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Pradman K Qasba - One of the best experts on this subject based on the ideXlab platform.
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β 1 4 galactosyltransferase and Lactose Synthase molecular mechanical devices
Biochemical and Biophysical Research Communications, 2002Co-Authors: Boopathy Ramakrishnan, Elizabeth Boeggeman, Pradman K QasbaAbstract:Abstract Recent structural investigations on the β-1,4-galactosyltransferase-1 (Gal-T1) and Lactose Synthase (LS) have revealed that they are akin to an exquisite mechanical device with two well-coordinated flexible loops that are contained within the Gal-T1 catalytic domain. The smaller one has a Trp residue (Trp314) flanked by glycine residues. The larger one comprises amino acid residues 345 to 365. Upon substrate binding, the Trp314 side chain moves to lock the sugar nucleotide in the binding site, while the large loop undergoes a conformational change, masking the sugar nucleotide binding site, and creates (i) the oligosaccharide binding cavity; (ii) a protein–protein interacting site for the enzyme's partner, α-lactalbumin (LA); and (iii) a metal ion binding site. Only in conformation II do Gal-T1 and LA form the LS complex, enabling Gal-T1 to choose the new substrate glucose. LA holds and puts Glc right in the acceptor binding site of Gal-T1, which then maximizes the interactions with Glc, thereby making it a preferred acceptor for the LS reaction. The interaction of LA with Gal-T1 in conformation II also stabilizes the sugar–nucleotide–enzyme complex, kinetically enhancing the sugar transfer, even from the less preferred sugar nucleotides. The conformational change that masks the sugar nucleotide binding site can also be induced by the acceptor alone, thus making it possible for the protein to act as a specific lectin.
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a-lactalbumin stimulates milk b-1,4galactosyltransferase I to transfer glucose from UDp-Glc to N-acetylglucosamine: Crystal structure of b4Gal-t1.LA complex with UDP-Glc
Journal of Biological Chemistry, 2001Co-Authors: Boopathy Ramakrishnan, P.s. Shah, Pradman K QasbaAbstract:beta-1,4-Galactosyltransferase 1 (Gal-T1) transfers gaLactose (Gal) from UDP-Gal to N-acetylglucosamine (GlcNAc), which constitutes its normal galactosyltransferase (Gal-T) activity. In the presence of alpha -lactalbumin (LA), it transfers Gal to Glc, which is its Lactose Synthase (LS) activity. It also transfers glucose (Glc) from UDP-Glc to GlcNAc, constituting the glucosyltransferase (Glc-T) activity, albeit at an efficiency of only 0.3-0.4% of Gal-T activity. In the present study, we show that LA increases this activity almost 30-fold. It also enhances the Glc-T activity toward various N-acyl substituted glucosamine acceptors. Steady state kinetic studies of Glc-T reaction show that the Km for the donor and acceptor substrates are high in the absence of LA. In the presence of LA, the Km for the acceptor substrate is reduced 30-fold, whereas for UDP-Glc it is reduced only 5-fold. In order to understand this property, we have determined the crystal structures of the Gal-T1·LA complex with UDP-Glc·Mn2+ and with N-butanoyl-glucosamine (N-butanoyl-GlcN), a preferred sugar acceptor in the Glc-T activity. The crystal structures reveal that although the binding of UDP-Glc is quite similar to UDP-Gal, there are few significant differences observed in the hydrogen bonding interactions between UDP-Glc and Gal-T1. Based on the present kinetic and crystal structural studies, a possible explanation for the role of LA in the Glc-T activity has been proposed.
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crystal structure of Lactose Synthase reveals a large conformational change in its catalytic component the β1 4 galactosyltransferase i
Journal of Molecular Biology, 2001Co-Authors: Boopathy Ramakrishnan, Pradman K QasbaAbstract:Abstract The Lactose Synthase (LS) enzyme is a 1:1 complex of a catalytic component, β1,4-galactosyltransferse (β4Gal-T1) and a regulatory component, α-lactalbumin (LA), a mammary gland-specific protein. LA promotes the binding of glucose (Glc) to β4Gal-T1, thereby altering its sugar acceptor specificity from N-acetylglucosamine (GlcNAc) to glucose, which enables LS to synthesize Lactose, the major carbohydrate component of milk. The crystal structures of LS bound with various substrates were solved at 2 A resolution. These structures reveal that upon substrate binding to β4Gal-T1, a large conformational change occurs in the region comprising residues 345 to 365. This repositions His347 in such a way that it can participate in the coordination of a metal ion, and creates a sugar and LA-binding site. At the sugar-acceptor binding site, a hydrophobic N-acetyl group-binding pocket is found, formed by residues Arg359, Phe360 and Ile363. In the Glc-bound structure, this hydrophobic pocket is absent. For the binding of Glc to LS, a reorientation of the Arg359 side-chain occurs, which blocks the hydrophobic pocket and maximizes the interactions with the Glc molecule. Thus, the role of LA is to hold Glc by hydrogen bonding with the O-1 hydroxyl group in the acceptor-binding site on β4Gal-T1, while the N-acetyl group-binding pocket in β4Gal-T1 adjusts to maximize the interactions with the Glc molecule. This study provides details of a structural basis for the partially ordered kinetic mechanism proposed for Lactose Synthase.
Boopathy Ramakrishnan - One of the best experts on this subject based on the ideXlab platform.
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β 1 4 galactosyltransferase and Lactose Synthase molecular mechanical devices
Biochemical and Biophysical Research Communications, 2002Co-Authors: Boopathy Ramakrishnan, Elizabeth Boeggeman, Pradman K QasbaAbstract:Abstract Recent structural investigations on the β-1,4-galactosyltransferase-1 (Gal-T1) and Lactose Synthase (LS) have revealed that they are akin to an exquisite mechanical device with two well-coordinated flexible loops that are contained within the Gal-T1 catalytic domain. The smaller one has a Trp residue (Trp314) flanked by glycine residues. The larger one comprises amino acid residues 345 to 365. Upon substrate binding, the Trp314 side chain moves to lock the sugar nucleotide in the binding site, while the large loop undergoes a conformational change, masking the sugar nucleotide binding site, and creates (i) the oligosaccharide binding cavity; (ii) a protein–protein interacting site for the enzyme's partner, α-lactalbumin (LA); and (iii) a metal ion binding site. Only in conformation II do Gal-T1 and LA form the LS complex, enabling Gal-T1 to choose the new substrate glucose. LA holds and puts Glc right in the acceptor binding site of Gal-T1, which then maximizes the interactions with Glc, thereby making it a preferred acceptor for the LS reaction. The interaction of LA with Gal-T1 in conformation II also stabilizes the sugar–nucleotide–enzyme complex, kinetically enhancing the sugar transfer, even from the less preferred sugar nucleotides. The conformational change that masks the sugar nucleotide binding site can also be induced by the acceptor alone, thus making it possible for the protein to act as a specific lectin.
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a-lactalbumin stimulates milk b-1,4galactosyltransferase I to transfer glucose from UDp-Glc to N-acetylglucosamine: Crystal structure of b4Gal-t1.LA complex with UDP-Glc
Journal of Biological Chemistry, 2001Co-Authors: Boopathy Ramakrishnan, P.s. Shah, Pradman K QasbaAbstract:beta-1,4-Galactosyltransferase 1 (Gal-T1) transfers gaLactose (Gal) from UDP-Gal to N-acetylglucosamine (GlcNAc), which constitutes its normal galactosyltransferase (Gal-T) activity. In the presence of alpha -lactalbumin (LA), it transfers Gal to Glc, which is its Lactose Synthase (LS) activity. It also transfers glucose (Glc) from UDP-Glc to GlcNAc, constituting the glucosyltransferase (Glc-T) activity, albeit at an efficiency of only 0.3-0.4% of Gal-T activity. In the present study, we show that LA increases this activity almost 30-fold. It also enhances the Glc-T activity toward various N-acyl substituted glucosamine acceptors. Steady state kinetic studies of Glc-T reaction show that the Km for the donor and acceptor substrates are high in the absence of LA. In the presence of LA, the Km for the acceptor substrate is reduced 30-fold, whereas for UDP-Glc it is reduced only 5-fold. In order to understand this property, we have determined the crystal structures of the Gal-T1·LA complex with UDP-Glc·Mn2+ and with N-butanoyl-glucosamine (N-butanoyl-GlcN), a preferred sugar acceptor in the Glc-T activity. The crystal structures reveal that although the binding of UDP-Glc is quite similar to UDP-Gal, there are few significant differences observed in the hydrogen bonding interactions between UDP-Glc and Gal-T1. Based on the present kinetic and crystal structural studies, a possible explanation for the role of LA in the Glc-T activity has been proposed.
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crystal structure of Lactose Synthase reveals a large conformational change in its catalytic component the β1 4 galactosyltransferase i
Journal of Molecular Biology, 2001Co-Authors: Boopathy Ramakrishnan, Pradman K QasbaAbstract:Abstract The Lactose Synthase (LS) enzyme is a 1:1 complex of a catalytic component, β1,4-galactosyltransferse (β4Gal-T1) and a regulatory component, α-lactalbumin (LA), a mammary gland-specific protein. LA promotes the binding of glucose (Glc) to β4Gal-T1, thereby altering its sugar acceptor specificity from N-acetylglucosamine (GlcNAc) to glucose, which enables LS to synthesize Lactose, the major carbohydrate component of milk. The crystal structures of LS bound with various substrates were solved at 2 A resolution. These structures reveal that upon substrate binding to β4Gal-T1, a large conformational change occurs in the region comprising residues 345 to 365. This repositions His347 in such a way that it can participate in the coordination of a metal ion, and creates a sugar and LA-binding site. At the sugar-acceptor binding site, a hydrophobic N-acetyl group-binding pocket is found, formed by residues Arg359, Phe360 and Ile363. In the Glc-bound structure, this hydrophobic pocket is absent. For the binding of Glc to LS, a reorientation of the Arg359 side-chain occurs, which blocks the hydrophobic pocket and maximizes the interactions with the Glc molecule. Thus, the role of LA is to hold Glc by hydrogen bonding with the O-1 hydroxyl group in the acceptor-binding site on β4Gal-T1, while the N-acetyl group-binding pocket in β4Gal-T1 adjusts to maximize the interactions with the Glc molecule. This study provides details of a structural basis for the partially ordered kinetic mechanism proposed for Lactose Synthase.
Hironobu Hashimoto - One of the best experts on this subject based on the ideXlab platform.
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udp n acetyl 5 thio galactosamine is a substrate of Lactose Synthase
Bioorganic & Medicinal Chemistry Letters, 1997Co-Authors: Osamu Tsuruta, Go Shinohara, Hideya Yuasa, Hironobu HashimotoAbstract:Abstract Uridine 5′-(N-acetyl-5-thio-galactosaminyl diphosphate) (UDP-5SGalNAc) was synthesized from an N-acetylgalactosamine derivative via ring opening-recyclization approach. UDP-5SGalNAc was active as a donor substrate for Lactose Synthase, the complex of galactosyltransferase and lactalbumin, giving the disaccharide mimic (5SGalNAcβ[1→4]GlcNAc) which has a sulfur in the ring of the non-reducing sugar. The initial rate of the formation of the disaccharide mimic was 0.23 % that for the natural disaccharide (GalNAcβ[1→4]GlcNAc).
Keith Brew - One of the best experts on this subject based on the ideXlab platform.
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Composition of the milks of the bottlenose dolphin (Tursiops truncatus) and the florida manatee (Trichechus manatus latirostris)
Comparative Biochemistry and Physiology Part A: Physiology, 2003Co-Authors: Syed Pervaiz, Keith BrewAbstract:Abstract 1. 1. Milk samples from four individual bottlenose dolphins ( Tursiops truncatus ) and two Florida manatees ( Trichechus manatus latirostris ) of known lactation stages were analyzed for protein, carbohydrate and lipid composition, as well as for activity levels of α-lactalbumin, the regulatory protein of Lactose Synthase. 2. 2. The milk from both species had relatively high protein and lipid levels, as reported previously for other marine mammals. The major proportion of the lipid was in the form of triglycerides. 3. 3. Dolphin milk contained an average of 2.2% neutral sugars, which was essentially all in the form of Lactose, as determined by several criteria. 4. 4. Manatee milk samples contained 0.6% of neutral sugars, and a larger proportion (about 2%) of amino sugars. Lactose was not detected by enzymatic assay or paper chromatography, but HPLC analysis indicated the presence of low levels of Lactose together with two components that were tentatively identified as oligosaccharides. 5. 5. α-Lactalbumin activity, determined by assay with bovine galactosyltransferase, was found in both dolphin and manatee milk. The level in dolphin milk was comparable with that found in bovine and other milk, but the level in the manatee was less than 10% of that in the dolphin.
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Functional Site in α-Lactalbumin Encompasses a Region Corresponding to a Subsite in Lysozyme and Parts of Two Adjacent Flexible Substructures†
Biochemistry, 1996Co-Authors: Vladimir Malinovskii, Jie Tian, Jay A. Grobler, Keith BrewAbstract:: Aromatic cluster 1 of alpha-lactalbumin (LA), a substructure adjacent to the cleft, is important for its interaction with galactosyltransferase (GT) and effects on glucose binding in the Lactose Synthase complex [Grobler, J. A., Wang, M., Pike, A. K., & Brew, K. (1994) J. Biol. Chem. 269, 5106-5114]. The full extent of the functional region in LA has been probed by mutagenesis of residues that are near aromatic cluster 1 or within the cleft that corresponds to the active site in the homologous type c lysozymes. The conserved residues Val42, Gln54, and Ile59, which correspond to residues of lysozyme that act in substrate binding in subsites C to E, together with residues adjacent to aromatic cluster 1, were found to be not required for activity. In contrast, replacing Leu110, a component of the region corresponding to lysozyme subsite F, with His or Glu greatly reduces the affinity of LA for GT while the introduction of Arg lowers the synergism of LA and glucose binding to GT and also reduces the affinity of LA for GT. Substitutions for Ala106, which is adjacent to Leu110 in the structure, also perturb activity. The region of the cleft corresponding to subsite F is important for function in LA as well as in lysozyme since other components of this subsite, His32 and Phe31, are also crucial for LA activity. The qualitatively different effects of various substitutions for Leu110 may be mediated by their influence on His32 or by changes in the structure of the Lactose Synthase complex.
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crystal structures of guinea pig goat and bovine α lactalbumin highlight the enhanced conformational flexibility of regions that are significant for its action in Lactose Synthase
Structure, 1996Co-Authors: A C W Pike, Keith Brew, Ravi K AcharyaAbstract:Abstract Background: The regulation of milk Lactose biosynthesis is highly dependent on the action of a specifier protein, α-lactalbumin (LA). Together with a glycosyltransferase, LA forms the enzyme complex Lactose Synthase. LA promotes the binding of glucose to the complex and facilitates the biosynthesis of Lactose. To gain further insight into the molecular basis of LA function in Lactose Synthase we have determined the structures of three species variants of LA. Results The crystal structures of guinea-pig, goat and a recombinant form of bovine LA have been determined using molecular replacement techniques. Overall, the structures are very similar and reflect their high degree of amino acid sequence identity (66–94%). Nonetheless, the structures show that a portion of the molecule (residues 105–110), known to be important for function, exhibits a variety of distinct conformers. This region lies adjacent to two residues (Phe31 and His32) that have been implicated in monosaccharide binding by Lactose Synthase and its conformation has significant effects on the environments of these functional groups. The crystal structures also demonstrate that residues currently implicated in LA's modulatory properties are located in a region of the structure that has relatively high thermal parameters and is therefore probably flexible in vivo . Conclusion LA's proposed interaction site for the catalytic component of the Lactose Synthase complex is primarily located in the flexible C-terminal portion of the molecule. This general observation implies that conformational adjustments may be important for the formation and function of Lactose Synthase.
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study by mutagenesis of the roles of two aromatic clusters of alpha lactalbumin in aspects of its action in the Lactose Synthase system
Journal of Biological Chemistry, 1994Co-Authors: Jay A. Grobler, A C W Pike, Mei Wang, Keith BrewAbstract:Abstract A new system for the bacterial expression of a variant of bovine alpha-lactalbumin has been developed. Eighteen mutant proteins containing single site substitutions in a cluster of predominantly aromatic residues adjacent to the cleft (aromatic cluster I) and in the hydrophobic box were expressed. The proteins were extracted from inclusion bodies and treated to generate native folding and disulfide bonds to provide appropriately folded protein samples for nine of the mutants. These were characterized with respect to kinetic parameters reflecting aspects of alpha-lactalbumin activity in modulating the specificity of bovine galactosyltransferase. In aromatic cluster I, changes at tryptophan 118 or glutamine 117 were found to specifically reduce affinity for galactosyltransferase, whereas substitutions for phenylalanine 31 or histidine 32 have major effects on the ability to promote glucose binding (13-200-fold) and lesser effects on galactosyltransferase affinity (1.5-70-fold). Substitutions in the hydrophobic box were found to affect folding rather than activity. Thus, the binding of alpha-lactalbumin to galactosyltransferase and its ability to promote glucose binding can be separately perturbed and are associated with distinct but adjacent structures. Aromatic cluster I is directly involved in activity whereas the hydrophobic box appears to have a structural rather than functional role.
Osamu Tsuruta - One of the best experts on this subject based on the ideXlab platform.
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udp n acetyl 5 thio galactosamine is a substrate of Lactose Synthase
Bioorganic & Medicinal Chemistry Letters, 1997Co-Authors: Osamu Tsuruta, Go Shinohara, Hideya Yuasa, Hironobu HashimotoAbstract:Abstract Uridine 5′-(N-acetyl-5-thio-galactosaminyl diphosphate) (UDP-5SGalNAc) was synthesized from an N-acetylgalactosamine derivative via ring opening-recyclization approach. UDP-5SGalNAc was active as a donor substrate for Lactose Synthase, the complex of galactosyltransferase and lactalbumin, giving the disaccharide mimic (5SGalNAcβ[1→4]GlcNAc) which has a sulfur in the ring of the non-reducing sugar. The initial rate of the formation of the disaccharide mimic was 0.23 % that for the natural disaccharide (GalNAcβ[1→4]GlcNAc).
