibitory effect on OGT enzyme activity. D422A produced a 50–100% increase in activity, whereas all of the other acidic amino acid substitutions (D407A, D438A, E482A, and D488A) caused an ∼95–99% reduction in enzyme activity. The Spindly mutation of Arabidopsis is in a conserved region of OGT corresponding to Gly538. Mutation of Gly538 to Ser produced a dramatic reduction in enzyme activity: 1% compared with wild type. This is the first demonstration that the Spindly mutation directly lowers OGT activity and points to the relevance of this residue in the mammalian OGT enzyme. Mutation to Ala of a neighboring residue, Tyr539, decreased OGT activity by 99%. Because glycine is not usually associated with catalytic activity in glycosyltransferases, three other Gly residues were substituted to Ser to determine whether this phenomenon was specific to Gly538 or whether other Gly residues had an effect on enzyme activity. Surprisingly, all three of these Gly residues reduced the enzyme activity by more than 98%. Finally, the remaining mutation, F460A, caused inactivation of the enzyme. All of the above results were confirmed using an alternative, scintillation-based assay. The OGT glycosylation assay was performed in ScintiStrip wells coated with Nup62, and incorporation of [C14]GlcNAc was measured by scintillation counting (TABLE TWO).TABLE TWOActivity of mutants compared with wild type mOGT (scintillation assay) The ability of OGT mutants to glycosylate nuclear pore protein Nup62 was compared with wild type mOGT. Incorporation of [C14]GlcNAc into Nup62 was determined by scintillation counting.CD ICD IIConstructActivityConstructActivity%%mOGT100F721A10G402S<1W735A<1D407A<1W748A<1D422A80F752A110Y434A65F776A10D438A<1L796A<1G453S5W812A<1F460A<1C836S10G472A<1C839S<1E482A5W878A<1D488A<1G538S<1E568A<1 Open table in a new tab Site-directed Mutagenesis of CD II Amino Acid Residues—The CD II domain of OGT is predicted to be a lectin domain, to be an allosteric regulatory site, or to be directly involved in binding of the donor sugar. Because aromatic residues have been previously implicated in binding reactions of lectins and glycosyltransferases with lectin-like domains (49Beattie B.K. Prentice G.A. Merrill A.R. Biochemistry. 1996; 35: 15134-15142Crossref PubMed Scopus (39) Google Scholar), all of the aromatic amino acid residues in CD II were mutated. Three Phe residues and four Trp residues were mutated to Ala. Two Cys residues (Cys836 and Cys839) were also mutated. A complete list of CD II mutations can be found in TABLE ONE. As with CD I mutant constructs, enzyme activity of CD II mutant constructs was determined by the O-GlcNAc transferase assay, and the results were visualized by fluorography of incorporated [C14]GlcNAc into Nup62 (Fig. 3B). The bands were quantified, and the resulting values were normalized as percentages of mOGT activity (Fig. 3A). All of the data were normalized to the concentration of OGT in each sample (Fig. 3C). All of the CD II mutant constructs that contained a Trp to Ala substitution (W735A, W748A, W812A, and W878A) showed a dramatic reduction in enzyme activity. OGT activity of these mutants was reduced by 96–100%. Of the three Phe residues to be mutated, one caused an ∼98% reduction in enzyme activity. The two other Phe residues had much less effect on enzyme activity, with F752A having a similar level of activity as wild type and F776A being reduced to ∼24% of normal activity. N-Ethylmaleimide and Alloxan inhibit OGT presumably by interfering with critical cysteine residues. Furthermore, dithiothreitol (a disulfide bond reducing agent) is known to increase the activity of wild type OGT (45Lubas W.A. Hanover J.A. J. Biol. Chem. 2000; 275: 10983-10988Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar), the importance of disulfide bonds, and their effect on producing active enzyme. The mechanism underlying this increase in activation is unknown; however, dithiothreitol is known to effect disulfide bonds between adjacent Cys residues or Cys residues that are in close proximity to one another. Two candidate residues for this type of interaction were Cys836 and Cys839. Each of these Cys residues were substituted with a Ser, and their OGT activity was determined. Both mutant enzymes caused a reduction in the level of enzyme activity. The C836S mutant showed variable activity, ranging from an ∼40–90% reduction in activity, whereas C839S showed an ∼93% reduction in activity. These results suggest that both Cys residues may be important for function of the enzyme; however, the difference in the level of OGT activity indicates that these residues may not interact with each other. OGT was placed into the glycogen phosphorylase superfamily (glycogen phosphorylase/glycosyltransferase family (GTGPF)) by Wrabl and Grishin (50Wrabl J.O. Grishin N.V. J. Mol. Biol. 2001; 314: 365-374Crossref PubMed Scopus (112) Google Scholar), based on its predicted three-dimensional structure consisting of two Rossman-type fold domains. Enzymes in this family share a common motif; however, the importance of amino acids in the GTGPF motif and the effect that these residues have on catalytic activity have not been studied. Two of the mutations produced are within this motif, L796A and W812A. As mentioned above, all Trp residues reduced OGT activity to background levels. The second mutation of Leu796 to Ala was not predicted to have a large impact on enzyme activity; however, this substitution caused a 99–100% reduction in the level of activity when compared with wild type. These results suggest that at least some of the amino acid residues found within the GTGPF motif are important for enzyme activity. However, examination of this region shows that many of these residues are highly conserved across many of the different OGT species and therefore may be important for maintaining the structure of the enzyme. These residues do not, however, show a great degree of primary sequence homology with glycogen phosphorylase, the enzyme with which the GTGPF superfamily classification was determined. Once again, the above results were confirmed using an alternative, scintillation-based assay (TABLE TWO). Kinetic Analysis of mOGT and Mutant Constructs D422A, Y434A, and F752A—Having shown that three of the mutations made to OGT could transfer a similar or increased amount of sugar nucleotide to Nup62, we next wanted to compare the kinetic properties of each with the native enzyme. Enzyme activity with respect to increasing concentrations of radiolabeled [C14]UDP-GlcNAc was measured by the O-GlcNAc transferase assay, and the results are presented in TABLE THREE. Wild type mOGT exhibited a Km value of ∼0.8 μm and a Vmax value of ∼125 nmol/mg/min (TABLE THREE and Ref. 45Lubas W.A. Hanover J.A. J. Biol. Chem. 2000; 275: 10983-10988Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar). OGT mutant D422A appeared to have an overall increase in activity (Fig. 2B), which was found to be due to a lower Km value than wild type (0.29 μm) and an almost 14-fold higher Vmax value (1580 nmol/mg/min). OGT mutant Y434A had approximately double the Km value of wild type (1.9 μm) and a 5-fold increase in the Vmax value (717.4 nmol/mg/min; TABLE THREE). Finally, the F752A OGT mutant had a lower Km value (0.15 μm) and a higher Vmax value (660 nmol/mg/min; TABLE THREE). For comparison, the kinetics of F776A were determined. This mutation was not inactive but showed a ∼76% reduction in enzyme activity. F776A had a Km value of 0.44 μm and a Vmax value of 53 nmol/mg/min (TABLE THREE).TABLE THREEKinetic properties of OGT and active mutants The kinetic parameters of activating mutations D422A, Y434A, and F752A were analyzed and compared with mOGT. The Km and Vmax values were determined with respect to UDP-GlcNAc, using the concentration range 1.5–12 μm.ConstructKm (mm)Vmax (nmol/mg/min)mOGT0.8125D422A0.291580Y434A1.9717.4F752A0.15660F776A0.4453 Open table in a new tab To make certain that the mutant proteins used for kinetic analysis were not aggregated and maintained their multimeric status, native gel electrophoresis was performed (Fig. 4). mOGT, D422A, Y434A, F752A, and F776A were compared with a monomeric form of OGT (35Jinek M. Rehwinkel J. Lazarus B.D. Izaurralde E. Hanover J.A. Conti E. Nat. Struct. Mol. Biol. 2004; 11: 1001-1
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