Characterization of a Metal-independent CAZy Family 6 Glycosyltransferase from Bacteroides ovatus
2009; Elsevier BV; Volume: 284; Issue: 37 Linguagem: Inglês
10.1074/jbc.m109.033878
ISSN1083-351X
Autores Tópico(s)Bacteriophages and microbial interactions
ResumoThe myriad functions of complex carbohydrates include modulating interactions between bacteria and their eukaryotic hosts. In humans and other vertebrates, variations in the activity of glycosyltransferases of CAZy family 6 generate antigenic variation between individuals and species that facilitates resistance to pathogens. The well characterized vertebrate glycosyltransferases of this family are multidomain membrane proteins with C-terminal catalytic domains. Genes for proteins homologous with their catalytic domains are found in at least nine species of anaerobic commensal bacteria and a cyanophage. Although the bacterial proteins are strikingly similar in sequence to the catalytic domains of their eukaryotic relatives, a metal-binding Asp-X-Asp sequence, present in a wide array of metal ion-dependent glycosyltransferases, is replaced by Asn-X-Asn. We have cloned and expressed one of these proteins from Bacteroides ovatus, a bacterium that is linked to inflammatory bowel disease. Functional characterization shows it to be a metal-independent glycosyltransferase with a 200-fold preference for UDP-GalNAc as substrate relative to UDP-Gal. It efficiently catalyzes the synthesis of oligosaccharides similar to human blood group A and may participate in the synthesis of the bacterial O-antigen. The kinetics for GalNAc transfer to 2′-fucosyl lactose are characteristic of a sequential mechanism, as observed previously for this family. Mutational studies indicate that despite the lack of a metal cofactor, there are pronounced similarities in structure-function relationships between the bacterial and vertebrate family 6 glycosyltransferases. These two groups appear to provide an example of horizontal gene transfer involving vertebrates and prokaryotes. The myriad functions of complex carbohydrates include modulating interactions between bacteria and their eukaryotic hosts. In humans and other vertebrates, variations in the activity of glycosyltransferases of CAZy family 6 generate antigenic variation between individuals and species that facilitates resistance to pathogens. The well characterized vertebrate glycosyltransferases of this family are multidomain membrane proteins with C-terminal catalytic domains. Genes for proteins homologous with their catalytic domains are found in at least nine species of anaerobic commensal bacteria and a cyanophage. Although the bacterial proteins are strikingly similar in sequence to the catalytic domains of their eukaryotic relatives, a metal-binding Asp-X-Asp sequence, present in a wide array of metal ion-dependent glycosyltransferases, is replaced by Asn-X-Asn. We have cloned and expressed one of these proteins from Bacteroides ovatus, a bacterium that is linked to inflammatory bowel disease. Functional characterization shows it to be a metal-independent glycosyltransferase with a 200-fold preference for UDP-GalNAc as substrate relative to UDP-Gal. It efficiently catalyzes the synthesis of oligosaccharides similar to human blood group A and may participate in the synthesis of the bacterial O-antigen. The kinetics for GalNAc transfer to 2′-fucosyl lactose are characteristic of a sequential mechanism, as observed previously for this family. Mutational studies indicate that despite the lack of a metal cofactor, there are pronounced similarities in structure-function relationships between the bacterial and vertebrate family 6 glycosyltransferases. These two groups appear to provide an example of horizontal gene transfer involving vertebrates and prokaryotes. The structures of complex glycans are determined by the specificities of the glycosyltransferases (GTs) 2The abbreviations used are:GTglycosyltransferaseGT6CAZy family 6GTA and GTBhuman histo-blood group A and B synthase, respectivelyα3GTUDP-galactose, N-acetyllactosamine, α1,3-galactosyltransferaseLacNAcN-acetyllactosamine. that catalyze their biosynthesis. GTs fall into two groups that differ in mechanism, based on whether the anomeric configuration of the donor substrate (α for most UDP-sugars) is retained or inverted in the product (1Unligil U.M. Rini J.M. Curr. Opin. Struct. Biol. 2000; 10: 510-517Crossref PubMed Scopus (340) Google Scholar, 2Breton C. Snajdrová L. Jeanneau C. Koca J. Imberty A. Glycobiology. 2006; 16: 29R-37RCrossref PubMed Scopus (480) Google Scholar, 3Lairson L.L. Henrissat B. Davies G.J. Withers S.G. Annu. Rev. Biochem. 2008; 77: 521-555Crossref PubMed Scopus (1250) Google Scholar). They are classified into 90 different families in the CAZy data base based on sequence similarities (4Campbell J.A. Davies G.J. Bulone V. Henrissat B. Biochem. J. 1997; 326: 929-939Crossref PubMed Scopus (621) Google Scholar, 5Coutinho P.M. Deleury E. Davies G.J. Henrissat B. J. Mol. Biol. 2003; 328: 307-317Crossref PubMed Scopus (899) Google Scholar), but the majority of those that have been structurally characterized fall into one of two fold types, designated GT-A and GT-B (2Breton C. Snajdrová L. Jeanneau C. Koca J. Imberty A. Glycobiology. 2006; 16: 29R-37RCrossref PubMed Scopus (480) Google Scholar). The retaining GTs of CAZy family 6 (GT6) have a GT-A fold and catalyze the transfer of either galactose or GalNAc into an α-linkage with the 3-OH group of β-linked galactose or GalNAc. GT6 includes the histo-blood group A and B GTs (GTA and GTB), the α-galactosyltransferase (α3GT) that catalyzes the synthesis of the xenoantigen or α-gal epitope, Forssman glycolipid synthase, isogloboside 3 synthase, and their homologues from other vertebrates (6Turcot-Dubois A.L. Le Moullac-Vaidye B. Despiau S. Roubinet F. Bovin N. Le Pendu J. Blancher A. Glycobiology. 2007; 17: 516-528Crossref PubMed Scopus (44) Google Scholar). GT6 enzymes from vertebrates are type-2 membrane proteins with N-terminal cytosolic domains, a transmembrane helix, a spacer, and a C-terminal catalytic domain (6Turcot-Dubois A.L. Le Moullac-Vaidye B. Despiau S. Roubinet F. Bovin N. Le Pendu J. Blancher A. Glycobiology. 2007; 17: 516-528Crossref PubMed Scopus (44) Google Scholar). Crystallographic studies of recombinant catalytic domains of GTA, GTB, and α3GT have provided detailed information about their interactions with substrates, metal cofactor, and inhibitors (7Gastinel L.N. Bignon C. Misra A.K. Hindsgaul O. Shaper J.H. Joziasse D.H. EMBO J. 2001; 20: 638-649Crossref PubMed Scopus (177) Google Scholar, 8Boix E. Swaminathan G.J. Zhang Y. Natesh R. Brew K. Acharya K.R. J. Biol. Chem. 2001; 276: 48608-48614Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 9Patenaude S.I. Seto N.O. Borisova S.N. Szpacenko A. Marcus S.L. Palcic M.M. Evans S.V. Nat. Struct. Biol. 2002; 9: 685-690Crossref PubMed Scopus (200) Google Scholar). Most GT-A fold GTs, including those in the GT6 family, require divalent metal ions, such as Mn2+, for catalytic activity; their metal dependence is linked to a shared DXD sequence motif. Residues of this motif interact with the metal ion and both the ribose and phosphates of the donor substrate to produce an appropriate substrate orientation and conformation for catalysis and to stabilize the UDP leaving group (3Lairson L.L. Henrissat B. Davies G.J. Withers S.G. Annu. Rev. Biochem. 2008; 77: 521-555Crossref PubMed Scopus (1250) Google Scholar, 7Gastinel L.N. Bignon C. Misra A.K. Hindsgaul O. Shaper J.H. Joziasse D.H. EMBO J. 2001; 20: 638-649Crossref PubMed Scopus (177) Google Scholar, 8Boix E. Swaminathan G.J. Zhang Y. Natesh R. Brew K. Acharya K.R. J. Biol. Chem. 2001; 276: 48608-48614Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 9Patenaude S.I. Seto N.O. Borisova S.N. Szpacenko A. Marcus S.L. Palcic M.M. Evans S.V. Nat. Struct. Biol. 2002; 9: 685-690Crossref PubMed Scopus (200) Google Scholar, 10Zhang Y. Wang P.G. Brew K. J. Biol. Chem. 2001; 276: 11567-11574Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar). glycosyltransferase CAZy family 6 human histo-blood group A and B synthase, respectively UDP-galactose, N-acetyllactosamine, α1,3-galactosyltransferase N-acetyllactosamine. Mammalian members of GT6 are responsible for variations in glycan structures between different species and individuals as the result of selective enzyme inactivation in certain species (α3GT, Forssman glycolipid synthase, and isogloboside 3 synthase) or the inheritance of multiple alleles at one locus that encode enzymes with different substrate specificity (GTA and GTB) or are inactive (11Galili U. Shohet S.B. Kobrin E. Stults C.L. Macher B.A. J. Biol. Chem. 1988; 263: 17755-17762Abstract Full Text PDF PubMed Google Scholar, 12Xu H. Storch T. Yu M. Elliott S.P. Haslam D.B. J. Biol. Chem. 1999; 274: 29390-29398Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar, 13Yamamoto F. Clausen H. White T. Marken J. Hakomori S. Nature. 1990; 345: 229-233Crossref PubMed Scopus (869) Google Scholar, 14Keusch J.J. Manzella S.M. Nyame K.A. Cummings R.D. Baenziger J.U. J. Biol. Chem. 2000; 275: 25308-25314Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar). The presence of circulating antibodies against glycan structures that are subject to interspecies and individual variability has been linked to resistance to pathogens that also carry the glycans; these antibodies are thought to arise from exposure to potential pathogens, including enveloped viruses and bacteria that carry structurally similar glycans (11Galili U. Shohet S.B. Kobrin E. Stults C.L. Macher B.A. J. Biol. Chem. 1988; 263: 17755-17762Abstract Full Text PDF PubMed Google Scholar). In addition to the well characterized enzymes discussed previously, atypical members of the GT6 family have been identified in mammals that have sequence changes in highly conserved regions of the active site, including the DXD motif (6Turcot-Dubois A.L. Le Moullac-Vaidye B. Despiau S. Roubinet F. Bovin N. Le Pendu J. Blancher A. Glycobiology. 2007; 17: 516-528Crossref PubMed Scopus (44) Google Scholar). However, no glycosyltransferase activity was detected in recombinant forms of two of these, and their functions are unclear (6Turcot-Dubois A.L. Le Moullac-Vaidye B. Despiau S. Roubinet F. Bovin N. Le Pendu J. Blancher A. Glycobiology. 2007; 17: 516-528Crossref PubMed Scopus (44) Google Scholar). Although GT6 members are widely distributed among vertebrates, no homologues have been found in other eukaryotes (6Turcot-Dubois A.L. Le Moullac-Vaidye B. Despiau S. Roubinet F. Bovin N. Le Pendu J. Blancher A. Glycobiology. 2007; 17: 516-528Crossref PubMed Scopus (44) Google Scholar). However, GT6 members have been identified in several bacterial species (15Sullivan M.B. Coleman M.L. Weigele P. Rohwer F. Chisholm S.W. PLoS Biol. 2005; 3: e144Crossref PubMed Scopus (400) Google Scholar, 16Yi W. Shao J. Zhu L. Li M. Singh M. Lu Y. Lin S. Li H. Ryu K. Shen J. Guo H. Yao Q. Bush C.A. Wang P.G. J. Am. Chem. Soc. 2005; 127: 2040-2041Crossref PubMed Scopus (87) Google Scholar, 17Yi W. Shen J. Zhou G. Li J. Wang P.G. J. Am. Chem. Soc. 2008; 130: 14420-14421Crossref PubMed Scopus (28) Google Scholar). GT6 enzymes from Escherichia coli O86, and Helicobacter mustelae that appear to function in the biosynthesis of the lipopolysaccharide O-antigen have been cloned and expressed by Wang and co-workers (16Yi W. Shao J. Zhu L. Li M. Singh M. Lu Y. Lin S. Li H. Ryu K. Shen J. Guo H. Yao Q. Bush C.A. Wang P.G. J. Am. Chem. Soc. 2005; 127: 2040-2041Crossref PubMed Scopus (87) Google Scholar, 17Yi W. Shen J. Zhou G. Li J. Wang P.G. J. Am. Chem. Soc. 2008; 130: 14420-14421Crossref PubMed Scopus (28) Google Scholar) and found to have specificities similar to those of human GTB and GTA, respectively. These enzymes have been applied in the enzymatic synthesis of oligosaccharides. Other homologues are encoded by Hemophilus somnus, Psychroacter sp., PRwf-1 (15Sullivan M.B. Coleman M.L. Weigele P. Rohwer F. Chisholm S.W. PLoS Biol. 2005; 3: e144Crossref PubMed Scopus (400) Google Scholar), Francisella philomiragia, and three Bacteroides species, Bacteroides ovatus, Bacteroides caccae, and Bacteroides stercoris, as well as a cyanophage, PSSM-2 (15Sullivan M.B. Coleman M.L. Weigele P. Rohwer F. Chisholm S.W. PLoS Biol. 2005; 3: e144Crossref PubMed Scopus (400) Google Scholar). Genes for other homologues from unidentified species are present in the marine metagenome (18Venter J.C. Remington K. Heidelberg J.F. Halpern A.L. Rusch D. Eisen J.A. Wu D. Paulsen I. Nelson K.E. Nelson W. Fouts D.E. Levy S. Knap A.H. Lomas M.W. Nealson K. White O. Peterson J. Hoffman J. Parsons R. Baden-Tillson H. Pfannkoch C. Rogers Y.H. Smith H.O. Science. 2004; 304: 66-74Crossref PubMed Scopus (3155) Google Scholar, 19Yooseph S. Sutton G. Rusch D.B. Halpern A.L. Williamson S.J. Remington K. Eisen J.A. Heidelberg K.B. Manning G. Li W. Jaroszewski L. Cieplak P. Miller C.S. Li H. Mashiyama S.T. Joachimiak M.P. van Belle C. Chandonia J.M. Soergel D.A. Zhai Y. Natarajan K. Lee S. Raphael B.J. Bafna V. Friedman R. Brenner S.E. Godzik A. Eisenberg D. Dixon J.E. Taylor S.S. Strausberg R.L. Frazier M. Venter J.C. PLoS Biol. 2007; 5: e16Crossref PubMed Scopus (611) Google Scholar) and human gut metagenome (20Gill S.R. Pop M. Deboy R.T. Eckburg P.B. Turnbaugh P.J. Samuel B.S. Gordon J.I. Relman D.A. Fraser-Liggett C.M. Nelson K.E. Science. 2006; 312: 1355-1359Crossref PubMed Scopus (3136) Google Scholar, 21Kurokawa K. Itoh T. Kuwahara T. Oshima K. Toh H. Toyoda A. Takami H. Morita H. Sharma V.K. Srivastava T.P. Taylor T.D. Noguchi H. Mori H. Ogura Y. Ehrlich D.S. Itoh K. Takagi T. Sakaki Y. Hayashi T. Hattori M. DNA Res. 2007; 14: 169-181Crossref PubMed Scopus (642) Google Scholar). The phage and bacterial enzymes are substantially truncated at the N terminus relative to the catalytic domains of vertebrate GT6 representatives and are smaller than the reported minimal functional unit of a primate α3GT (22Henion T.R. Macher B.A. Anaraki F. Galili U. Glycobiology. 1994; 4: 193-201Crossref PubMed Scopus (92) Google Scholar). When bacterial and vertebrate GT6 amino acid sequences are aligned (Fig. 1 and supplemental Figs. S1 and S2), it can be seen that the metal-binding DXD of the eukaryotic GTs is replaced by NXN (where X is Ala, Gly, or Ser) in the bacterial homologues. The cyanophage GT6 member and related proteins in the marine metagenome, however, retain the DXD motif. This conspicuous difference in the bacterial proteins is particularly interesting, since, in the mammalian enzymes, the aspartates of the DXD and adjacent residues are crucial for catalytic activity (10Zhang Y. Wang P.G. Brew K. J. Biol. Chem. 2001; 276: 11567-11574Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar, 23Persson M. Letts J.A. Hosseini-Maaf B. Borisova S.N. Palcic M.M. Evans S.V. Olsson M.L. J. Biol. Chem. 2007; 282: 9564-9570Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar). B. ovatus is a Gram-negative commensal bacterium that inhabits the distal mammalian gut and has been implicated in the pathology of inflammatory bowel disease in humans (24Saitoh S. Noda S. Aiba Y. Takagi A. Sakamoto M. Benno Y. Koga Y. Clin. Diagn. Lab. Immunol. 2002; 9: 54-59PubMed Google Scholar). The B. ovatus genome contains two genes that encode GT6 representatives (Fig. 1). We selected one of these for initial investigation, and designate it BoGT6a (family 6 glycosyltransferase 1 of Bacteroides). The gene for this protein was amplified by PCR and cloned and expressed in His-tagged form in E. coli BL21(DE3). Assays with a variety of substrates show that its substrate specificity is similar to that of human GTA. Previous studies of the activities of bacterial enzymes were conducted in the presence of Mn2+ (16Yi W. Shao J. Zhu L. Li M. Singh M. Lu Y. Lin S. Li H. Ryu K. Shen J. Guo H. Yao Q. Bush C.A. Wang P.G. J. Am. Chem. Soc. 2005; 127: 2040-2041Crossref PubMed Scopus (87) Google Scholar, 17Yi W. Shen J. Zhou G. Li J. Wang P.G. J. Am. Chem. Soc. 2008; 130: 14420-14421Crossref PubMed Scopus (28) Google Scholar), but we find that the B. ovatus enzyme does not require divalent metal ions for activity and is fully active in EDTA. Despite this striking difference, BoGT6a is similar to its metal-dependent relatives in catalytic properties; also, the effects of amino acid substitutions for residues corresponding to several that act in substrate binding and catalysis in vertebrate GT6 glycosyltransferases suggest that they have similar structure-function relationships. These results indicate that the metal cofactor is not a conserved feature in the GT6 family. They also raise questions about the catalytic mechanism of prokaryotic GT6 members and the evolutionary relationship between bacterial, phage, and vertebrate enzymes. Freeze-dried B. ovatus cells were purchased from ATCC. Forward and reverse synthetic oligonucleotide primers (Invitrogen) that introduce NdeI and BamHI coding sites were designed to amplify full-length and C-terminally truncated forms of the gene: forward, 5′-AAA AAA CAT ATG (NdeI) AGA ATT GGT ATA TTA TAT ATC TGT ACT GGC-3′; reverse full-length, 5′-AAA AAA GGA TCC (BamHI) TCA ATC AGC CGA TTT AAA TTT TTG GCA GAT TAG-3′; reverse truncated, 5′-AAA AAA GGA TCC (BamHI) TCA GTT TTT TCT TCG CAA TAA TTC ATG CCC GCC-3′. B. ovatus cells (1 mg) were suspended in water (100 μl). A PCR mixture (49 μl) containing 1 μl of the B. ovatus cell suspension, 1 nmol each of the forward and reverse primers, dNTPs, and 5 μl of Thermo Buffer for Polymerase (New England Biolabs) was heated at 100 °C for 10 min. The mixture was allowed to cool to room temperature, and 2 units (1 μl) of Vent polymerase (New England Biolabs) was added. The B. ovatus gene was amplified under the following conditions: 94 °C for 3 min, followed by 30 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1.5 min with a final incubation at 72 °C for 10 min. The PCR product was gel-purified (Qiagen) and digested with 40 units (2 μl) each of NdeI and BamHI in 6 μl of BamHI buffer (New England Biolabs) and 1 μl of 100× bovine serum albumin at 37 °C for 3 h. The digestion product was gel-purified and mixed in a 1:1 ratio with pET42b vector that had been previously digested with NdeI and BamHI, together with 2.5 μl of 10× T4 ligase buffer and 800 units (2 μl) of T4 DNA ligase in a total volume of 25 μl. The mixture was incubated in a thermocycler under the following conditions: 30 cycles of 10 °C for 3 min, 12 °C for 3 min, 14 °C for 3 min, 16 °C for 3 min, and 18 °C for 1 min followed by a final incubation at 65° for 10 min. 10 μl of the ligation product was transformed into E. coli DH5α-competent cells using heat shock. Transformants were grown in 1 ml of SOC medium with rapid shaking (250 rpm) at 37 °C for 60 min before being plated on LB agar plates containing 50 μg/ml kanamycin. The plates were placed incubated at 37 °C for 18–20 h. A single colony was inoculated in 8 ml of LB (kan) medium and grown overnight, and the vector DNA (pET42b_BoGT6a) was extracted and sequenced (Davis Sequencing, LLC). Cultures of E. coli BL21(DE3) cells were transformed with pET42b_BoGT6a and grown in LB medium containing 50 μg/ml kanamycin with rapid shaking (250 rpm) at 37 °C. The temperature was reduced to 24 °C when the A600 nm of the culture reached 0.8–1.0, and incubation was continued overnight to allow leaky expression at the lower temperature. Bacterial cells were harvested by centrifugation at 4,000 rpm for 20 min, and the cell pellets were washed with 20% sucrose containing 20 mm Tris-HCl, pH 8.0, suspended in 30 ml of lysis buffer (50 mm Tris-HCl buffer, pH 8.0, containing 1 mm EDTA and 0.1 m NaCl), and disrupted using a French press. Insoluble material was removed by centrifugation at 30,000 rpm for 20 min, and the supernatant was applied to an Ni2+-nitrilotriacetic acid column (Qiagen), which had been equilibrated with 50 volumes of 20 mm Tris-HCl, pH 7.9, containing 0.5 m NaCl. The column was subsequently washed with 10 volumes of 20 mm Tris buffer containing 0.5 m NaCl and 5 mm imidazole, pH 7.9, followed by 20 volumes of 20 mm Tris buffer containing 0.5 m NaCl and 60 mm imidazole, pH 7.9, and finally eluted with 10 volumes of 20 mm Tris-HCl, pH 7.9, containing 0.5 m NaCl and 500 mm imidazole. Fractions containing the purified protein were dialyzed against two changes of 50 volumes of 20 mm Tris-HCl, pH 7.9, containing 0.1 m NaCl and 2 mm dithiothreitol; 10 mm EDTA was added to the buffer for storage. All steps in enzyme purification were conducted at 4 °C. Seven mutants (D95N, D97N, A155M, A155Q, E192Q, R299A, and K231A) (see Fig. 2) were constructed using the PCR megaprimer method with previously described modifications (25Zhang Y. Deshpande A. Xie Z. Natesh R. Acharya K.R. Brew K. Glycobiology. 2004; 14: 1295-1302Crossref PubMed Scopus (20) Google Scholar) with pET42b_BoGT6a as a template. In the first amplification, a 50-μl mixture of (forward) mutagenic primers (Invitrogen) template, T7 terminator (Invitrogen), dNTPs (Eppendorf), Thermo Buffer, and Vent polymerase (New England Biolabs) was used under the following incubation conditions: 3 min at 94 °C followed by 30 cycles of 94 °C for 1 min, 60 °C for 1 min, 72 °C for 1 min, followed by 10 min at 72 °C. The product was gel-purified (Qiagen) and used as the reverse primer in a second reaction that also included T7 promoter, dNTPs, template, Thermo Buffer, and Vent polymerase under the following conditions: 94 °C for 3 min followed by 30 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min and a final 10-min incubation at 72 °C. The double mutant, R244A/R245A, was constructed by PCR in one step. The amplification reaction contained the (reverse) mutagenic primer, template, T7 promoter, dNTPs, Thermo Buffer, and Vent polymerase and was incubated under the following conditions: 3 min at 94 °C followed by 30 cycles of 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min with a final 10-min incubation at 72 °C. The following primers were used for mutagenesis: N95D (forward), 5′-CTATTTTTCTTCGATGCCAATCTCTTATTCACC-3′; N97D (forward), 5′-CTATTTTTCTTCAATGCCGATCTCTTATTCACC-3′; A155M (forward), 5′-CGATATTATTACATGGGAGGGCTTTCAGGTGGA-3′; A155Q (forward), 5′-CGATATTATTACCAGGGAGGGCTTTCAGGTGGA-3′; E192Q (forward), 5′-CCAATTTGGCACGACCAATCTCTAATCAATAAA-3′; R229A (forward), 5′-CCAATAATCCTCATTGCAGACAAAAATAAA-3′; K231A (forward), 5′-ATCCTCATTCGAGACGCAAATAAACCCCAATAT-3′; R244A/R245A (reverse), 5′-AAAAAAGGATCCTCAGTTTTTTGCTGCCAATAATTCATGCCCGCC-3′. The native molecular size of BoGT6a was investigated by medium pressure gel filtration with a column (10 × 300 mm) of Superdex 200 (Tricorn; GE Healthcare), equilibrated and eluted with 0.1 m sodium phosphate buffer, pH 6.8, containing 0.4 m NaCl and 10 mm sodium azide. Protein samples were applied in 100 μl of sample buffer. The column was calibrated with standard proteins of known molecular weight, thyroglobulin (670,000), catalase (250,000), immunoglobulin G (158,000), transferrin (79,500), ovalbumin (44,000), myoglobin (17,000), and α-lactalbumin (14,000), and the molecular weight was estimated from a regression analysis of a plot of elution volume versus log(molecular weight) of the standards. Glycosyltransferase activities were measured using a radiochemical assay (10Zhang Y. Wang P.G. Brew K. J. Biol. Chem. 2001; 276: 11567-11574Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar) with the potential acceptor substrates 2′-fucosyllactose, fucosyl α1-2 galactose, blood group H type II trisaccharide (Fucα1-2Galβ1-4GlcNAc) (V-Labs), N-acetyl-lactosamine, and Forssman synthase acceptor (GalNAc β1-3Gal α1-4Gal), by following the transfer of radioactive sugars from UDP-[3H]N-acetylgalactosamine (UDP-GalNAc), UDP-[3H]Gal, or UDP-[3H]Glc (Sigma). Standard assays contained 0.021 μg of enzyme, 50 mm Tris-HCl buffer, pH 7.0, 10 mm EDTA, and 0.1% bovine serum albumin in a total volume of 50 μl and were incubated at 37 °C for 10–15 min. For more detailed characterization of enzyme kinetics, acceptor concentrations were 0.1–0.5 mm for 2′-fucosyllactose, 0.1–3.0 mm for Fucα1-2Gal, and 0.2–1.0 mm for Fucα1-2Galβ1-4GlcNAc. The concentration of UDP-[3H]GalNAc (specific activity, 666 cpm/nmol) was varied from 0.06 to 0.3 mm. Reactions from which the acceptor substrate is omitted served as controls. A higher concentration of enzyme (3 μg) and a longer incubation time (20 min) were used in assays of UDP-GalNAc hydrolase activity. Because the BoGT6a-catalyzed reactions could not be terminated by the addition of EDTA or by cooling on ice (since BoGT6a has a significant level of activity at 0 °C), the reaction mixture was immediately applied to a 1-ml Dowex (1 × 800) anion exchange column, followed by 0.5 ml of water to wash the tube. The product was subsequently eluted with 1 ml of water to elute the product; the eluate was collected in a plastic vial, mixed with 10 ml of Ecolume (ICN Biomedicals, Costa Mesa, CA); and radioactivity was measured with a liquid scintillation counter (LKB). Kinetic data were analyzed by non-linear regression using Sigma PlotTM. Data were fitted to Michaelis-Menten equations for a single substrate reaction (Equation 1) and a general two substrate sequential reaction (Equation 2),V=VmSS+Km(Eq. 1) V=VmABKiaKb+KaB+KbA+AB(Eq. 2) where, in Equation 1, [S] represents the concentration of the varied substrate, and Vm and Km are the apparent maximum velocity and Michaelis constants. In Equation 2, [A] is the concentration of UDP-GalNAc, and [B] is the concentration of 2′-fucosyllactose. Ka and Kb are the cognate Michaelis constants, and Kia is the dissociation constant substrate A. Data were also fitted to variants of Equation 2 lacking a Ka or Kia term, respectively. Mutant enzymes were characterized by varying each substrate individually at a fixed concentration of the second substrate, and the data were fitted to Equation 1 to give apparent Km and Vm values. The sequences of 11 bacterial GT6 family members are in the following data bases: NR data base, H. somnus, E. coli O86, and Psychrobacter sp. PRf-1; Whole-Genome Shotgun Reads, B. ovatus (two sequences), B. stercoris, B. caccae, and Subdoligranulum variable (two sequences). The single putative viral protein sequence from Prochlorococcus cyanophage PSSM-2 is also in the NR data base. The bacterial sequences are also listed in Interpro (available on the World Wide Web), entry IPR005076. An alignment of the GT6 protein sequences from the three Bacteroides species with that from the cyanophage PSSM-2 and the catalytic domains of human GTA and bovine α3GT generated using MUSCLE (available on the World Wide Web) is shown in Fig. 1, and an alignment of all of the bacterial GTs is shown in supplemental Fig. S1. The Environmental Samples data base contains six additional sequences from the Human Gut Metagenome that encode proteins that are similar to the bacterial GT6s (including one that is identical with the B. stercoris sequence) and more than 90 sequences with high levels of sequence similarity to the putative cyanophage protein are found in the Marine Metagenome. There are high levels of sequence similarity between the bacterial, phage, and vertebrate GT6s, amounting to 35% amino acid sequence identity for some pairwise comparisons, and relatively few gaps need to be inserted to align their sequences. Sections of sequence identified as parts of the active site in α3GT, GTA, and GTB by structural and mutational studies (25Zhang Y. Deshpande A. Xie Z. Natesh R. Acharya K.R. Brew K. Glycobiology. 2004; 14: 1295-1302Crossref PubMed Scopus (20) Google Scholar, 26Zhang Y. Swaminathan G.J. Deshpande A. Boix E. Natesh R. Xie Z. Acharya K.R. Brew K. Biochemistry. 2003; 42: 13512-13521Crossref PubMed Scopus (53) Google Scholar, 27Tumbale P. Jamaluddin H. Thiyagarajan N. Acharya K.R. Brew K. Glycobiology. 2008; 18: 1036-1043Crossref PubMed Scopus (14) Google Scholar) are enclosed in boxes in Fig. 1 (A–F). These are similar to ligand binding regions assigned by Heissigerova et al. (28Heissigerova H. Breton C. Moravcova J. Imberty A. Glycobiology. 2003; 13: 377-386Crossref PubMed Scopus (29) Google Scholar) in vertebrate GT6s. The sequences within and adjacent to some of these regions are well conserved, but in regions D and E, the sequences are less conserved because these regions are responsible for specificity differences for acceptor and donor substrates (6Turcot-Dubois A.L. Le Moullac-Vaidye B. Despiau S. Roubinet F. Bovin N. Le Pendu J. Blancher A. Glycobiology. 2007; 17: 516-528Crossref PubMed Scopus (44) Google Scholar, 9Patenaude S.I. Seto N.O. Borisova S.N. Szpacenko A. Marcus S.L. Palcic M.M. Evans S.V. Nat. Struct. Biol. 2002; 9: 685-690Crossref PubMed Scopus (200) Google Scholar, 27Tumbale P. Jamaluddin H. Thiyagarajan N. Acharya K.R. Brew K. Glycobiology. 2008; 18: 1036-1043Crossref PubMed Scopus (14) Google Scholar). In the vertebrate GT6 glycosyltransferases, an additional region at the C terminus is also important for activity (region G; supplemental Fig. S2). However, because the C-terminal regions of the vertebrate and bacterial enzymes do not align well, we have not marked this region in Fig. 1. Despite their strong overall similarity in sequence, the bacterial and phage proteins are shorter than the catalytic domains of the vertebrate enzymes by 47 residues. The truncation is at the N terminus and includes, in the known structures of the vertebrate enzymes, GTA, GTB, and α3GT, an α-helix, and a β strand (8Boix E. Swaminathan G.J. Zhang Y. Natesh R. Brew K. Acharya K.R. J. Biol. Chem. 2001; 276: 48608-48614Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar, 9Patenaude S.I. Seto N.O. Borisova S.N. Szpacenko A. Marcus S.L. Palcic M.M. Evans S.V. Nat. Struct. Biol. 2002; 9: 685-690Crossref PubMed Scopus (200) Google Scholar). This region does not appear to be a separate domain or subdomain, since it has a large number of interactions with the rest of the catalytic domain. The C-terminal regions of the bact
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