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• W2156197297 abstract "The glycosyltransferase termed MshA catalyzes the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to 1-l-myo-inositol-1-phosphate in the first committed step of mycothiol biosynthesis. The structure of MshA from Corynebacterium glutamicum was determined both in the absence of substrates and in a complex with UDP and 1-l-myo-inositol-1-phosphate. MshA belongs to the GT-B structural family whose members have a two-domain structure with both domains exhibiting a Rossman-type fold. Binding of the donor sugar to the C-terminal domain produces a 97° rotational reorientation of the N-terminal domain relative to the C-terminal domain, clamping down on UDP and generating the binding site for 1-l-myo-inositol-1-phosphate. The structure highlights the residues important in binding of UDP-N-acetylglucosamine and 1-l-myo-inositol-1-phosphate. Molecular models of the ternary complex suggest a mechanism in which the β-phosphate of the substrate, UDP-N-acetylglucosamine, promotes the nucleophilic attack of the 3-hydroxyl group of 1-l-myo-inositol-1-phosphate while at the same time promoting the cleavage of the sugar nucleotide bond. The glycosyltransferase termed MshA catalyzes the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine to 1-l-myo-inositol-1-phosphate in the first committed step of mycothiol biosynthesis. The structure of MshA from Corynebacterium glutamicum was determined both in the absence of substrates and in a complex with UDP and 1-l-myo-inositol-1-phosphate. MshA belongs to the GT-B structural family whose members have a two-domain structure with both domains exhibiting a Rossman-type fold. Binding of the donor sugar to the C-terminal domain produces a 97° rotational reorientation of the N-terminal domain relative to the C-terminal domain, clamping down on UDP and generating the binding site for 1-l-myo-inositol-1-phosphate. The structure highlights the residues important in binding of UDP-N-acetylglucosamine and 1-l-myo-inositol-1-phosphate. Molecular models of the ternary complex suggest a mechanism in which the β-phosphate of the substrate, UDP-N-acetylglucosamine, promotes the nucleophilic attack of the 3-hydroxyl group of 1-l-myo-inositol-1-phosphate while at the same time promoting the cleavage of the sugar nucleotide bond. Members of the Actinomycetales family function in diverse and important roles to humans. On the positive side, Streptomyces species produce over two-thirds of the clinically useful antibiotics of natural origin (1Demain A.L. Appl. Microbiol. Biotechnol. 1999; 52: 455-463Crossref PubMed Scopus (335) Google Scholar) and Corynebacteria species are heavily utilized in industrial synthesis (1Demain A.L. Appl. Microbiol. Biotechnol. 1999; 52: 455-463Crossref PubMed Scopus (335) Google Scholar, 2Hermann T. J. Biotechnol. 2003; 104: 155-172Crossref PubMed Scopus (513) Google Scholar), whereas on the negative side many Actinomycetales are pathogens, for example, Mycobacterium are the causative agents of the diseases tuberculosis (3Dye C. Scheele S. Dolin P. Pathania V. Raviglione M.C. J. Am. Med. Assoc. 1999; 282: 677-686Crossref PubMed Scopus (2729) Google Scholar) and leprosy, infecting millions worldwide each year (3Dye C. Scheele S. Dolin P. Pathania V. Raviglione M.C. J. Am. Med. Assoc. 1999; 282: 677-686Crossref PubMed Scopus (2729) Google Scholar, 4Britton W.J. Lockwood D.N. Lancet. 2004; 363: 1209-1219Abstract Full Text Full Text PDF PubMed Scopus (649) Google Scholar). An intimate knowledge of biochemistry common to all Actinomycetales allows their utilization as chemistry toolboxes while mitigating their pathogenic nature. For most organisms, maintenance of the appropriate reducing environment in the cell is required for proper cellular function, and this is usually achieved through the synthesis and cellular balance of low molecular weight thiols such as glutathione (5Hand C.E. Honek J.F. J. Nat. Prod. 2005; 68: 293-308Crossref PubMed Scopus (128) Google Scholar). Although glutathione is the predominate thiol in Gram-negative bacteria and eukaryotes, in the Actinomycetales, the major thiol is instead mycothiol (MSH), 2The abbreviations used are: MSH, mycothiol or 1-d-myoinosityl-2-(n-acetyl-l-cysteinyl)amido-2-deoxy-α-d-glucopyranoside; CgMshA, C. glutamicum MshA; 1-l-Ins-1-P, 1-l-myo-inositol 1-phosphate; GlcNAc-Ins-P, 3-phospho-1-d-myo-inosityl-2-acetamido-2-dexoy-α-d-glucopyranoside; GT, glycosyltransferase; Af_INO, inositol-1-phosphate synthase from A. fulgidus; man, mannose; TEA, triethanolamine; CAZy, Carbohydrate-Active Enzymes data base. 1-d-myoinosityl-2-(n-acetyl-l-cysteinyl)amido-2-deoxy-α-d-glucopyranoside (6Newton G.L. Arnold K. Price M.S. Sherrill C. Delcardayre S.B. Aharonowitz Y. Cohen G. Davies J. Fahey R.C. Davis C. J. Bacteriol. 1996; 178: 1990-1995Crossref PubMed Google Scholar). The synthetic pathway for MSH is believed to be conserved in all Actinomycetales species and requires at least four enzymes (MshA through MshD, Fig. 1). The first enzyme, MshA, catalyzes the transfer of N-acetylglucosamine from UDP-N-acetylglucosamine (UDP-GlcNAc) to 1-l-myo-inositol 1-phosphate (1-l-Ins-1-P) to produce 3-phospho-1-d-myo-inosityl-2-acetamido-2-deoxy-α-d-glucopyranoside (GlcNAc-Ins-P) (7Newton G.L. Ta P. Bzymek K.P. Fahey R.C. J. Biol. Chem. 2006; 281: 33910-33920Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar, 8Newton G.L. Koledin T. Gorovitz B. Rawat M. Fahey R.C. Av-Gay Y. J. Bacteriol. 2003; 185: 3476-3479Crossref PubMed Scopus (75) Google Scholar). A yet to be discovered phosphatase is proposed to dephosphorylate GlcNAc-Ins-P to produce GlcNAc-Ins (7Newton G.L. Ta P. Bzymek K.P. Fahey R.C. J. Biol. Chem. 2006; 281: 33910-33920Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). GlcNAc-Ins is then deacetylated (MshB) and subsequently cysteinylated (MshC) at the amino group to produce Cys-GlcN-Ins, which then is acetylated (MshD) to produce AcCys-GlcN-Ins or MSH (9Newton G.L. Av-Gay Y. Fahey R.C. J. Bacteriol. 2000; 182: 6958-6963Crossref PubMed Scopus (99) Google Scholar, 10Koledin T. Newton G.L. Fahey R.C. Arch. Microbiol. 2002; 178: 331-337Crossref PubMed Scopus (75) Google Scholar, 11Newton G.L. Fahey R.C. Arch. Microbiol. 2002; 178: 388-394Crossref PubMed Scopus (156) Google Scholar, 12Anderberg S.J. Newton G.L. Fahey R.C. J. Biol. Chem. 1998; 273: 30391-30397Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar, 13Sareen D. Steffek M. Newton G.L. Fahey R.C. Biochemistry. 2002; 41: 6885-6890Crossref PubMed Scopus (96) Google Scholar). In Mycobacterium smegmatis, mutants blocked in MSH biosynthesis exhibit enhanced sensitivity to cellular stress reagents, including hydrogen peroxide and rifampicin (14Newton G.L. Ta P. Fahey R.C. J. Bacteriol. 2005; 187: 7309-7316Crossref PubMed Scopus (57) Google Scholar, 15Rawat M. Newton G.L. Ko M. Martinez G.J. Fahey R.C. Av-Gay Y. Antimicrob. Agents Chemother. 2002; 46: 3348-3355Crossref PubMed Scopus (164) Google Scholar), whereas in Mycobacterium tuberculosis (Erdman strain) mutants blocked in MSH production were not viable (16Sareen D. Newton G.L. Fahey R.C. Buchmeier N.A. J. Bacteriol. 2003; 185: 6736-6740Crossref PubMed Scopus (114) Google Scholar). Of the four genes, mshA and mshC were found to be critical to the production of MSH and therefore viability of the organism (16Sareen D. Newton G.L. Fahey R.C. Buchmeier N.A. J. Bacteriol. 2003; 185: 6736-6740Crossref PubMed Scopus (114) Google Scholar, 17Buchmeier N. Fahey R.C. FEMS Microbiol. Lett. 2006; 264: 74-79Crossref PubMed Scopus (53) Google Scholar). Interruption of mshB or mshD was either complemented by a promiscuous cellular activity or the product of interrupted synthesis acted as a poor analog of MSH (14Newton G.L. Ta P. Fahey R.C. J. Bacteriol. 2005; 187: 7309-7316Crossref PubMed Scopus (57) Google Scholar, 18Buchmeier N.A. Newton G.L. Fahey R.C. J. Bacteriol. 2006; 188: 6245-6252Crossref PubMed Scopus (82) Google Scholar). Therefore MshA and MshC are important potential drug targets for treatment of tuberculosis and other Actinomycetales infections. The three-dimensional structures of both MshB (19Maynes J.T. Garen C. Cherney M.M. Newton G. Arad D. Av-Gay Y. Fahey R.C. James M.N. J. Biol. Chem. 2003; 278: 47166-47170Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar) and MshD (20Vetting M.W. Yu M. Rendle P.M. Blanchard J.S. J. Biol. Chem. 2006; 281: 2795-2802Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar, 21Vetting M.W. Roderick S.L. Yu M. Blanchard J.S. Prot. Sci. 2003; 12: 1954-1959Crossref PubMed Scopus (62) Google Scholar) have been determined; however, there have been no reports of structures for MshA or MshC. Glycosyltransferases (GTs), such as MshA, can be grouped based on sequence into what is currently a total of 90 distinct families (CAZy, Carbohydrate-Active Enzymes data base) (22Campbell J.A. Davies G.J. Bulone V.V. Henrissat B. Biochem. J. 1998; 329: 719Crossref PubMed Scopus (52) Google Scholar, 23Coutinho P.M. Deleury E. Davies G.J. Henrissat B. J. Mol. Biol. 2003; 328: 307-317Crossref PubMed Scopus (931) Google Scholar). GTs can also be grouped based on structure, with most GTs determined to date having one of two folds, termed GT-A and GT-B (24Bourne Y. Henrissat B. Curr. Opin. Struct. Biol. 2001; 11: 593-600Crossref PubMed Scopus (365) Google Scholar). Recently two new GT folds have been described for CAZY family GT51 (25Lovering A.L. de Castro L.H. Lim D. Strynadka N.C. Science. 2007; 315: 1402-1405Crossref PubMed Scopus (238) Google Scholar) and GT66 (26Igura M. Maita N. Kamishikiryo J. Yamada M. Obita T. Maenaka K. Kohda D. EMBO J. 2008; 27: 234-243Crossref PubMed Scopus (146) Google Scholar). Finally GTs can be termed “retaining” or “inverting” based on the configuration of the sugar nucleotide-derived carbohydrate in the final product. There is no absolute correlation between the fold (GT-A or GT-B) and the stereochemical outcome of the reaction as examples of both inverting and retaining GTs have been found in both families; however, all members of a CAZy family are expected to have the same fold (23Coutinho P.M. Deleury E. Davies G.J. Henrissat B. J. Mol. Biol. 2003; 328: 307-317Crossref PubMed Scopus (931) Google Scholar, 27Breton C. Snajdrova L. Jeanneau C. Koca J. Imberty A. Glycobiology. 2006; 16: 29R-37RCrossref PubMed Scopus (504) Google Scholar). Based on sequence similarity MshA is grouped into CAZy family GT4, all of which have been found to be retaining glycosyltransferases and have the GT-B fold. GT4 is the second largest CAZy data base family (6563 CAZY entries at the time of this writing) and have a diverse set of donor and acceptor molecules. Only recently a limited number of GT4 family members have been structural characterized, including WaaG, which transfers glucose from UDP-glucose onto l-glycero-d-mannoheptose II; AviGT4, which is involved in avilamycin A biosynthesis; and PimA, a phosphatidylinositol mannosyltransferase (28Martinez-Fleites C. Proctor M. Roberts S. Bolam D.N. Gilbert H.J. Davies G.J. Chem. Biol. 2006; 13: 1143-1152Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 29Guerin M.E. Kordulakova J. Schaeffer F. Svetlikova Z. Buschiazzo A. Giganti D. Gicquel B. Mikusova K. Jackson M. Alzari P.M. J. Biol. Chem. 2007; 282: 20705-20714Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Here we present the structure of unliganded MshA from Cornybacterium glutamicum and its complex with UDP and 1-l-Ins-1-P. Enzymatic studies demonstrate that UDP-GlcNAc and 1-l-Ins-1-P are substrates for CgMshA, and that the product is GlcNAc-Ins-P, as was previously determined for MshA from crude lysates of Mycobacterium smegmatis (7Newton G.L. Ta P. Bzymek K.P. Fahey R.C. J. Biol. Chem. 2006; 281: 33910-33920Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Initial velocity studies indicate a sequential mechanism with UDP-GlcNAc almost certainly binding first followed by 1-l-Ins-1-P. The structural and kinetic data are most consistent with a mechanism involving the β-phosphoryl group of the substrate, UDP-GlcNAc, acting as the general base and not a mechanism involving a covalent enzyme-N-acetylglucosamine intermediate. The crystal structure highlights residues involved in binding and catalysis and serves as a starting point for inhibitor design. Cloning—The MshA gene (residues 1–418) was amplified from C. glutamicum genomic DNA (ATCC 13032) using PCR (primers 5-TTTTTCATATGCGCGTAGCTATGATTTCC-3′ and 5′-TTTTTCTCGAGTTAGCCGTGATGCGTTTCAC-3′) and blunt-end-cloned into pCR-BLUNT (Invitrogen). An internal Nde1 site was removed using PCR mutagenesis and primers containing the desired alteration (primers, 5′-CCGGATACCTATAGGCAcATGGCAGAGGAACTGGGC-3′ and 5′-GCCCAGTTCCTCTGCCATgTGCCTATAGGTATCCGG-3′). The CgMshA gene was subcloned into the Nde1 and HindIII sites of pET28a(+) utilizing a HindIII site of the pCR-BLUNT vector as the downstream restriction site. The CgMshA gene was also cloned into the Nde1 and XhoI sites of pET29a(+) utilizing PCR amplification and the CgMshA: pET28a(+) vector as a template (primers 5′-TTTTTCATATGCGCGTAGCTATGATTTCC-3′ and 5′-ATCCCGCTCTCGAGGCCGTGATGCGTTTCACC-3′). Expression—Overexpression vectors containing the CgMshA gene were transformed into Rosetta2 cells (Invitrogen) and grown in 8-ml overnight cultures of MDG media (30Studier F.W. Protein Expression Purif. 2005; 41: 207-234Crossref PubMed Scopus (4193) Google Scholar). These cultures were then made 10% in glycerol and stored at -80 °C in 2-ml aliquots. A 100-ml starter culture of MDG media was inoculated with one 2-ml storage stock and grown at 37 °C to late-log phase. This was used to inoculate 4 liters of ZYP5052 autoinduction media in six 2-liter baffled flasks (30Studier F.W. Protein Expression Purif. 2005; 41: 207-234Crossref PubMed Scopus (4193) Google Scholar). The cultures were grown at 23 °C with 300 rpm shaking and were harvested at saturation (36–48 h, A600 > 12). All growth media contained 30 μg/ml chloramphenicol and 200 μg/ml kanamycin. Purification—Frozen cell paste was resuspended in three times its volume of buffer A (100 mm TEA, pH 7.8, 200 mm (NH4)2SO4, 10% glycerol, 15 mm imidazole), and lysozyme was added to 1 mg/ml. After incubation at 4 °C for 30 min the cell slurry was disrupted by sonication and was spun at 25,000 × g for 30 min. The supernatant was applied to a nickel-Sepharose HP (GE-Healthcare) column equilibrated against buffer A. Bound proteins were eluted with a gradient of 15–300 mm imidazole over 10 column volumes. Fractions with greater than 20% of the peak activity were pooled, made 1 m in (NH4)2SO4, and applied to a Phenyl-Sepharose HP (GE-Healthcare) column equilibrated against buffer B (20 mm TEA, pH 7.8, 1 m (NH4)2SO4, 10% glycerol, 0.5 mm EDTA, 1 mm β-mercaptoethanol). Bound proteins were separated with a 1 to 0 m (NH4)2SO4 gradient over 10 column volumes with properly folded CgMshA eluting between 500 and 300 mm (NH4)2SO4. Pooled fractions were concentrated by ultrafiltration (Amicon) to a concentration of 20–40 mg/ml and stored at -80 °C. Crystallization and Phasing—Crystallization of CgMshA was by vapor diffusion under silicon oil (Fisher) utilizing 96-well round bottom assay plates stored open to room humidity at 18 °C. Crystals of APO CgMshA with a hexagonal bipyramidal morphology were obtained from drops containing 2 μl of protein (CgMshA:pET28a(+), 15 mg/ml, 400 mm (NH4)2SO4, 10% glycerol, 0.5 mm EDTA, 1 mm β-mercaptoethanol) with 2 μl of precipitant (20% polyethylene glycol 4000, 100 mm Tris, pH 8.5, 200 mm LiSO4). Crystals were soaked in a stabilization solution of 40% polyethylene glycol 4000, 100 mm Tris, pH 8.5, 200 mm LiSO4, prior to vitrification by plunging in liquid nitrogen. Data were collected on an MSC R-AXIS-IV++ image plate detector using CuKα radiation from a Rigaku RU-H3R x-ray generator and processed using MOSFLM and SCALA (31Evans P. Acta Crystallogr. D Biol. Crystallogr. 2006; 62: 72-82Crossref PubMed Scopus (3794) Google Scholar, 32Leslie A.G. Acta Crystallogr. D Biol. Crystallogr. 2006; 62: 48-57Crossref PubMed Scopus (972) Google Scholar). The space group was determined to be P31 with approximate cell dimensions of a = b = 79.7 Å, and c = 148.4 Å. There is a molecular dimer per asymmetric unit with a solvent content of 59%. The structure of APO CgMshA was determined by SIRAS using a single mercury derivitized crystal, which was prepared by soaking a crystal for 2 h in the stabilization solution with the addition of saturated p-chloromercuribenzoate. Heavy atom positions and initial phases were determined using PHENIX (33Adams P.D. Gopal K. Grosse-Kunstleve R.W. Hung L.W. Ioerger T.R. McCoy A.J. Moriarty N.W. Pai R.K. Read R.J. Romo T.D. Sacchettini J.C. Sauter N.K. Storoni L.C. Terwilliger T.C. J. Synchrotron Rad. 2004; 11: 53-55Crossref PubMed Scopus (280) Google Scholar). The solvent flattened SIRAS map was of sufficient quality for ARP/WARP (34Perrakis A. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 448-455Crossref PubMed Scopus (484) Google Scholar) to autobuild a majority of the structure. Iterative rounds of modeling building within the molecular graphics program COOT (35Emsley P. Cowtan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 2126-2132Crossref PubMed Scopus (23628) Google Scholar) followed by refinement against the data using REFMAC (36Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar) were used to build the remainder of the structure. The final rounds included TLS refinement. A second crystal form of CgMshA was obtained in drops that contained 2 μl of protein (CgMshA:pET29a(+), 40 mg/ml, 400 mm (NH4)2SO4, 10% glycerol, 0.5 mm EDTA, 1 mm β-mercaptoethanol, 10 mm UDP-GlcNAc) combined with 2 μl of precipitant (0.8 m sodium citrate, pH 5.5). This crystal form was cryoprotected by soaking in 1.2 m sodium citrate, pH 5.5, 10% glycerol, and 200 mm (NH4)2SO4 prior to vitrification by plunging in liquid nitrogen. For the UDP·1-l-Ins-1-P dataset the (NH4)2SO4 was replaced with 50 mm 1-l-Ins-1-P, and the crystals were soaked for 1 h. Data were collected at Brookhaven National Laboratories at beamline X25 and processed using MOSFLM (32Leslie A.G. Acta Crystallogr. D Biol. Crystallogr. 2006; 62: 48-57Crossref PubMed Scopus (972) Google Scholar) and SCALA (31Evans P. Acta Crystallogr. D Biol. Crystallogr. 2006; 62: 72-82Crossref PubMed Scopus (3794) Google Scholar). The space group was determined to be I422 with approximate cell dimensions of a = b = 223.9 Å, and c = 125.0 Å. This crystal form has two molecules per asymmetric unit with a solvent content of 71.5%. Phasing of the UDP dataset utilized the molecular replacement program AMORE (37Navaza J. Acta Crystallogr. Sect. A. 1994; 50: 157-163Crossref Scopus (5030) Google Scholar) and the APO form of CgMshA as the model. Searches with the complete APO structure and or either of the N- or C-terminal domains did not produce a convincing molecular replacement solution. However, utilizing a dimerized N-terminal domain produced two solutions that were significantly above other solutions despite (or possibly because of) the fact that both of the molecular dimers in this crystal form are acted upon by crystallographic symmetry. Molecular replacement searches for the missing C-terminal domain after placement of the N-terminal domains were unsuccessful. The recently determined structure of PimA (PDB ID: 2GEJ) was used as a guide for the structure of a closed GT-B:GT-4 glycosyltransferase family member (29Guerin M.E. Kordulakova J. Schaeffer F. Svetlikova Z. Buschiazzo A. Giganti D. Gicquel B. Mikusova K. Jackson M. Alzari P.M. J. Biol. Chem. 2007; 282: 20705-20714Abstract Full Text Full Text PDF PubMed Scopus (112) Google Scholar). Utilizing electron density maps phased with only the two placed N-terminal domains, and the PimA structure as a guide, there was enough secondary structure visible in a skeletonized map to manually place both C-terminal domains, which were then successfully refined into their correct positions with rigid body refinement. Molecular constraints for UDP and 1-l-Ins-1-P were calculated using the PRODRG server (38Schuttelkopf A.W. van Aalten D.M. Acta Crystallogr. Sect. D Biol. Crystallogr. 2004; 60: 1355-1363Crossref PubMed Scopus (4360) Google Scholar). Refinement of this crystal form utilized REFMAC with the final rounds incorporating TLS refinement (36Murshudov G.N. Vagin A.A. Dodson E.J. Acta Crystallogr. Sect. D Biol. Crystallogr. 1997; 53: 240-255Crossref PubMed Scopus (13914) Google Scholar). DLS measurements were performed with a 4 mg/ml CgMshA solution in 250 mm (NH4)2SO4, 50 mm TEA, pH 7.8, at 25 °C on a DynaPro MS/X instrument (Protein Solutions). Data collection and deconvolution were performed using the DYMAMICS 6.2.05 software (Protein Solutions). Purified CgMshA was subjected to gel-filtration chromatography at a flow rate of 0.5 ml/min on a Superose-12 column (10/300, Amersham Biosciences). The column was pre-equilibrated with 50 mm TEA, pH 7.8, 200 mm (NH4)2SO4, 10% glycerol. The following proteins were used as molecular mass standards: thyroglobulin, 669 kDa; bovine γ-globulin, 158 kDa; chicken albumin, 44 kDa; equine myoglobin, 17.0 kDa; and vitamin B12, 1.3 kDa. Production of 1-l-Ins-1-P—The gene for inositol-1-phosphate synthase from the hyperthermophile Archaeoglobus fulgidus (Af_INO) was cloned as described by a previous group (39Chen L. Zhou C. Yang H. Roberts M.F. Biochemistry. 2000; 39: 12415-12423Crossref PubMed Scopus (64) Google Scholar). Briefly, the Af_INO gene was PCR-amplified from genomic DNA and ligated into pET23a(+) vector utilizing Nde1 and HindIII restriction sites. The resultant plasmid was transformed into Escherichia coli strain BL21DE3/pLysS, and the cells were grown and protein was expressed by autoinduction in an identical fashion as CgMshA (see above), except that the antibiotic was ampicillin (100 μg/ml), and the cells were grown at 37 °C for 12–18 h. Cells were sonicated in three times the weight/volume of buffer A (50 mm Tris, pH 8.5), and spun at 25,000 × g to remove cell debris. The supernatant, contained in a steel vessel, was heated in an 80 °C water bath, with constant stirring for 5 min. The vessel was then placed on ice and cooled to 4 °C as quickly as possible by constant stirring. The precipitated proteins were removed by centrifugation (25,000 × g). The supernatant was applied to a 2.6 × 12 cm Poros-HQ anion exchange (POROS MEDIA) column, equilibrated with buffer A. Bound proteins were eluted with a 0 to 0.25 m (NH4)2SO4 gradient over ten column volumes. Fractions, which related to peak concentrations of Af_INO, by SDS gels, were pooled, made 1 m in (NH4)2SO4 and applied to a 2.6 × 12 cm phenyl-Sepharose (GE-Healthcare) hydrophobic exchange column equilibrated with buffer A plus 1 m (NH4)2SO4. Bound proteins were eluted with a 1.0–0 m (NH4)2SO4 gradient in buffer A over ten column volumes, pooled based on purity by SDS gels, and dialyzed overnight in buffer A. Purified Af_INO was concentrated to 10 mg/ml by Amicon ultrafiltration, snap frozen in liquid nitrogen, and stored at -80 °C. 1-l-Ins-1-P was synthesized using Af_INO and glucose 6-phosphate. The starting solution was 125 mm glucose 6-phosphate, 0.625 mm ZnCl2, 1.25 mm NAD+, and 3.6 mg of Af_INO in 50 mm Tris, pH 7.5 (NH4OH). The reaction was held at 85 °C with a heat block, and every 45 min the NAD was increased by 0.5 mm and another 2 mg of Af_INO was added. The progress of the reaction was monitored utilizing the CgMshA/pyruvate kinase/lactate dehydrogenase assay with substoichiometric 1-l-Ins-1-P (potential from reaction, 0.20 mm) to NADH (0.25 mm). The completed reaction (∼2–3 h) was diluted to 100 mm (taking into account additions during the reaction) and stored at -80 °C to be used directly in assays for MshA activity. Measurement of Enzymatic Activity—The production of UDP was measured using a coupled assay system and monitoring the reaction at 340 nm. Standard conditions were 30 nm enzyme, 50 mm TEA, pH 7.8, 200 μm NADH, 500 μm phosphoenol pyruvate, 10 mm MgCl2, 20 units of pyruvate kinase (ammonium sulfate suspension), and 55 units of lactate dehydrogenase (ammonium sulfate suspension) in a 1-ml reaction at 25 °C. Except for the enzyme, all components were mixed in the cuvette and allowed to equilibrate for 2 min. Reactions were initiated by the addition of enzyme. The amount of coupling enzymes was sufficient to not limit the rate of reaction and was required to minimize a lag in the assays. Data Analysis—To determine the basic kinetic parameters for each substrate, initial velocity plots at saturating concentrations of one substrate were fit to the Michaelis-Menten equation (Equation 1) where Vmax is the maximal velocity, A is the concentration of the varied substrate, and Ka is the Michaelis constant for substrate A. When both substrates were varied, intersecting initial velocity plots were fit to Equation 2. V=Vmax[A]Ka+[A]Eq. 1 V=Vmax[A][B]KiaKb+Kb[A]+Ka[B]+[A][B]Eq. 2 Data described with single variable equations were analyzed using KaleidaGraph (Synergy Software). Data described using equations with multiple independent variables were analyzed using GraFit (Erithacus Software). Isolation of GlcNAc-inositol 3-Phosphate—CgMshA (80 μg) was added to a 1-ml solution containing 100 mm TEA, pH 7.8, 100 mm 1-l-Ins-1-P, and 100 mm UDP-GlcNAc. The production of UDP was monitored using a high-performance liquid chromatography-based assay and a UV detector (260 nm). The separation of compounds was accomplished using a 1-ml Mono Q ion-exchange column (GE-Healthcare) with the following programmed gradient: 0–5 min (0% B), 20 min (35% B), 25–30 min (100% B), 33 min (0%B) where Buffer A is 20 mm ammonium bicarbonate and Buffer B is 600 mm ammonium bicarbonate and a flow rate of 1 ml/min. Retention times for UDP-GlcNAc and UDP were 12 and 20 min, respectively. When the reaction had run to completion (∼5 h), the entire reaction was injected and fractionated using the above high-performance liquid chromatography method. Fractions (1 ml) were treated with alkaline phosphatase and then assayed for inorganic phosphate using a malachite green phosphate assay kit (Bioassay Systems). Fractions 9–12 were found to contain phosphate but did not correlate to a peak at 260 nm. These fractions were pooled and lyophilized. The lyophilized white powder was analyzed by 1H NMR, 31P NMR, and by mass spectrometry. Electrospray ionization-mass spectrometry yielded a value of m/z 462.36 (M-H)-1 (C14H25NO14P-1 calculated 462.10). Expression and Purification—Initial screens of full-length clones of MshA from Mycobacterium tuberculosis, Streptomyces coeliclor, Nocardia farcina, and C. glutamicum as N-terminally hexahistidine-tagged constructs all resulted in high overexpression, but only the MshA from C. glutamicum (CgMshA) resulted in large amounts of soluble protein. Typically, yields of soluble, active CgMshA using autoinduction media were 200 mg/liter. After nickel-nitrilotriacetic acid affinity chromatography, purification on Phenyl Sepharose hydrophobic exchange media was critical to separating active, monodisperse protein from inactive, polydisperse protein. Ammonium sulfate (>100 mm) was required to maintain activity, whereas protein kept in a minimal ionic strength buffer was subject to degradation and loss of activity. The initial construct of MshA utilized an N-terminal thrombin-cleavable 6× His tag to facilitate purification. Attempts at thrombin cleavage resulted in nonspecific degradation, so the N-terminal tag was not removed. The N-terminally tagged protein was utilized for the initial structure determination of unliganded MshA. A second C-terminally tagged MshA was used to determine the structure of MshA in complex with UDP and 1-l-Ins-1-P. Enzymatic Characterization—From the initial velocity plots for 1-l-Ins-1-P and UDP-GlcNAc, both substrates exhibited Michaelis-Menten kinetics, and no curvature was seen in the Lineweaver-Burk linear replots (supplemental Fig. S1). The kinetic parameters for each substrate were determined from fits of the data to Equation 1. A value of 12.5 ± 0.2 s-1 was determined for the kcat, and the Michaelis constants for 1-l-Ins-1-P and UDP-GlcNAc were 240 ± 10 and 210 ± 20 μm, respectively. The Km values are consistent with those determined previously for MshA from Mycobacterium smegmatis crude lysates (7Newton G.L. Ta P. Bzymek K.P. Fahey R.C. J. Biol. Chem. 2006; 281: 33910-33920Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). To determine whether MshA proceeds through a sequential or ping-pong mechanism, an initial velocity plot was made by varying both substrates. The plot shows intersecting lines, and the data fit well to Equation 2, consistent with a sequential mechanism (supplemental Fig. S2). The pseudo-disaccharide phosphate monoester product of the reaction was purified using ion-exchange chromatography. 31P and 1H NMR spectroscopy of the product was consistent with GlcNAc-Ins-P (supplemental Figs. S3 and S4). A coupling constant of 3.3 Hz was determined for the anomeric proton confirming that the product retains the alpha sugar configuration (inset, supplemental Fig. S4). GT-B Fold Monomer Structure—The structure of unliganded CgMshA (APO form) was determined by single isomorphous replacement with anomalous scattering utilizing a CuKα home source and a mercury derivative to 2.1-Å resolution (Table 1). The structure of CgMshA has two domains each exhibiting the β/α/β Rossmann-fold type typical of the GT-B fold superfamily (Fig. 2). The N-terminal domain has an 8-stranded β-sheet (β8, β7, β6, β5, β1, β2, β4, and β3) that is bounded by six α-helices (α1–α5 and α14). The C-terminal d" @default.
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• W2156197297 date "2008-06-01" @default.
• W2156197297 modified "2023-09-29" @default.
• W2156197297 title "Structural and Enzymatic Analysis of MshA from Corynebacterium glutamicum" @default.
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• W2156197297 doi "https://doi.org/10.1074/jbc.m801017200" @default.
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