EC Number | Cloned (Comment) | Organism |
---|---|---|
2.4.1.132 | gene ALG2, cloning from Saccharomyces cerevisiae strain W303a, recombinant expression of wild-type and mutant N-terminal thioredoxin (Trx)-His6-tagged enzymes in Escherichia coli strain DE3 | Saccharomyces cerevisiae |
2.4.1.257 | gene ALG2, cloning from Saccharomyces cerevisiae strain W303a, recombinant expression of wild-type and mutant N-terminal thioredoxin (Trx)-His6-tagged enzymes in Escherichia coli strain DE3 | Saccharomyces cerevisiae |
EC Number | Protein Variants | Comment | Organism |
---|---|---|---|
2.4.1.132 | E335A | site-directed mutagenesis, Trx-scAlg2E335A produces only no final product and only 32% of intermediate Man2Gn2 compared to wild-type enzyme | Saccharomyces cerevisiae |
2.4.1.132 | E343A | site-directed mutagenesis, inactive mutant | Saccharomyces cerevisiae |
2.4.1.132 | F337A | site-directed mutagenesis, Trx-scAlg2F337A produces 26% Man3Gn2 product compared to wild-type enzyme | Saccharomyces cerevisiae |
2.4.1.132 | G377R | site-directed mutagenesis, a temperature-sensitive alg2-1 mutant containing a single missense mutation, catalytically inactive | Saccharomyces cerevisiae |
2.4.1.132 | H336A | site-directed mutagenesis, Trx-scAlg2H336A produces 8% Man3Gn2 product compared to wild-type enzyme | Saccharomyces cerevisiae |
2.4.1.132 | additional information | site-directed mutagenesis of conserved EX7E motif. Trx-scAlg2E335A, mutated in the first E, has significantly decreased activity, producing no final product and only 32% of intermediate Man2Gn2. Trx-scAlg2E343A, mutated in the second E, has no detectable activity. The intervening amino acids of the EX7E are also important, though less than either E335 or E343. Trx-scAlg2H336A and Trx-scAlg2F337A produce 8% and 26% of Man3Gn2 product, respectively, compared to wild-type. Cells deleted for ALG2 are inviable, a plasmid shuffling technique is used to measure complementation. Mutant alg2 alleles display intraallelic complementation. Mutations (changed to proline) in five of the glycines (G19, G20, G256, G357, G358) result in complete loss of activity, while two of them (G17, G257) are significantly decreased | Saccharomyces cerevisiae |
2.4.1.132 | V62G | site-directed mutagenesis, Trx-scAlg2V62G produces 25% Man3Gn2 product compared to wild-type enzyme. The HA-tagged mutant allele (3HAscAlg2V62G) fails to complement the lethality of the alg2DELTA LSY2 when grown on 5-FOA | Saccharomyces cerevisiae |
2.4.1.257 | E335A | site-directed mutagenesis, Trx-scAlg2E335A produces only no final product and only 32% of intermediate Man2Gn2 compared to wild-type enzyme | Saccharomyces cerevisiae |
2.4.1.257 | E343A | site-directed mutagenesis, inactive mutant | Saccharomyces cerevisiae |
2.4.1.257 | F337A | site-directed mutagenesis, Trx-scAlg2F337A produces 26% Man3Gn2 product compared to wild-type enzyme | Saccharomyces cerevisiae |
2.4.1.257 | G377R | site-directed mutagenesis, a temperature-sensitive alg2-1 mutant containing a single missense mutation, catalytically inactive | Saccharomyces cerevisiae |
2.4.1.257 | H336A | site-directed mutagenesis, Trx-scAlg2H336A produces 8% Man3Gn2 product compared to wild-type enzyme | Saccharomyces cerevisiae |
2.4.1.257 | additional information | site-directed mutagenesis of conserved EX7E motif. Trx-scAlg2E335A, mutated in the first E, has significantly decreased activity, producing no final product and only 32% of intermediate Man2Gn2. Trx-scAlg2E343A, mutated in the second E, has no detectable activity. The intervening amino acids of the EX7E are also important, though less than either E335 or E343. Trx-scAlg2H336A and Trx-scAlg2F337A produce 8% and 26% of Man3Gn2 product, respectively, compared to wild-type. Cells deleted for ALG2 are inviable, a plasmid shuffling technique is used to measure complementation. Mutant alg2 alleles display intraallelic complementation. Mutations (changed to proline) in five of the glycines (G19, G20, G256, G357, G358) result in complete loss of activity, while two of them (G17, G257) are significantly decreased | Saccharomyces cerevisiae |
2.4.1.257 | V62G | site-directed mutagenesis, Trx-scAlg2V62G produces 25% Man3Gn2 product compared to wild-type enzyme. The HA-tagged mutant allele (3HAscAlg2V62G) fails to complement the lethality of the alg2DELTA LSY2 when grown on 5-FOA | Saccharomyces cerevisiae |
EC Number | Localization | Comment | Organism | GeneOntology No. | Textmining |
---|---|---|---|---|---|
2.4.1.132 | endoplasmic reticulum | - |
Saccharomyces cerevisiae | 5783 | - |
2.4.1.132 | membrane | - |
Saccharomyces cerevisiae | 16020 | - |
2.4.1.132 | microsome | - |
Saccharomyces cerevisiae | - |
- |
2.4.1.257 | endoplasmic reticulum | - |
Saccharomyces cerevisiae | 5783 | - |
2.4.1.257 | membrane | - |
Saccharomyces cerevisiae | 16020 | - |
2.4.1.257 | microsome | - |
Saccharomyces cerevisiae | - |
- |
EC Number | Metals/Ions | Comment | Organism | Structure |
---|---|---|---|---|
2.4.1.132 | Mg2+ | required | Saccharomyces cerevisiae | |
2.4.1.257 | Mg2+ | required | Saccharomyces cerevisiae |
EC Number | Natural Substrates | Organism | Comment (Nat. Sub.) | Natural Products | Comment (Nat. Pro.) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
2.4.1.132 | GDP-alpha-D-mannose + D-Man-beta-(1->4)-D-GlcNAc-beta-(1->4)-D-GlcNAc-diphosphodolichol | Saccharomyces cerevisiae | - |
GDP + D-Man-alpha-(1->3)-D-Man-beta-(1->4)-D-GlcNAc-beta-(1->4)-D-GlcNAc-diphosphodolichol | - |
? | |
2.4.1.132 | GDP-alpha-D-mannose + D-Man-beta-(1->4)-D-GlcNAc-beta-(1->4)-D-GlcNAc-diphosphodolichol | Saccharomyces cerevisiae ATCC 204508 | - |
GDP + D-Man-alpha-(1->3)-D-Man-beta-(1->4)-D-GlcNAc-beta-(1->4)-D-GlcNAc-diphosphodolichol | - |
? | |
2.4.1.257 | GDP-alpha-D-mannose + alpha-D-Man-(1->3)-beta-D-Man-(1->4)-beta-D-GlcNAc-(1->4)-alpha-D-GlcNAc-diphosphodolichol | Saccharomyces cerevisiae | - |
GDP + alpha-D-Man-(1->3)-[alpha-D-Man-(1->6)]-beta-D-Man-(1->4)-beta-D-GlcNAc-(1->4)-alpha-D-GlcNAc-diphosphodolichol | - |
? | |
2.4.1.257 | GDP-alpha-D-mannose + alpha-D-Man-(1->3)-beta-D-Man-(1->4)-beta-D-GlcNAc-(1->4)-alpha-D-GlcNAc-diphosphodolichol | Saccharomyces cerevisiae ATCC 204508 | - |
GDP + alpha-D-Man-(1->3)-[alpha-D-Man-(1->6)]-beta-D-Man-(1->4)-beta-D-GlcNAc-(1->4)-alpha-D-GlcNAc-diphosphodolichol | - |
? |
EC Number | Organism | UniProt | Comment | Textmining |
---|---|---|---|---|
2.4.1.132 | Saccharomyces cerevisiae | P43636 | - |
- |
2.4.1.132 | Saccharomyces cerevisiae ATCC 204508 | P43636 | - |
- |
2.4.1.257 | Saccharomyces cerevisiae | P43636 | - |
- |
2.4.1.257 | Saccharomyces cerevisiae ATCC 204508 | P43636 | - |
- |
EC Number | Purification (Comment) | Organism |
---|---|---|
2.4.1.132 | recombinant N-terminal thioredoxin (Trx)-His6-tagged wild-type and mutant ALG2s from Escherichia coli strain DE3 membranes, preparation of proteoliposomes | Saccharomyces cerevisiae |
2.4.1.257 | recombinant N-terminal thioredoxin (Trx)-His6-tagged wild-type and mutant ALG2s from Escherichia coli strain DE3 membranes, preparation of proteoliposomes | Saccharomyces cerevisiae |
EC Number | Substrates | Comment Substrates | Organism | Products | Comment (Products) | Rev. | Reac. |
---|---|---|---|---|---|---|---|
2.4.1.132 | GDP-alpha-D-mannose + D-Man-beta-(1->4)-D-GlcNAc-beta-(1->4)-D-GlcNAc-diphosphodolichol | - |
Saccharomyces cerevisiae | GDP + D-Man-alpha-(1->3)-D-Man-beta-(1->4)-D-GlcNAc-beta-(1->4)-D-GlcNAc-diphosphodolichol | - |
? | |
2.4.1.132 | GDP-alpha-D-mannose + D-Man-beta-(1->4)-D-GlcNAc-beta-(1->4)-D-GlcNAc-diphosphodolichol | - |
Saccharomyces cerevisiae ATCC 204508 | GDP + D-Man-alpha-(1->3)-D-Man-beta-(1->4)-D-GlcNAc-beta-(1->4)-D-GlcNAc-diphosphodolichol | - |
? | |
2.4.1.132 | GDP-alpha-D-mannose + Man-(beta1,4)-Gn-(beta1,4)-Gn-PP-phytanyl | synthesis of acceptor phytanyl oligosaccharide, Man1Gn2-PPhy, from Gn-(beta1,4)-Gn-PP-phytanyl (Gn2-PPhy) using yeast Alg1. Recombinant scAlg2 transfers 2 Man residues to the beta1,4-Man of the Man1Gn2-PPhy substrate with alpha1,6 and alpha1,3-linkages, yielding Man-(alpha1,3)[Man-(alpha1,6)]-Man1Gn2-PPhy, cf. EC 2.4.1.257 | Saccharomyces cerevisiae | GDP + Man-(alpha1,3)[Man-(alpha1,6)]-Man1Gn2-PPhy | - |
? | |
2.4.1.132 | GDP-alpha-D-mannose + Man-(beta1,4)-Gn-(beta1,4)-Gn-PP-phytanyl | synthesis of acceptor phytanyl oligosaccharide, Man1Gn2-PPhy, from Gn-(beta1,4)-Gn-PP-phytanyl (Gn2-PPhy) using yeast Alg1. Recombinant scAlg2 transfers 2 Man residues to the beta1,4-Man of the Man1Gn2-PPhy substrate with alpha1,6 and alpha1,3-linkages, yielding Man-(alpha1,3)[Man-(alpha1,6)]-Man1Gn2-PPhy, cf. EC 2.4.1.257 | Saccharomyces cerevisiae ATCC 204508 | GDP + Man-(alpha1,3)[Man-(alpha1,6)]-Man1Gn2-PPhy | - |
? | |
2.4.1.132 | additional information | unique bifunctionality of Alg2 during lipid-linked oligosaccharide (LLO) synthesis | Saccharomyces cerevisiae | ? | - |
- |
|
2.4.1.132 | additional information | unique bifunctionality of Alg2 during lipid-linked oligosaccharide (LLO) synthesis | Saccharomyces cerevisiae ATCC 204508 | ? | - |
- |
|
2.4.1.257 | 2 GDP-alpha-D-mannose + beta-D-Man-(1->4)-beta-D-GlcNAc-(1->4)-GlcNAc-PP-phytanyl | synthesis of acceptor phytanyl oligosaccharide, Man1Gn2-PPhy, from beta-D-GlcNAc-(1->4)-GlcNAc-PP-phytanyl (Gn2-PPhy) using yeast Alg1. Recombinant scAlg2 transfers 2 Man residues to the beta1,4-Man of the Man1Gn2-PPhy substrate with alpha1,6 and alpha1,3-linkages, yielding alpha-D-Man-(1->3)[alpha-D-Man-(1->6)]-beta-D-Man-(1->4)-beta-D-GlcNAc-(1->4)-alpha-D-GlcNAc-PP-phytanyl | Saccharomyces cerevisiae | 2 GDP + alpha-D-Man-(1->3)[alpha-D-Man-(1->6)]-beta-D-Man-(1->4)-beta-D-GlcNAc-(1->4)-alpha-D-GlcNAc-PP-phytanyl | - |
? | |
2.4.1.257 | GDP-alpha-D-mannose + alpha-D-Man-(1->3)-beta-D-Man-(1->4)-beta-D-GlcNAc-(1->4)-alpha-D-GlcNAc-diphosphodolichol | - |
Saccharomyces cerevisiae | GDP + alpha-D-Man-(1->3)-[alpha-D-Man-(1->6)]-beta-D-Man-(1->4)-beta-D-GlcNAc-(1->4)-alpha-D-GlcNAc-diphosphodolichol | - |
? | |
2.4.1.257 | GDP-alpha-D-mannose + alpha-D-Man-(1->3)-beta-D-Man-(1->4)-beta-D-GlcNAc-(1->4)-alpha-D-GlcNAc-diphosphodolichol | - |
Saccharomyces cerevisiae ATCC 204508 | GDP + alpha-D-Man-(1->3)-[alpha-D-Man-(1->6)]-beta-D-Man-(1->4)-beta-D-GlcNAc-(1->4)-alpha-D-GlcNAc-diphosphodolichol | - |
? | |
2.4.1.257 | additional information | unique bifunctionality of Alg2 during lipid-linked oligosaccharide (LLO) synthesis | Saccharomyces cerevisiae | ? | - |
- |
|
2.4.1.257 | additional information | unique bifunctionality of Alg2 during lipid-linked oligosaccharide (LLO) synthesis | Saccharomyces cerevisiae ATCC 204508 | ? | - |
- |
EC Number | Synonyms | Comment | Organism |
---|---|---|---|
2.4.1.132 | Alg2 | - |
Saccharomyces cerevisiae |
2.4.1.132 | Alg2 mannosyltransferase | - |
Saccharomyces cerevisiae |
2.4.1.132 | Alg2 MTase | - |
Saccharomyces cerevisiae |
2.4.1.132 | More | see also EC 2.4.1.257 | Saccharomyces cerevisiae |
2.4.1.132 | MTase | - |
Saccharomyces cerevisiae |
2.4.1.132 | scAlg2 | - |
Saccharomyces cerevisiae |
2.4.1.257 | Alg2 | - |
Saccharomyces cerevisiae |
2.4.1.257 | Alg2 mannosyltransferase | - |
Saccharomyces cerevisiae |
2.4.1.257 | Alg2 MTase | - |
Saccharomyces cerevisiae |
2.4.1.257 | More | see also EC 2.4.1.132 | Saccharomyces cerevisiae |
2.4.1.257 | MTase | - |
Saccharomyces cerevisiae |
2.4.1.257 | scAlg2 | - |
Saccharomyces cerevisiae |
EC Number | Temperature Optimum [°C] | Temperature Optimum Maximum [°C] | Comment | Organism |
---|---|---|---|---|
2.4.1.132 | 30 | - |
assay at | Saccharomyces cerevisiae |
2.4.1.257 | 30 | - |
assay at | Saccharomyces cerevisiae |
EC Number | pH Optimum Minimum | pH Optimum Maximum | Comment | Organism |
---|---|---|---|---|
2.4.1.132 | 6 | - |
assay at | Saccharomyces cerevisiae |
2.4.1.257 | 6 | - |
assay at | Saccharomyces cerevisiae |
EC Number | General Information | Comment | Organism |
---|---|---|---|
2.4.1.132 | malfunction | cells deleted for ALG2 are inviable. Mutant alg2 alleles display intraallelic complementation | Saccharomyces cerevisiae |
2.4.1.132 | metabolism | the fourth and fifth steps of lipid-linked oligosaccharide (LLO) synthesis are catalyzed by Alg2, an unusual mannosyltransferase (MTase) with two different MTase activities | Saccharomyces cerevisiae |
2.4.1.132 | additional information | the conserved C-terminal EX7E motif, N-terminal cytosolic tail, and 3G-rich loop motifs in Alg2 play crucial roles for these activities, both in vitro and in vivo. Alg2 immunoprecipitates from extracts of yeast microsomal membranes also displays both alpha1,3- and alpha1,6-mannosyltransferase (MTase) activities. The conserved Val62 residue is required for yeast Alg2 function. The first E (E335) and His-336 are partially required for alpha1,6-mannosylation, and importance of both E335 and E343 of the EX7E domain for Alg2 function in vivo. Identification of three conserved G-rich motifs in scAlg2, located in the N-terminal cytosolic short tail, in the middle of Alg2, and in the C-terminal domain. Residues G17, G19, and G20 are within the N-terminal cytosolic tail of Alg2, importance of this domain for Alg2 function | Saccharomyces cerevisiae |
2.4.1.132 | physiological function | asparagine (N)-linked glycosylation requires the ordered, stepwise synthesis of lipid-linked oligosaccharide (LLO) precursor Glc3Man9GlcNAc2-diphosphate-dolichol (Glc3Man9Gn2-PDol) on the endoplasmic reticulum. The fourth and fifth steps of LLO synthesis are catalyzed by Alg2, an unusual mannosyltransferase (MTase) with two different MTase activities. Alg2 adds both an alpha1,3- and alpha1,6-mannose ontoManGlcNAc2-PDol to form the trimannosyl core Man3GlcNAc2-PDol. Alg2-dependent Man3GlcNAc2-PDol production relies on net-neutral lipids with a propensity to form bilayers | Saccharomyces cerevisiae |
2.4.1.257 | malfunction | cells deleted for ALG2 are inviable. Mutant alg2 alleles display intraallelic complementation | Saccharomyces cerevisiae |
2.4.1.257 | metabolism | the fourth and fifth steps of lipid-linked oligosaccharide (LLO) synthesis are catalyzed by Alg2, an unusual mannosyltransferase (MTase) with two different MTase activities | Saccharomyces cerevisiae |
2.4.1.257 | additional information | the conserved C-terminal EX7E motif, N-terminal cytosolic tail, and 3G-rich loop motifs in Alg2 play crucial roles for these activities, both in vitro and in vivo. Alg2 immunoprecipitates from extracts of yeast microsomal membranes also displays both alpha1,3- and alpha1,6-mannosyltransferase (MTase) activities. The conserved Val62 residue is required for yeast Alg2 function. The first E (E335) and His-336 are partially required for alpha1,6-mannosylation, and importance of both E335 and E343 of the EX7E domain for Alg2 function in vivo. Identification of three conserved G-rich motifs in scAlg2, located in the N-terminal cytosolic short tail, in the middle of Alg2, and in the C-terminal domain. Residues G17, G19, and G20 are within the N-terminal cytosolic tail of Alg2, importance of this domain for Alg2 function | Saccharomyces cerevisiae |
2.4.1.257 | physiological function | asparagine (N)-linked glycosylation requires the ordered, stepwise synthesis of lipid-linked oligosaccharide (LLO) precursor Glc3Man9GlcNAc2-diphosphate-dolichol (Glc3Man9Gn2-PDol) on the endoplasmic reticulum. The fourth and fifth steps of LLO synthesis are catalyzed by Alg2, an unusual mannosyltransferase (MTase) with two different MTase activities. Alg2 adds both an alpha1,3- and alpha1,6-mannose onto ManGlcNAc2-PDol to form the trimannosyl core Man3GlcNAc2-PDol. Alg2-dependent Man3GlcNAc2-PDol production relies on net-neutral lipids with a propensity to form bilayers | Saccharomyces cerevisiae |