5 Department of Biomedical Sciences, Florida Atlantic University, Boca Raton, FL 33341; 6 Department of Biology and Biochemistry, University of Bath, Claverton Down, Bath BA2 7AY, UK
Received on May 28, 2004; revised on June 24, 2004; accepted on June 25, 2004
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Abstract |
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Key words: catalysis / crystal structure / glycosyltransferase / mutation / substrate binding
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Introduction |
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UDP-galactose ß galactosyl 1-3-galactosyltransferase (
3GT, E.C. 2.4.1.151) is a retaining GT that catalyzes the synthesis of
1,3-galactosyl ß-OR structures in glycoconjugates (van den Eijnden et al., 1983
) and is a component of trans-Golgi membranes. An inactive form of this enzyme is produced in humans and their closest relatives among the primates (Galili et al., 1988
; Galili and Swansen, 1991
). The absence of active
3GT allows the production of natural antibodies (13% of circulating IgG) to the product of its action, the
-Gal epitope, which provide defenses against pathogens (Avila et al., 1989
) and viruses arising from mammalian hosts that have active
3GT (Takeuchi et al., 1996
).
3GT is a member of a family of homologous retaining galactosyl- and N-acetylgalactosaminyl-transferases that form
-1,3 linkages to ß-galactosyl and ß-N-acetylgalactosaminyl residues in glycoconjugates but differ in specificity: the histo-blood group A and B glycosyltransferases (Yamamato et al., 1990
), Forssman glycolipid (Gb5) synthase (Haslam and Baenziger, 1996
), and isogloboside 3 (iGb3) synthase (Keusch et al., 2000
). These enzymes, like most eukaryotic glycosyltransferases, are all type II membrane proteins with short cytosolic N-terminal domains, a membrane spanning region, a stem, and C-terminal catalytic domains (Paulson and Colley, 1989
). Structures have been determined for recombinant truncated catalytic domains of bovin e
3GT (Boix et al., 2001
, 2002
; Gastinel et al., 2001
) and human histo-blood group A and B glycosyltransferases (Patenaude et al., 2002
).
Two distinct structural states have been described for 3GT, a lower-resolution tetragonal form (Form I), in which the C-terminus of the enzyme is disordered (Gastinel et al., 2001
), and a higher-resolution monoclinic form (Form II), which has a distinct and highly ordered conformation for the C-terminal region (Boix et al., 2001
). The structured C-terminus was found by mutagenesis to be important for the catalytic activity of the enzyme (Boix et al., 2001
). We have reported Form II structures for
3GT in complexes with Mn2+ and UDP at 1.53 Å resolution (Boix et al., 2001
), Mn2+/UDP-galactose (UDP-Gal), Mn2+/UDP-glucose (UDP-Glc), and with both Mn2+/UDP and the acceptor substrates lactose and N-acetyllactosamine (Boix et al., 2002
). The structures of these complexes identify four tryptophans, residues 249, 250, 314, and 356, in the active site of
3GT that interact with acceptor substrates (see Table I and Figure 1). Molecular modelling studies by Heissigerová et al. (2003)
suggest that these specific tryptophans and the corresponding residues in other members of the
3GT family may be important in acceptor substrate selectivity.
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Results |
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The side chain of Trp250 is located near the glucose or GlcNAc component of acceptor substrates in their complexes with enzyme (Boix et al., 2002). However, unlike Trp249, the indole ring is approximately perpendicular to the pyranose ring, lying at the edge of the entrance of lactose binding groove. Specific interactions of Trp250 with acceptor substrates are an H-bond between the indole ring N and the 3'-OH of both lactose and LacNAc and packing interactions of the indole ring with the 2'-acetamido group of LacNAc. There is no corresponding interaction with lactose, but the tryptophan makes about seven additional nonpolar contacts with both acceptors. Mutants were expressed with conservative substitutions of Tyr and Phe, corresponding to residues present in Forssman glycolipid synthase and the blood group enzymes (Table II).
Trp314 is conserved in all homolog of 3GT. In
3GT, the side chain has more than 10 hydrophobic interactions with the ß-galactosyl moiety of the acceptor and the N of the indole ring is within H-bonding distance of O6 of the glucose moiety of lactose. Also, in the enzyme complexes with UDP-Gal and UDP-Glc (in which the substrate is cleaved) it has 10 and 8 hydrophobic contacts, respectively, with the monosaccharide ring. In the complex with UDP-Gal, the carbonyl OH H-bonds with the 2- and 3-OH groups of the galactose (Table I; Boix et al., 2002
).
Trp356 is part of the acceptor binding site making six to seven nonpolar van der Waals contacts with acceptor substrates. It is adjacent to the C-terminal section of the polypeptide chain that undergoes a rearrangement between the Form I and Form II structures and is linked with UDP binding (Boix et al., 2001). This residue was mutated to Thr, the amino acid present at the corresponding site in the histo blood group A and B transferases (Table II).
All of the 3GT mutants were expressed in soluble active form in good yield and showed similar solubility to the wild-type enzyme.
Effects of mutations on activity
Trp249 to Gly
Acceptor substrate (lactose) binding is so strongly perturbed by this mutation that the relationship between velocity and lactose concentration up to 100 mM at 1 mM UDP-Gal is linear. The limited solubility of lactose in water precludes the use of higher lactose concentrations in assays. Although kcat and Kb with lactose as substrate cannot be determined for this mutant, the value of kcat/Kb (apparent), determined from the slope of v versus [lactose] plot is 80-fold lower than that of the wild-type enzyme (Table III). The lack of curvature indicates that the Km for lactose must exceed 200 mM. It was also not possible to achieve saturation with higher-affinity acceptor substrates including LacNAc and p-NO2-phenyl-LacNAc because of their limited solubility in water. The apparent Km for UDP-Gal, determined at a fixed nonsaturating concentration of lactose (25 mM), is similar to the wild-type enzyme (Table III), and the apparent kcat determined at 25 mM lactose is 0.26 s1, a value that is much lower than the true kcat because of the low acceptor concentration relative to the Km. The kcat and Km for UDP-Gal hydrolysis are essentially unchanged (Table III), and it is possible that kcat for the transferase reaction is also unchanged, in which case the Km for lactose would be about 1.5 M. Isothermal titration calorimetry studies (see Boix et al., 2002) indicate that the free energy and enthalpy of binding of UDP and UDP-Gal to the enzyme in the presence of Mn2+ were not significantly changed by the mutation. However, unlike wild-type
3GT, the binding of lactose in the presence of UDP and Mn2+ to the Trp249Gly mutant was not detected, suggesting that the negative enthalpy of binding and/or affinity of lactose for this inhibitory enzyme complex are reduced (data not shown).
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Both of these mutations therefore increase the overall rate of product-release steps and lower the on-rate of UDP-Gal binding three- to fourold. This interpretation is consistent with the slightly increased kcat for UDP-Gal hydrolysis and may reflect a local structural change resulting from the mutation.
The Km of 3GT for LacNAc is about 12-fold lower than the Km for lactose, which could reflect contributions to the free energy of binding arising from interactions of the LacNAc 2-acetamido group with Trp250. The substitution of Tyr or Phe, residues with smaller side chains than Trp, may perturb these interactions and affect the binding of LacNAc but not lactose. To test this hypothesis, apparent Km and kcat were determined with LacNAc for the wild-type enzyme and the two mutants (at 1 mM UDP-Gal). The values of these parameters were: wild type: Km, 2.67 ± 0.27 mM, kcat, 4.59 ± 0.18 s1; Trp250Phe: Km, 1.85 ± 0.25 mM, kcat, 5.97 ± 0.26 s1; Trp250Tyr: Km, 2.76 ± 0.24 mM; kcat, 6.19 ± 0.22 s1. The apparent kinetic parameters for the wild-type enzyme are similar to true values determined in experiments in which the concentrations of both UDP-Gal and LacNAc were varied: Km 1.6 ± 0.14 mM; kcat 5.6 ± 0.2 s1. These results indicate that the substitutions for Trp250 do not selectively disfavor the binding of LacNAc relative to lactose.
Trp314 to Tyr
Galactosyltransferase activity is greatly reduced in the Trp314Tyr mutant but UDP-Gal hydrolase activity is essentially unchanged relative to the wild-type enzyme (Table III). As discussed previously for a different mutant of 3GT (Zhang et al., 2003
), the high proportion of hydrolase activity makes it difficult to determine kinetic parameters with high precision using the radiochemical assay, and we determined apparent kinetic parameters for the transferase reaction. These indicate that the main effect of the mutation is to reduce the kcat for the galactosyltransferase reaction 30-fold but the kinetic parameters for UDP-Gal hydrolase activity are insignificantly affected. These effects are closely similar to those in the previously described Gln247Glu mutant (Zhang et al., 2003
).
Trp356 to Thr
This mutant has a reduced level of overall catalytic activity. The loss in hydrolase activity reflects a 15-fold reduction in kcat but, for the galactosyltransferase reaction, a reduction in kcat and an increase in the Km for lactose (determined at 1 mM UDP-Gal) contribute to a greater reduction in activity. There is some uncertainty in the precise values of the parameters for the transferase reaction because the highest substrate concentration in the assay (100 mM) is less than the calculated value of Kb (126 mM), but it is clear from the results that kcat is reduced 1015-fold for both reactions and acceptor substrate affinity is about 5-fold lower than for the wild-type enzyme (Table III).
Structures of the Trp249Gly and Trp314Tyr mutants
The Form II structure of the complex of Trp249Gly with UDP and Mn2+ was determined at 2.07 Å resolution (Table IV). The mutation has no significant effect on the overall structure of the enzyme and the active site. The root mean square deviation from the structure of the wild-type enzyme is 0.11 Å, and there are only minor structural changes in the vicinity of the mutation that are shown by superimposing the structures of the mutant and wild-type enzymes (Figure 2). Two specific structural changes can be directly attributed to the mutation; first, the loss of the Trp side chain disrupts an interaction between Trp249 and Asp340 and, second, a new structured water molecule is present in the mutant enzyme located in the space produced by removal of the Trp side chain (Figure 2).
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Discussion |
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The present results indicate that although tryptophans 249, 250, 314, and 356 contribute to the binding sites for acceptors in these structures, they make very distinct contributions to the catalytic properties of the enzyme. The Trp249Gly mutation specifically perturbs the Km for acceptor but appears to have little effect on kcat, indicating that mutation of Trp249 has similar effects on acceptor binding in the Michaelis complex and transition state. This is consistent with the relatively distant location of this residue from the catalytic center of the enzyme, its role in interacting with the Glc or GlcNAc component of the acceptor, and the absence of any significant change in overall structure arising from the mutation. It is interesting that the corresponding residue in human blood group A and B glycosyltransferases is one of the four sites at which these closely related enzymes differ in sequence (Gly and Ser, see Table II). This residue also influences acceptor substrate affinity in these enzymes because substitution of Ser for Gly in the truncated catalytic domain of the blood group A glycosyltransferases produces a threefold increase in the Km for acceptor substrate but insignificant changes in kcat or donor substrate specificity (Seto et al., 1999).
Substitutions of Tyr or Phe for Trp250 produce small changes in catalytic activity, specifically, a small increase in kcat for both the hydrolase and transferase activities and an increased Km for UDP-Gal for the transferase activity. These changes may reflect a small change in active site accessibility that produces increased rates of product release and reduces the on-rate for UDP-Gal binding.
The enzyme mutant with a Trp314 to Tyr substitution has properties that are similar to those of a previously described Gln247 to Glu variant (Zhang et al., 2003). The kcat for the transferase reaction is reduced, but hydrolase activity is essentially unchanged; the mutation also produces a small reduction in the Km for acceptor. Both the Trp314 to Tyr and Gln247 to Glu mutations reduce the ratio of the transferase to hydrolase activity
40-fold (Table III). Therefore, the Trp314Tyr mutation selectively destabilizes the transition state for the transferase reaction, as shown by its effect on catalytic efficiency for this reaction (Table III), but does not affect acceptor binding in the Michaelis complex or any aspect of UDP-Gal hydrolysis. Trp314 interacts with both acceptor substrates in their UDP complexes and with the monosaccharides in the enzyme complexes with UDP-Gal and UDP-Glc (Boix et al., 2002
). The mutation does not alter the structure of the enzyme or the binding of UDP, lactose or LacNAc. These results suggest that Trp314 has a role in stabilizing the bisubstrate transition state for the transfer of galactose to a disaccharide substrate but not for transfer to water.
The effects of the mutation of Trp356 to Thr differs from the others investigated here in reducing the kcat for the both transferase and hydrolase reactions. This effect appears to be linked to its location immediately adjacent to the C-terminal region of 3GT (residues 358365) that undergoes a conformational change between the Form I and II structures (Boix et al., 2001
; Gastinel et al., 2001
). A previously described
3GT mutant with a substitution of Lys for Arg365, a residue that interacts with O2 of the
-P of UDP, showed large reductions in kcat and catalytic efficiency for the transferase reaction (Zhang et al., 2003
) and a similar reduction in UDP-Gal hydrolyase activity to that seen in the Trp356Thr mutant (Table III). This suggests that the conformational change is linked to the ability of
3GT to catalyze galactose transfer to a carbohydrate acceptor or to water, possibly through the enclosure of the catalytic site and bound UDP-Gal by the transconformation and formation of an appropriate environment for catalysis (Zhang et al., 2003
). However, Trp356 also interacts with the acceptor substrate, and the Thr mutation also increases the Km for lactose. Thus mutations of different tryptophans influence substrate specificity in this glycosyltransferase through their effects on substrate affinity (Trp249), transition state stabilization (Trp314), or both (Trp356).
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Materials and methods |
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Steady-state kinetic studies with wild-type and mutant enzymes were performed with a radiochemical assay as previously described (Zhang et al., 2001). For characterizing the transferase activity of most mutants, enzyme activity was measured in the presence of 10 mM MnCl2 at a series of UDP-Gal concentrations (generally 0.12.0 mM) and different lactose concentrations. The data were analyzed by fitting to the rate equation for a symmetrical sequential initial velocity pattern (Zhang et al., 2001
) to give values for kcat, Ka (Km for UDP-Gal), Kia (Kd for UDP-Gal from E.UDP-Gal complex) and Kb (Km for acceptor) (Zhang et al., 2001
, 2003
). As discussed in the text, in some cases the concentration of UDP-Gal was varied at a single fixed concentration of lactose (generally 25 mM) and lactose was varied at a single fixed concentration of UDP-Gal, generally 1.0 or 1.2 mM. Analysis of these data by fitting to the standard single-substrate Michaelis-Menten equation provided apparent values for Km for each substrate and two Vm values. The apparent Vm value, obtained by varying [lactose] at fixed [UDP-Gal] was adjusted by multiplying by (1 + [UDP-Gal]/Ka), where Ka is the apparent Km for UDP-Gal to get an improved estimate of the true Vm and kcat. This method was used in the case of mutants with relatively high UDP-Gal hydrolase activities, as discussed next.
An enzyme concentration was 25 µg/ml was used for galactosyltransferase assays and UDP-Gal hydrolase activities were determined at 1020-fold higher concentrations (Zhang et al., 2003). The transferase and hydrolase activities of the less active Trp356Thr mutant were determined at enzyme concentrations and incubation times of 1.6 µM/15 min and 6.4 µM/60 min.
X-ray crystallography
Crystals of the Trp249Gly and Trp314Tyr mutants were grown at 16°C by the vapor diffusion hanging drop method as described elsewhere (Boix et al., 2001) by mixing 2 µl of the protein at 5 mg/ml in 20 mM 2-(N-morpholino) ethanesulfonic acidNaOH buffer, pH 6.0, 10% glycerol, containing 10 mM UDP and 0.1 mM MnCl2, with an equal volume of a reservoir solution containing 5% PEG 6000 and 0.1 M TrisHCl, pH 8.0. Single crystals appeared after 34 days. Crystals of Trp314Tyr complexes with lactose and LacNAc were obtained by soaking in 100 mM solution of lactose and LacNAc, respectively. Before data collection, all crystals were flash-cooled at 100 K in a cryoprotectant containing 10% PEG 6000, 0.1 M Tris-HCl, pH 8.0, 25% glycerol, and appropriate ligands. High-resolution data sets (space group P21 with 2 molecules in the asymmetric unit) were collected at the Synchrotron Radiation Source (Daresbury, UK) (stations PX9.6 and PX14.1 using a CCD-ADSC detector system). Raw data images were indexed and scaled using the DENZO and SCALEPACK modules of the HKL suite (Otwinowski and Minor, 1997
), see Table IV.
The cell dimensions for all the data sets were isomorphous with the previously reported Form II 3GTUDP structure (Boix et al., 2001
), PDB entry 1K4V (Table IV). Therefore these coordinates were used as a starting model. Crystallographic refinement was performed using the CNS program package (Brünger et al., 1998
). Multiple rounds of bulk solvent correction, energy minimization, individual isotropic B-factor refinement, simulated annealing, and model building using the O program (Jones et al., 1991
) were carried until the Rfree value was optimized. Appropriate ligands (UDP, lactose, LacNAc, and Mn2+ ion) were incorporated into each model after careful observation of the respective electron density map. Water molecules were gradually included into the model at positions corresponding to peaks in the |Fo| |Fc| electron density map with heights greater than 3 sigma and at H-bond distance from appropriate atoms. The final refinement statistics are given in Table IV.
For the Trp314Tyr-lactose complex structure at 1.7 Å resolution, further refinement was carried out using SHELX-97 (Sheldrick and Schneider, 1997). Initial conjugate gradient least squares refinement in SHELXL was carried out restraining all the 1,2- and 1,3-distances with Engh and Huber (1991)
restraints. The atomic displacement parameters were kept isotropic, and clear alternate conformations for some of the side chains were modeled. The multiple conformation site occupancy factors were refined constraining their sum to unity. Occupancy of atoms in molecules was constrained as 1.0 or 0.5 depending on their peak heights in the electron density maps. Geometric restraints were set to default values as specified in the program documentation. Final analysis of the Ramachandran (
) plot showed that all residues lie in the allowed regions for all three structures. The final refinement statistics are included in Table IV. The atomic coordinates for
3GT Trp249Gly mutant, Trp314Tyr mutantlactose complex, and Trp314Tyr mutant-N-acetyl-lactosamine complex (codes 1VZT, 1VZU, and 1VZX, respectively) have been deposited in the RCSB Protein Data Bank. All figures were generated using MOLSCRIPT (Kraulis, 1991
).
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Acknowledgements |
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Footnotes |
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1 Present address: Department of Protein Engineering, Genentech, 1 DNA Way, South San Francisco, CA 94080
2 Present address: CDFD, Hyderabad 500 076, India
3 Present address: ICGEB, Aruna Asaf Ali Marg, New Delhi 110 067, India
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