Effect of C-domain N-glycosylation and deletion on rat pancreaticα-amylase secretion and activity

2002 ◽  
Vol 362 (2) ◽  
pp. 259-264 ◽  
Author(s):  
Yannick DOYON ◽  
William HOME ◽  
Philippe DAULL ◽  
Denis LeBEL
Keyword(s):  
Enzyme Activity ◽  
Animal Cell ◽  
Brefeldin A ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Amylase Secretion ◽  
Wild Type ◽  
Terminal Domain ◽  
Relative Independence ◽  
Asparagine Residue

Even though all animal α-amylases include glycosylation sequons (Asn-Xaa-Thr/Ser) in their sequences, amylases purified from natural sources are not quantitatively glycosylated. When wild-type rat pancreatic α-amylase, which contains two glycosylation sequons, was expressed in animal cell lines the protein displayed a very low rate of glycosylation (approx. 2%), even after Brefeldin A treatment to increase the contact with the glycosylation machinery. Site-directed mutagenesis of the first glycosylation sequon (Asn410 → Gln) resulted in 90% of the protein being glycosylated at the second glycosylation sequon (Asn459). Mutation of the second sequon completely inhibited glycosylation. In order to ascertain if the interference in the glycosylation of Asn459 that was eliminated by the Asn410 →Gln mutation could be due to the position of the asparagine residue in the Cys448-Cys460 disulphide bridge, these cysteine residues were mutated to serine residues. The resulting mutant was found to be 100% glycosylated. All mutants with mutations in the C-domain had specific activities identical to that of the wild-type enzyme, indicating that enzymic activity is independent of the structure and modification of the C-terminal domain. To further test the independence of the C-domain with respect to the two N-terminal domains of the protein, which harbour the catalytic site, the last seven of the ten β-strands that make up the β-sandwich configuration of the domain were deleted. The truncated protein was not secreted from cells and all enzyme activity was destroyed. These observations show that Asn459 is the only site that can be glycosylated in wild-type amylase, and confirm the relative independence of the C-terminal domain of α-amylase with respect to enzyme activity. In addition, they also establish that the C-terminal domain is absolutely essential for the correct post-translational folding of the enzyme that is responsible for its activity and allows for its secretion.

scholarly journals Importance of the two tryptophan residues in the Streptomyces R61 exocellular dd-peptidase

1992 ◽  
Vol 282 (2) ◽  
pp. 361-367 ◽  
Author(s):  
C Bourguignon-Bellefroid ◽  
J M Wilkin ◽  
B Joris ◽  
R T Aplin ◽  
C Houssier ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Beta Lactams ◽  
Beta Lactamases ◽  
Type Protein ◽  
Wild Type Protein ◽  
Tryptophan Residues

Modification of the Streptomyces R61 DD-peptidase by N-bromosuccinimide resulted in a rapid loss of enzyme activity. In consequence, the role of the enzyme's two tryptophan residues was investigated by site-directed mutagenesis. Trp271 was replaced by Leu. The modification yielded a stable enzyme whose structural and catalytic properties were similar to those of the wild-type protein. Thus the Trp271 residue, though almost invariant among the beta-lactamases of classes A and C and the low-Mr penicillin-binding proteins, did not appear to be essential for enzyme activity. Mutations of the Trp233 into Leu and Ser strongly decreased the enzymic activity, the affinity for beta-lactams and the protein stability. Surprisingly, the benzylpenicilloyl-(W233L)enzyme deacylated at least 300-fold more quickly than the corresponding acyl-enzyme formed with the wild-type protein and gave rise to benzylpenicilloate instead of phenylacetylglycine. This mutant DD-peptidase thus behaved as a weak beta-lactamase.

scholarly journals Lys-197 and Asp-414 are critical residues for binding of ATP/Mg2+ by rat brain inositol 1,4,5-trisphosphate 3-kinase

1993 ◽  
Vol 291 (3) ◽  
pp. 811-816 ◽  
Author(s):  
D Communi ◽  
K Takazawa ◽  
C Erneux
Keyword(s):  
Enzyme Activity ◽  
Amino Acid ◽  
Rat Brain ◽  
Catalytic Domain ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Calmodulin Binding ◽  
Inositol 1,4,5 Trisphosphate

Rat brain inositol 1,4,5-trisphosphate (InsP3) 3-kinase A was expressed in Escherichia coli in order to identify the amino acid residues involved in substrate ATP/Mg2+ binding. Two amino acid regions that are conserved in the catalytic domain of InsP3 3-kinase isoenzymes A and B had characteristics consistent with two ATP/Mg(2+)-binding motives. Site-directed mutagenesis was performed on residues Lys-197, Lys-207 and Asp-414 to generate three mutant enzymes, referred to as C5 K197I, C5 K207I and C5 D414N. Comparison of the wild-type and mutant proteins with regard to enzymic activity revealed that C5 K197I exhibited 10% of control enzyme activity, C5 D414N was totally inactive and C5 K207I was fully active. The reduced levels of enzyme activity for C5 K197I and C5 D414N were correlated with an altered ability of the mutant enzymes to bind ATP/Mg2+, as determined by ATP-agarose affinity chromatography. Neither Ca2+/calmodulin binding nor InsP3 binding appeared to be affected. Mutant C5 K207I showed the same characteristics as the wild-type enzyme. Taken together, these results strongly indicated (i) that amino acid residues Lys-197 and Asp-414 are necessary for InsP3 3-kinase activity and form part of the ATP/Mg(2+)-binding domain, and (ii) that amino acid residues Lys-197, Lys-207 and Asp-414 are not involved in either InsP3 binding or enzyme stimulation by Ca2+/calmodulin.

scholarly journals Structural and biochemical characterization of bifunctional XynA

2020 ◽  
Author(s):  
Wei Xie ◽  
Qi Yu ◽  
Yun Liu ◽  
Ruoting Cao ◽  
Ruiqing Zhang ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Enzyme Activity ◽  
Catalytic Mechanism ◽  
Biochemical Characterization ◽  
Cellulase Activity ◽  
Cost Savings ◽  
Site Directed Mutagenesis ◽  
Enzyme Activity Assay ◽  
Great Cost ◽  
Terminal Domain

AbstractXylan and cellulose are the two major constituents in numerous types of lignocellulosic biomass, representing a promising resource for biofuels and other biobased industries. The efficient degradation of lignocellulose requires the synergistic actions of cellulase and xylanase. Thus, bifunctional enzyme incorporated xylanase/cellulase activity has attracted considerable attention since it has great cost savings potential. Recently, a novel GH10 family enzyme XynA identified from Bacillus sp. is found to degrade both cellulose and xylan. To understand its molecular catalytic mechanism, here we first solve the crystal structure of XynA at 2.3 Å. XynA is characterized with a classic (α/β)8 TIM-barrel fold (GH10 domain) flanked by the flexible N-terminal domain and C-terminal domain. Circular dichroism, protein thermal shift and enzyme activity assays reveal that conserved residues Glu182 and Glu280 are both important for catalytic activities of XynA, which is verified by the crystal structure of XynA with E182A/E280A double mutant. Molecular docking studies of XynA with xylohexaose and cellohexaose as well as site-directed mutagenesis and enzyme activity assay demonstrat that Gln250 and His252 are indispensible to cellulase and bifunctional activity, separately. These results elucidate the structural and biochemical features of XynA, providing clues for further modification of XynA for industrial application.

scholarly journals The effects of the site-directed removal of N-glycosylation sites from β-1,4-N-acetylgalactosaminyltransferase on its function

1995 ◽  
Vol 312 (1) ◽  
pp. 273-280 ◽  
Author(s):  
M Haraguchi ◽  
S Yamashiro ◽  
K Furukawa ◽  
K Takamiya ◽  
H Shiku ◽  
...  
Keyword(s):  
Enzyme Activity ◽  
Intracellular Localization ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Kinetics Analysis ◽  
Glycosylation Sites ◽  
In The Wild ◽  
Polymerase Chain ◽  
The Stability

The amino acid sequence deduced from the cloned human cDNA of beta-1,4-N-acetylgalactosaminyltransferase (GalNAc-T; EC 2.4.1.92) gene predicted three potential sites for N-linked glycosylation. Although many glycosyltransferases isolated contain from 2 to 6 N-glycosylation sites, their significance has not been adequately demonstrated. To clarify the roles of N-glycosylation in GalNAc-T function, we generated a series of mutant cDNAs, in which some or all of the glycosylation recognition sites were eliminated by polymerase chain reaction (PCR)-mediated site-directed mutagenesis. Using transcription/translation in vitro, we confirmed that all potential N-glycosylation sites could be used. Although cell lines transfected with mutant cDNAs showed equivalent levels of GalNAc beta 1-->4(NeuAc alpha 2-->3)Gal beta 1-->4Glc-Cer (GM2) to that of the wild-type, the extracts from mutant cDNA transfectants demonstrated lower enzyme activity than in the wild-type. The decrease in enzyme activity was more evident as the number of deglycosylated sites increased, with about 90% decrease in a totally deglycosylated mutant. The enzyme kinetics analysis revealed no significant change of Km among wild-type and mutant cDNA products. The intracellular localization of GalNAc-T expressed in transfectants with wild-type or mutant cDNAs also showed a similar perinuclear pattern (Golgi pattern). These results suggest that N-linked carbohydrates on GalNAc-T are required for regulating the stability of the enzyme structure.

Effects of N224 glycosylation in Saccharomycopsis fibuligera R64 α-amylase on enzyme activity and stability

2021 ◽  
Vol 17 ◽  
Author(s):  
Yovin Sugijo ◽  
Tina Dewi Rosahdi ◽  
Fernita Puspasari ◽  
Wangsa Tirta Ismaya ◽  
Khomaini Hasan ◽  
...  
Keyword(s):  
Thermal Stability ◽  
Enzyme Activity ◽  
Evolutionary Relationship ◽  
Glycosylation Site ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Saccharomycopsis Fibuligera ◽  
Glycosylation Sites ◽  
Starch Substrate ◽  
Relationship Of

Background: The amino acid sequence of an α-amylase of the yeast Saccharomycopsis fibuligera R64 (SfamyR64) contains the two putative N-linked glycosylation sites N153 and N224. N224 is hypothetically responsible for the binding of starch substrate because it is highly conserved among SfamyR64 homologs. Objective: To test whether N224 plays a key role in enzyme activity and stability. Methods: N224Q substitution was introduced by site-directed mutagenesis. The wild type and the mutant were independently over-produced in Pichia pastoris KM71. Activity of the wild type and of the mutant were compared, and their thermal-stability was assessed using heat treatments. The evolutionary relationship of SfamyR64 with its structural homologs with different glycosylation patterns was examined. Results: Activity of the N224Q mutant was approximately 80% lower than that of the wild type. The mutant showed no activity after 10 min of pre-incubation at 50 °C, whereas the wild type SfamyR64 showed activity until 30 min of treatment. Sfamy appeared to have evolved earlier than its structural homolog. Conclusion: SfamyR64 N224 is crucial for enzyme activity and thermal stability. This glycosylation site is unique for fungal and bacterial α-amylases.

scholarly journals Human xylosyltransferase I: functional and biochemical characterization of cysteine residues required for enzymic activity

2005 ◽  
Vol 386 (2) ◽  
pp. 227-236 ◽  
Author(s):  
Sandra MÜLLER ◽  
Manuela SCHÖTTLER ◽  
Sylvia SCHÖN ◽  
Christian PRANTE ◽  
Thomas BRINKMANN ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Biochemical Characterization ◽  
Active Form ◽  
Inhibitory Effect ◽  
Enzymic Activity ◽  
Site Directed Mutagenesis ◽  
Cysteine Residues ◽  
Thiol Groups ◽  
Wild Type ◽  
Free Thiol

XT-I (xylosyltransferase I) is the initial enzyme in the post-translational biosynthesis of glycosaminoglycan chains in proteoglycans. To gain insight into the structure–function relationship of the enzyme, a soluble active form of human XT-I was expressed in High Five insect cells with an apparent molecular mass of 90 kDa. Analysis of the electrophoretic mobility of the protein under non-reducing and reducing conditions indicated that soluble XT-I does not form homodimers through disulphide bridges. In addition, the role of the cysteine residues was investigated by site-directed mutagenesis combined with chemical modifications of XT-I by N-phenylmaleimide. Replacement of Cys471 or Cys574 with alanine led to a complete loss of catalytic activity, indicating the necessity of these residues for maintaining an active conformation of soluble recombinant XT-I by forming disulphide bonds. On the other hand, N-phenylmaleimide treatment showed no effect on wild-type XT-I but strongly inactivated the cysteine mutants in a dose-dependant manner, indicating that seven intramolecular disulphide bridges are formed in wild-type XT-I. The inhibitory effect of UDP on the XT-I activity of C561A (Cys561→Ala) mutant enzyme was significantly reduced compared with all other tested cysteine mutants. In addition, we tested for binding to UDP-agarose beads. The inactive mutants revealed no significantly different nucleotide-binding properties. Our study demonstrates that recombinant XT-I is organized as a monomer with no free thiol groups and strongly suggests that the catalytic activity does not depend on the presence of free thiol groups, furthermore, we identified five cysteine residues which are critical for enzyme activity.

scholarly journals Structure-function relationship of bifunctional XynA

2021 ◽  
Author(s):  
Wei Xie ◽  
Qi Yu ◽  
Ruiqin Zhang ◽  
Yun Liu ◽  
Ruoting Cao ◽  
...  
Keyword(s):  
Crystal Structure ◽  
Enzyme Activity ◽  
Catalytic Mechanism ◽  
Cellulase Activity ◽  
Cost Savings ◽  
Site Directed Mutagenesis ◽  
Enzyme Activity Assay ◽  
Great Cost ◽  
Terminal Domain ◽  
Gh10 Family

Xylan and cellulose are the two major constituents in numerous types of lignocellulose. Thus, bifunctional enzyme incorporated xylanase/cellulase activity has attracted considerable attention since it has great cost savings potential. Recently, a novel GH10 family enzyme XynA identified from Bacillus sp. was found to degrade both cellulose and xylan. To understand its molecular catalytic mechanism, here we first solve the crystal structure of XynA at 2.3 Å. XynA is characterized with a classic (α/β)8 TIM-barrel fold (GH10 domain) flanked by the flexible N-terminal domain and C-terminal domain. XynA has a longer N-terminal and C-terminal than most other GH10 family enzymes. The important thing is that the activity of our N-terminal truncated XynA_ΔN37 is significantly improved. And we found that the C-terminus is crucial to protein expression in solution. Protein thermal shift and enzyme activity assays reveal that conserved residues Glu182 and Glu280 are both important for catalytic activities of XynA, which is verified by the crystal structure of XynA with double mutant E182A/E280A. Molecular docking studies of XynA with xylohexaose and cellohexaose, together with site-directed mutagenesis and enzyme activity assay, demonstrate that Gln250 and His252 are indispensable to bifunctional activity. These results elucidate the structural and biochemical features of XynA, providing clues for further modification of XynA for industrial application.

scholarly journals Site-directed mutagenesis establishes cysteine-110 as essential for enzyme activity in human γ-glutamyl hydrolase

1999 ◽  
Vol 343 (3) ◽  
pp. 551-555 ◽  
Author(s):  
Karen J. CHAVE ◽  
John GALIVAN ◽  
Thomas J. RYAN
Keyword(s):  
Enzyme Activity ◽  
Structural Changes ◽  
Catalytic Mechanism ◽  
Iodoacetic Acid ◽  
Amino Acid Sequences ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Mutant Proteins ◽  
Key Enzyme

γ-Glutamyl hydrolase (GH), which hydrolyses the γ-glutamyl conjugates of folic acid, is a key enzyme in the maintenance of cellular folylpolyglutamate concentrations. The catalytic mechanism of GH is not known. Consistent with earlier reports that GH is sulphydryl-sensitive, we found that recombinant human GH is inhibited by iodoacetic acid, suggesting that at least one cysteine is important for activity [Rhee, Lindau-Shepard, Chave, Galivan and Ryan (1998) Mol. Pharmacol. 53, 1040-1046]. Using site-directed mutagenesis, the cDNA for human GH was altered to encode four different proteins each with one of four cysteine residues changed to alanine. Three of the mutant proteins had activities similar to wild-type GH and were inhibited by iodoacetic acid, whereas the C110A mutant had no activity. Cys-110 is conserved among the human, rat and mouse GH amino acid sequences. The wild-type protein and all four mutants had similar intrinsic fluorescence spectra, indicating no major structural changes had been introduced. These results indicate that Cys-110 is essential for enzyme activity and suggest that GH is a cysteine peptidase. These studies represent the first identification of the essential Cys residue in this enzyme and provide the beginning of a framework to determine the catalytic mechanism, important in defining GH as a therapeutic target.

scholarly journals Evidence that pyruvate dehydrogenase kinase belongs to the ATPase/kinase superfamily

1999 ◽  
Vol 344 (1) ◽  
pp. 47-53 ◽  
Author(s):  
Melissa BOWKER-KINLEY ◽  
Kirill M. POPOV
Keyword(s):  
Pyruvate Dehydrogenase ◽  
Catalytic Domain ◽  
Heat Shock Protein 90 ◽  
Enzymic Activity ◽  
Pyruvate Dehydrogenase Kinase ◽  
Site Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
C Terminus ◽  
Km Value

In this study the roles of invariant Asn-247, Asp-282, Gly-284, Gly-286 and Gly-319 of pyruvate dehydrogenase kinase were investigated by site-directed mutagenesis. Recombinant kinases, wild-type, Asn-247Ala, Asp-282Ala, Gly-284Ala, Gly-286Ala and Gly-319Ala, were expressed in bacteria, purified, and characterized. Three mutant kinases, Asn-247Ala, Asp-282Ala and Gly-286Ala, lacked any appreciable activity. Two other mutants, Gly-284Ala and Gly-319Ala, were catalytically active, with apparent Vmax values close to that of the wild-type kinase (67 and 85 versus 70 nmol/min per mg, respectively). The apparent Km value of Gly-319Ala for nucleotide substrate increased significantly (1500 versus 16 μM). In contrast, Gly-284Ala had only a slightly higher Km value than the wild-type enzyme (28 versus 16 μM). ATP-binding analysis showed that Asn-247Ala, Asp-282Ala and Gly-286Ala could not bind nucleotide. The Kd value of Gly-284Ala was slightly higher than that of the wild-type enzyme (7 versus 4 μM, respectively). In agreement with kinetic analysis, the Gly-319Ala mutant bound ATP so poorly that it was difficult to determine the binding constant. Despite the fact that Asn-247Ala, Asp-282Ala and Gly-286Ala lacked enzymic activity, they were still capable of binding the protein substrate, as shown by their negative-dominant effect in the competition assay with the wild-type kinase. The results of CD spectropolarimetry indicated that there were no major changes in the secondary structures of Asp-282Ala and Gly-286Ala. These results suggest strongly that the catalytic domain of pyruvate dehydrogenase kinase is located at the C-terminus. Furthermore, the catalytic domain is likely to be folded similarly to the catalytic domains of the members of ATPase/kinase superfamily [molecular chaperone heat-shock protein 90 (Hsp90), DNA gyrase B and histidine protein kinases].

scholarly journals Improvement in the Thermostability of d-Psicose 3-Epimerase from Agrobacterium tumefaciens by Random and Site-Directed Mutagenesis

2011 ◽  
Vol 77 (20) ◽  
pp. 7316-7320 ◽  
Author(s):  
Jin-Geun Choi ◽  
Yo-Han Ju ◽  
Soo-Jin Yeom ◽  
Deok-Kun Oh
Keyword(s):  
Enzyme Activity ◽  
Agrobacterium Tumefaciens ◽  
Operation Time ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Conversion Yield ◽  
Content Type ◽  
Aromatic Stacking

ABSTRACTThe S213C, I33L, and I33L S213C variants ofd-psicose 3-epimerase fromAgrobacterium tumefaciens, which were obtained by random and site-directed mutagenesis, displayed increases of 2.5, 5, and 7.5°C in the temperature for maximal enzyme activity, increases of 3.3-, 7.2-, and 29.9-fold in the half-life at 50°C, and increases of 3.1, 4.3, and 7.6°C in apparent melting temperature, respectively, compared with the wild-type enzyme. Molecular modeling suggests that the improvement in thermostability in these variants may have resulted from increased putative hydrogen bonds and formation of new aromatic stacking interactions. The immobilized wild-type enzyme with and without borate maintained activity for 8 days at a conversion yield of 70% (350 g/liter psicose) and for 16 days at a conversion yield of 30% (150 g/liter psicose), respectively. After 8 or 16 days, the enzyme activity gradually decreased, and the conversion yields with and without borate were reduced to 22 and 9.6%, respectively, at 30 days. In contrast, the activities of the immobilized I33L S213C variant with and without borate did not decrease during the operation time of 30 days. These results suggest that the I33L S213C variant may be useful as an industrial producer ofd-psicose.

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