Characterization of recombinant fusion constructs of human β1,4-galactosyltransferase 1 and the lipase pre-propeptide from Staphylococcus hyicus

Sauerzapfe, Birgit; Namdjou, Darius‐Jean; Schumacher, Thomas; Linden, N. Hoogerbrugge-van der; Křenek, Karel; Křen, Vladimı́r; Elling, Lothar

doi:10.1016/j.molcatb.2007.09.009

Cited by 38 publications

(83 citation statements)

References 44 publications

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“…A lower catalytic efficiency of the mutant enzyme was also observed using compound 2 as the acceptor substrate. In this case, in comparison with the wild-type enzyme construct (Sauerzapfe, Křenek, et al 2008), the Y284L mutant exhibited a 10% relative activity and a 3.5-fold higher K m value.…”

Section: Y284l Mutant Of Recombinant Human β14-galactosyltransferase-imentioning

confidence: 86%

“…However, the novel His 6 propeptide-catβ4GalT1 Y284L mutant of the human placental enzyme presented in this work is strictly specific for UDP-GalNAc, and UDP-Gal is not accepted at all, which is a novel feature compared to the bovine mutant enzyme (Table I). Kinetic analysis of the reaction of the UDP-GalNAc donor with the GlcNAcβ-Bn acceptor catalyzed by the β4GalT1 Y284L mutant showed an overall lower catalytic efficiency (V max /K m ) compared to an analogous reaction with the UDP-Gal donor and the wild-type enzyme construct (Sauerzapfe, Namdjou, et al 2008). A comparison of the mutant construct with the wild-type enzyme construct (Sauerzapfe, Namdjou, et al 2008) using the corresponding donor substrates (UDP-GalNAc and UDP-Gal, respectively) revealed a 59% relative activity (V max app ) for the respective donor substrates and 76% relative activity for the GlcNAcβ-Bn acceptor.…”

Section: Y284l Mutant Of Recombinant Human β14-galactosyltransferase-imentioning

confidence: 95%

“…Kinetic analysis of the reaction of the UDP-GalNAc donor with the GlcNAcβ-Bn acceptor catalyzed by the β4GalT1 Y284L mutant showed an overall lower catalytic efficiency (V max /K m ) compared to an analogous reaction with the UDP-Gal donor and the wild-type enzyme construct (Sauerzapfe, Namdjou, et al 2008). A comparison of the mutant construct with the wild-type enzyme construct (Sauerzapfe, Namdjou, et al 2008) using the corresponding donor substrates (UDP-GalNAc and UDP-Gal, respectively) revealed a 59% relative activity (V max app ) for the respective donor substrates and 76% relative activity for the GlcNAcβ-Bn acceptor. The K m values for the respective donor substrates and for the GlcNAcβ-Bn acceptor were 2.8-fold and 7-fold higher, respectively, with the mutant construct.…”

Section: Y284l Mutant Of Recombinant Human β14-galactosyltransferase-imentioning

confidence: 95%

“…For the expression of the gene of the mutant human placental β4GalT1, we used the strategy described in our recent work (Sauerzapfe, Namdjou, et al 2008). The fusion construct His 6 propeptide-catβ4GalT1 was expressed in the cytoplasm of E. coli.…”

Section: Y284l Mutant Of Recombinant Human β14-galactosyltransferase-imentioning

confidence: 99%

“…The relatively flexible substrate spectrum of bovine and human milk β4GalT1 was exploited in the synthesis of neo-glycoconjugates using nonnatural donor or acceptor substrates (Wong et al 1991;Křen et al 1994;Wong and Whitesides 1994;Zervosen and Elling 1996;Nishida et al 2000;Qian et al 2000;Brockhausen et al 2006). We have recently demonstrated that the affinity and activity of recombinant human β4GalT1 for hydrophobic glycosides of GlcNAc as acceptors are increased when it is fused to the propeptide of the lipase from Staphylococcus hyicus (Sauerzapfe, Křenek, et al 2008;Sauerzapfe, Namdjou, et al 2008).…”

Section: Introductionmentioning

confidence: 99%

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Synthesis of LacdiNAc-terminated glycoconjugates by mutant galactosyltransferase - A way to new glycodrugs and materials

Bojarová¹,

Křenek²,

Wetjen

et al. 2009

Glycobiology

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Human placental β1,4-galactosyltransferase-I (EC 2.4.1.38) transfers the galactosyl moiety from UDP-Gal to various GlcNAc or Glc acceptors in vivo. Here, we describe the construction of its Y284L mutant as a His 6 propeptidecatβ4GalT1 construct, in which the Gal-transferase activity was totally abolished in favor of its GalNAc-transferase activity. We used this mutant in the synthesis of three monoand bivalent LacdiNAc glycomimetics with good yields. These compounds proved to be powerful ligands of two activation receptors of natural killer cells, NKR-P1 and CD69. A synthetic bivalent tethered di-LacdiNAc is the best currently known precipitation agent for both of these receptors and has promising potential for the development of immunoactive glycodrugs.

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Section: Y284l Mutant Of Recombinant Human β14-galactosyltransferase-imentioning

confidence: 86%

Section: Y284l Mutant Of Recombinant Human β14-galactosyltransferase-imentioning

confidence: 95%

Section: Y284l Mutant Of Recombinant Human β14-galactosyltransferase-imentioning

confidence: 95%

Section: Y284l Mutant Of Recombinant Human β14-galactosyltransferase-imentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Synthesis of LacdiNAc-terminated glycoconjugates by mutant galactosyltransferase - A way to new glycodrugs and materials

Bojarová¹,

Křenek²,

Wetjen

et al. 2009

Glycobiology

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Enzymatic Transglycosylation

Tramice

Andreotti

Giordano

et al. 2009

Encyclopedia of Industrial Biotechnology

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The most important functions of carbohydrates are structural roles and energy storage, but carbohydrate parts of glycoproteins and glycolipids are involved in biological functions mainly related to cell recognition events. Carbohydrates are also important molecules in the technological domain. A copious amount of information concerning the synthesis and transformation of complex saccharides in living organisms exists nowadays: the enzymes responsible for the synthesis of glycosidic linkage have been recognized as glycosyltransferases. Glycoside hydrolases (glycosidases) are different enzymes responsible for the hydrolysis of glycosidic linkages, but they can be efficiently used in synthesis. Indeed, at the present time, genetic engineering of glycosidases has emerged as a powerful tool for scientists, in the synthetic field, with the aim to set up chemically, efficient biocatalysts. In this contribution, the glycosyl transfer between oxygen nucleophiles will be analyzed from the chemical and the biotechnological points of view. Chemical machinery for hydrolysis and synthesis of the enzyme glycoside hydrolases and glycosyltransferases will be analyzed, including issues inherent to donors and acceptors and biocatalyst natural sources; in particular, discussing the modern genetic efforts to prepare tailored enzymes with high synthetic ability. Case studies for laboratory and industrial importance are analyzed as well.

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Combinatorial One‐Pot Synthesis of Poly‐N‐acetyllactosamine Oligosaccharides with Leloir‐Glycosyltransferases

et al. 2011

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Poly-N-acetyllactosamine (Poly-LacNAc, [3Galb1,4GlcNAcb1] n ) glycans play an essential role in carbohydrate-protein interactions. The synthesis of poly-LacNAc, both chemical and enzymatic, is typically characterized by high losses of product during sequential synthesis, due to deprotection and/ or purification steps. In this work we present a onepot synthesis of poly-LacNAc oligosaccharides by combining recombinant glycosyltransferases. By fractionation of the poly-LacNAc glycan mixture we were able to isolate glycans with up to six N-acetyllactosamine (LacNAc) units. Activity measurements of the involved recombinant b1,4-galactosyltransferase-1 (b4GalT-1) and b1,3-N-acetylglucosaminyltransferase (b3GlcNAcT) with isolated glycan substrates of up to eight sugar units revealed a preference of b3GlcNAcT for the tetrasaccharide and no preference of b4GalT-1 for a specific glycan length.These findings led us to the optimization of combinatorial one-pot synthesis by variation of substrate and enzyme ratios, as well as starting the synthesis with various poly-LacNAc chain lengths. Consequently, we present here an optimized poly-LacNAc synthesis by the combination of two glycosyltransferases and a uridine-diphospho-glucose/N-acetylglucosamine 4'-epimerase as one-pot strategy resulting in long polyLacNAc glycans with up to six LacNAc units in high yields while minimizing reaction time and product loss. The obtained products are important ligands for the biofunctionalization of biomaterial surfaces and the construction of an artificial extracellular matrix for tissue engineering.

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Characterization of recombinant fusion constructs of human β1,4-galactosyltransferase 1 and the lipase pre-propeptide from Staphylococcus hyicus

Cited by 38 publications

References 44 publications

Synthesis of LacdiNAc-terminated glycoconjugates by mutant galactosyltransferase - A way to new glycodrugs and materials

Synthesis of LacdiNAc-terminated glycoconjugates by mutant galactosyltransferase - A way to new glycodrugs and materials

Enzymatic Transglycosylation

Combinatorial One‐Pot Synthesis of Poly‐N‐acetyllactosamine Oligosaccharides with Leloir‐Glycosyltransferases

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