Identification of the N-glycosylation sites on recombinant bovine CD38 expressed in Pichia pastoris: Their impact on enzyme stability and catalytic activity

Muller-Steffner, Hélène; Kuhn, Isabelle; Argentini, Manuela; Schuber, Francis

doi:10.1016/j.pep.2009.10.003

Cited by 21 publications

(15 citation statements)

References 37 publications

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“…A soluble recombinant form of the ecto-domain of human CD38 containing its four N-glycosylation sites (wild-type) was expressed as a secreted protein and affinity-purified as previously described for bovine CD38. 44 …”

Section: Expression and Purification Of Smnace And Human Cd38mentioning

confidence: 99%

Identification by high-throughput screening of inhibitors of Schistosoma mansoni NAD+ catabolizing enzyme

Kuhn

Kellenberger

Said-Hassane

et al. 2010

Bioorganic & Medicinal Chemistry

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Section: Expression and Purification Of Smnace And Human Cd38mentioning

confidence: 99%

Identification by high-throughput screening of inhibitors of Schistosoma mansoni NAD+ catabolizing enzyme

Kuhn

Kellenberger

Said-Hassane

et al. 2010

Bioorganic & Medicinal Chemistry

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“…It is missing seven residues at the C-terminus and an otherwise highly conserved disulfide bond [13]. Furthermore, bCD38 contains only two N -glycosylation motifs, of which just one is occupied at Asn201 [20], instead of the four observed in all others CD38. The impact of these differences on the structure and the mechanism of bCD38 is unknown.…”

Section: Introductionmentioning

confidence: 99%

Insights into the Mechanism of Bovine CD38/NAD+Glycohydrolase from the X-Ray Structures of Its Michaelis Complex and Covalently-Trapped Intermediates

et al. 2012

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Bovine CD38/NAD+glycohydrolase (bCD38) catalyses the hydrolysis of NAD+ into nicotinamide and ADP-ribose and the formation of cyclic ADP-ribose (cADPR). We solved the crystal structures of the mono N-glycosylated forms of the ecto-domain of bCD38 or the catalytic residue mutant Glu218Gln in their apo state or bound to aFNAD or rFNAD, two 2′-fluorinated analogs of NAD+. Both compounds behave as mechanism-based inhibitors, allowing the trapping of a reaction intermediate covalently linked to Glu218. Compared to the non-covalent (Michaelis) complex, the ligands adopt a more folded conformation in the covalent complexes. Altogether these crystallographic snapshots along the reaction pathway reveal the drastic conformational rearrangements undergone by the ligand during catalysis with the repositioning of its adenine ring from a solvent-exposed position stacked against Trp168 to a more buried position stacked against Trp181. This adenine flipping between conserved tryptophans is a prerequisite for the proper positioning of the N1 of the adenine ring to perform the nucleophilic attack on the C1′ of the ribofuranoside ring ultimately yielding cADPR. In all structures, however, the adenine ring adopts the most thermodynamically favorable anti conformation, explaining why cyclization, which requires a syn conformation, remains a rare alternate event in the reactions catalyzed by bCD38 (cADPR represents only 1% of the reaction products). In the Michaelis complex, the substrate is bound in a constrained conformation; the enzyme uses this ground-state destabilization, in addition to a hydrophobic environment and desolvation of the nicotinamide-ribosyl bond, to destabilize the scissile bond leading to the formation of a ribooxocarbenium ion intermediate. The Glu218 side chain stabilizes this reaction intermediate and plays another important role during catalysis by polarizing the 2′-OH of the substrate NAD+. Based on our structural analysis and data on active site mutants, we propose a detailed analysis of the catalytic mechanism.

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“…Apart from conferring an increased resistance to proteolysis, N-glycosylation can also enhance enzyme thermostability and alter catalytic activity (30)(31)(32)(33). The improved conformational stability of enzymes derived from the steric interactions between N-linked glycan and protein can decrease the enzyme flexibility or increase the rigidity of the enzyme structure (34,35).…”

mentioning

confidence: 99%

N -Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

Niu

Luo

Shi

et al. 2016

Appl Environ Microbiol

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N-Glycosylation can modulate enzyme structure and function. In this study, we identified two pepsin-resistant histidine acid phosphatase (HAP) phytases from Yersinia kristensenii (YkAPPA) and Yersinia rohdei (YrAPPA), each having an N-glycosylation motif, and one pepsin-sensitive HAP phytase from Yersinia enterocolitica (YeAPPA) that lacked an N-glycosylation site. Site-directed mutagenesis was employed to construct mutants by altering the N-glycosylation status of each enzyme, and the mutant and wild-type enzymes were expressed in Pichia pastoris for biochemical characterization. Compared with those of the N-glycosylation site deletion mutants and N-deglycosylated enzymes, all N-glycosylated counterparts exhibited enhanced pepsin resistance. Introduction of the N-glycosylation site into YeAPPA as YkAPPA and YrAPPA conferred pepsin resistance, shifted the pH optimum (0.5 and 1.5 pH units downward, respectively) and improved stability at acidic pH (83.2 and 98.8% residual activities at pH 2.0 for 1 h). Replacing the pepsin cleavage sites L197 and L396 in the immediate vicinity of the N-glycosylation motifs of YkAPPA and YrAPPA with V promoted their resistance to pepsin digestion when produced in Escherichia coli but had no effect on the pepsin resistance of N-glycosylated enzymes produced in P. pastoris. Thus, N-glycosylation may improve pepsin resistance by enhancing the stability at acidic pH and reducing pepsin's accessibility to peptic cleavage sites. This study provides a strategy, namely, the manipulation of N-glycosylation, for improvement of phytase properties for use in animal feed.A s much as 80% of the total phosphorus in cereal grains, oilseeds, and legumes exists in the form of phytate (myo-inositol hexakisphosphate), one of the most important functional ingredients in animal feed (1). However, phytate usually forms indigestible complexes with mineral cations and proteins, thereby causing reductions in nutrient utilization (2, 3). The dietary phytate undigested by monogastric animals is also released into the environment, where it causes phosphorus pollution (4, 5).The enzyme phytase catalyzes the sequential release of inorganic phosphate and lower myo-inositol phosphates from phytate (6); thus, inclusion of exogenous phytases in animal feeds as enzyme additives can enhance the nutrient uptake, lower the feedstuff production costs, and protect the environment in regions where intensive animal farming is conducted (7,8). Numerous phytases have been identified from microorganisms, animals, and plants (9-11), but only a few of them are commercially produced (12). The extensive application of phytases is limited by enzyme lability and protease sensitivity under high processing temperatures and low physiological pHs. Therefore, improving phytase stability at low pHs and high protease concentrations is desirable.Pepsin, a monomeric protein composed of two ␤-barrel-like domains, represents the main proteolytic enzyme in gastric juice (13). The acidic residues D32 and D215 are mainly responsible for proteoly...

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Identification of the N-glycosylation sites on recombinant bovine CD38 expressed in Pichia pastoris: Their impact on enzyme stability and catalytic activity

Cited by 21 publications

References 37 publications

Identification by high-throughput screening of inhibitors of Schistosoma mansoni NAD+ catabolizing enzyme

Identification by high-throughput screening of inhibitors of Schistosoma mansoni NAD+ catabolizing enzyme

Insights into the Mechanism of Bovine CD38/NAD+Glycohydrolase from the X-Ray Structures of Its Michaelis Complex and Covalently-Trapped Intermediates

N -Glycosylation Improves the Pepsin Resistance of Histidine Acid Phosphatase Phytases by Enhancing Their Stability at Acidic pHs and Reducing Pepsin's Accessibility to Its Cleavage Sites

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