Literature summary for 2.4.1.132 extracted from

Li, S.-T.; Wang, N.; Xu, X.-X.; Fujita, M.; Nakanishi, H.; Kitajima, T.; Dean, N.; Gao, X.-D.
Alternative routes for synthesis of N-linked glycans by Alg2 mannosyltransferase (2018), FASEB J., 32, 2492-2506 .

Search for more literature for 2.4.1.132

Cloned(Commentary)

Cloned (Comment)	Organism
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

Protein Variants

Protein Variants	Comment	Organism
E335A	site-directed mutagenesis, Trx-scAlg2E335A produces only no final product and only 32% of intermediate Man2Gn2 compared to wild-type enzyme	Saccharomyces cerevisiae
E343A	site-directed mutagenesis, inactive mutant	Saccharomyces cerevisiae
F337A	site-directed mutagenesis, Trx-scAlg2F337A produces 26% Man3Gn2 product compared to wild-type enzyme	Saccharomyces cerevisiae
G377R	site-directed mutagenesis, a temperature-sensitive alg2-1 mutant containing a single missense mutation, catalytically inactive	Saccharomyces cerevisiae
H336A	site-directed mutagenesis, Trx-scAlg2H336A produces 8% Man3Gn2 product compared to wild-type enzyme	Saccharomyces cerevisiae
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
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

Localization

Localization	Comment	Organism	GeneOntology No.	Textmining
endoplasmic reticulum	-	Saccharomyces cerevisiae	5783	-
membrane	-	Saccharomyces cerevisiae	16020	-
microsome	-	Saccharomyces cerevisiae	-	-

Metals/Ions

Metals/Ions	Comment	Organism	Structure
Mg2+	required	Saccharomyces cerevisiae

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
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	-	?
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	-	?

Organism

Organism	UniProt	Comment	Textmining
Saccharomyces cerevisiae	P43636	-	-
Saccharomyces cerevisiae ATCC 204508	P43636	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant N-terminal thioredoxin (Trx)-His6-tagged wild-type and mutant ALG2s from Escherichia coli strain DE3 membranes, preparation of proteoliposomes	Saccharomyces cerevisiae

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
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	-	?
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	-	?
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	-	?
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	-	?
additional information	unique bifunctionality of Alg2 during lipid-linked oligosaccharide (LLO) synthesis	Saccharomyces cerevisiae	?	-	-
additional information	unique bifunctionality of Alg2 during lipid-linked oligosaccharide (LLO) synthesis	Saccharomyces cerevisiae ATCC 204508	?	-	-

Synonyms

Synonyms	Comment	Organism
Alg2	-	Saccharomyces cerevisiae
Alg2 mannosyltransferase	-	Saccharomyces cerevisiae
Alg2 MTase	-	Saccharomyces cerevisiae
More	see also EC 2.4.1.257	Saccharomyces cerevisiae
MTase	-	Saccharomyces cerevisiae
scAlg2	-	Saccharomyces cerevisiae

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
30	-	assay at	Saccharomyces cerevisiae

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
6	-	assay at	Saccharomyces cerevisiae

General Information

General Information	Comment	Organism
malfunction	cells deleted for ALG2 are inviable. Mutant alg2 alleles display intraallelic complementation	Saccharomyces cerevisiae
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
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
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

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Release 2023.1 (January 2023)