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Literature summary for 2.4.1.359 extracted from

  • Franceus, J.; Decuyper, L.; Dhooghe, M.; Desmet, T.
    Exploring the sequence diversity in glycoside hydrolase family 13_18 reveals a novel glucosylglycerol phosphorylase (2018), Appl. Microbiol. Biotechnol., 102, 3183-3191 .
    View publication on PubMed
Search for more literature for 2.4.1.359

Application

Application Comment Organism
synthesis glucosylglycerol phosphorylase might be an attractive biocatalyst for the production of the osmolyte glucosylglycerol, which is currently produced on industrial scale by exploiting a side activity of the closely related sucrose phosphorylase Marinobacter adhaerens

Cloned(Commentary)

Cloned (Comment) Organism
expression in Escherichia coli Marinobacter adhaerens
gene gtfA, sequence comparisons and phylogenetic tree, recombinant expression of C-terminally His6-tagged wild-type and mutant enzymes in Escherichia coli strain BL21(DE3) Marinobacter adhaerens

Crystallization (Commentary)

Crystallization (Comment) Organism
homology modeling of structure. Residues Tyr194 and Gln336 each form a hydrogen bond with a hydroxyl group of glycerol, while Ala333 is situated at a suitable location for hydrophobic interaction with atom C1 Marinobacter adhaerens

Protein Variants

Protein Variants Comment Organism
A333D site-directed mutagenesis Marinobacter adhaerens
Q336A site-directed mutagenesis Marinobacter adhaerens
Q336A 2.6% residual activity Marinobacter adhaerens
Y194A 2.7% residual activity Marinobacter adhaerens
Y196A site-directed mutagenesis Marinobacter adhaerens

KM Value [mM]

KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
additional information
-
 additional information Michaelis-Menten kinetics Marinobacter adhaerens
2.5
-
 alpha-D-glucose 1-phosphate pH 6.5, 37°C Marinobacter adhaerens
2.5
-
 alpha-D-glucose 1-phosphate recombinant enzyme, pH 6.5, 37°C Marinobacter adhaerens
3.9
-
 2-O-(alpha-D-glucopyranosyl)glycerol pH 6.5, 37°C Marinobacter adhaerens
3.9
-
 2-O-(alpha-D-glucopyranosyl)glycerol recombinant enzyme, pH 6.5, 37°C Marinobacter adhaerens
7
-
 glycerol pH 6.5, 37°C Marinobacter adhaerens
7
-
 glycerol recombinant enzyme, pH 6.5, 37°C Marinobacter adhaerens
10
-
 phosphate pH 6.5, 37°C Marinobacter adhaerens
10
-
 2-O-(alpha-D-glucopyranosyl)glycerol recombinant enzyme, pH 6.5, 37°C Marinobacter adhaerens

Natural Substrates/ Products (Substrates)

Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2-O-(alpha-D-glucopyranosyl)glycerol + phosphate Marinobacter adhaerens
-
 alpha-D-glucose 1-phosphate + glycerol
-
 r
2-O-(alpha-D-glucopyranosyl)glycerol + phosphate Marinobacter adhaerens DSM 23420
-
 alpha-D-glucose 1-phosphate + glycerol
-
 r
2-O-(alpha-D-glucopyranosyl)glycerol + phosphate Marinobacter adhaerens HP15
-
 alpha-D-glucose 1-phosphate + glycerol
-
 r

Organism

Organism UniProt Comment Textmining
Marinobacter adhaerens E4PMA5
-
-
Marinobacter adhaerens E4PMA5 member of family GH13_18
-
Marinobacter adhaerens DSM 23420 E4PMA5
-
-
Marinobacter adhaerens DSM 23420 E4PMA5 member of family GH13_18
-
Marinobacter adhaerens HP15 E4PMA5
-
-

Purification (Commentary)

Purification (Comment) Organism
ecombinant His6-tagged wild-type and mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography and ultrafiltration Marinobacter adhaerens

Specific Activity [micromol/min/mg]

Specific Activity Minimum [µmol/min/mg] Specific Activity Maximum [µmol/min/mg] Comment Organism
12.7
-
 purified recombinant enzyme, pH 6.5, 37°C, phosphorolytic activity Marinobacter adhaerens
14.5
-
 pH 6.5, 37°C, acceptor substrate 1,2-propanediol Marinobacter adhaerens
17
-
 substrate 2-O-(alpha-D-glucopyranosyl)glycerol, pH 6.5, 37°C Marinobacter adhaerens
33
-
 pH 6.5, 37°C, acceptor substrate glycerol Marinobacter adhaerens
33
-
 purified recombinant wild-type enzyme, pH 6.5, 30°C Marinobacter adhaerens
45.2
-
 purified recombinant enzyme, pH 6.5, 37°C, synthetic activity Marinobacter adhaerens

Substrates and Products (Substrate)

Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2-O-(alpha-D-glucopyranosyl)glycerol + phosphate
-
 Marinobacter adhaerens alpha-D-glucose 1-phosphate + glycerol
-
 r
2-O-(alpha-D-glucopyranosyl)glycerol + phosphate
-
 Marinobacter adhaerens DSM 23420 alpha-D-glucose 1-phosphate + glycerol
-
 r
2-O-(alpha-D-glucopyranosyl)glycerol + phosphate
-
 Marinobacter adhaerens HP15 alpha-D-glucose 1-phosphate + glycerol
-
 r
alpha-D-glucose 1-phosphate + 1,2-propanediol
-
 Marinobacter adhaerens 2-O-(alpha-D-glucopyranosyl)propanediol + phosphate
-
 r
alpha-D-glucose 1-phosphate + 1,2-propanediol
-
 Marinobacter adhaerens DSM 23420 2-O-(alpha-D-glucopyranosyl)propanediol + phosphate
-
 r
alpha-D-glucose 1-phosphate + 1,2-propanediol
-
 Marinobacter adhaerens HP15 2-O-(alpha-D-glucopyranosyl)propanediol + phosphate
-
 r
alpha-D-glucose 1-phosphate + glycerol
-
 Marinobacter adhaerens 2-O-(alpha-D-glucopyranosyl)glycerol + phosphate
-
 r
alpha-D-glucose 1-phosphate + glycerol
-
 Marinobacter adhaerens DSM 23420 2-O-(alpha-D-glucopyranosyl)glycerol + phosphate
-
 r
alpha-D-glucose 1-phosphate + glycerol
-
 Marinobacter adhaerens HP15 2-O-(alpha-D-glucopyranosyl)glycerol + phosphate
-
 r
additional information no transglucosylation activity with fructose, fructose 6-phosphate or glycerate as acceptors Marinobacter adhaerens ?
-
 ?
additional information the enzyme catalyses the reversible phosphorolysis of 2-O-alpha-D-glucosylglycerol with retention of the anomeric configuration. Homology modelling, docking and mutagenesis are performed to pinpoint particular acceptor site residues (Tyr194, Ala333, Gln336) involved in the binding of glycerol. In the phosphorolytic direction, the enzyme is active on glucosylglycerol but not on sucrose or glucosylglycerate. A variety of other acceptors are surveyed as well, but significant activity can only be observed using 1,2-propanediol. No transglucosylation activity is detected when fructose, fructose 6-phosphate, or glycerate are supplied as acceptor Marinobacter adhaerens ?
-
-
additional information no transglucosylation activity with fructose, fructose 6-phosphate or glycerate as acceptors Marinobacter adhaerens DSM 23420 ?
-
 ?
additional information the enzyme catalyses the reversible phosphorolysis of 2-O-alpha-D-glucosylglycerol with retention of the anomeric configuration. Homology modelling, docking and mutagenesis are performed to pinpoint particular acceptor site residues (Tyr194, Ala333, Gln336) involved in the binding of glycerol. In the phosphorolytic direction, the enzyme is active on glucosylglycerol but not on sucrose or glucosylglycerate. A variety of other acceptors are surveyed as well, but significant activity can only be observed using 1,2-propanediol. No transglucosylation activity is detected when fructose, fructose 6-phosphate, or glycerate are supplied as acceptor Marinobacter adhaerens DSM 23420 ?
-
-
additional information no transglucosylation activity with fructose, fructose 6-phosphate or glycerate as acceptors Marinobacter adhaerens HP15 ?
-
 ?
additional information the enzyme catalyses the reversible phosphorolysis of 2-O-alpha-D-glucosylglycerol with retention of the anomeric configuration. Homology modelling, docking and mutagenesis are performed to pinpoint particular acceptor site residues (Tyr194, Ala333, Gln336) involved in the binding of glycerol. In the phosphorolytic direction, the enzyme is active on glucosylglycerol but not on sucrose or glucosylglycerate. A variety of other acceptors are surveyed as well, but significant activity can only be observed using 1,2-propanediol. No transglucosylation activity is detected when fructose, fructose 6-phosphate, or glycerate are supplied as acceptor Marinobacter adhaerens HP15 ?
-
-

Subunits

Subunits Comment Organism
? x * 55900, about, sequence calculation Marinobacter adhaerens

Synonyms

Synonyms Comment Organism
2-O-alpha-D-glucopyranosylglycerol:phosphate alpha-D-glucosyltransferase
-
 Marinobacter adhaerens
2-O-alpha-D-glucosylglycerol phosphorylase (retaining)
-
 Marinobacter adhaerens
GGoP
-
 Marinobacter adhaerens
glucosylglycerol phosphorylase
-
 Marinobacter adhaerens
GtfA
-
 Marinobacter adhaerens
MaGGoP
-
 Marinobacter adhaerens

Temperature Optimum [°C]

Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
37
-
-
 Marinobacter adhaerens

Temperature Range [°C]

Temperature Minimum [°C] Temperature Maximum [°C] Comment Organism
25 50 over 60% of maximal activity within this range Marinobacter adhaerens

Temperature Stability [°C]

Temperature Stability Minimum [°C] Temperature Stability Maximum [°C] Comment Organism
45
-
 10 min, no loss of activity Marinobacter adhaerens
50
-
 10 min, 46% residual activity Marinobacter adhaerens

Turnover Number [1/s]

Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
3 6 alpha-D-glucose 1-phosphate pH 6.5, 37°C Marinobacter adhaerens
3 6 alpha-D-glucose 1-phosphate recombinant enzyme, pH 6.5, 37°C Marinobacter adhaerens
14
-
 phosphate pH 6.5, 37°C Marinobacter adhaerens
14
-
 2-O-(alpha-D-glucopyranosyl)glycerol pH 6.5, 37°C Marinobacter adhaerens
14
-
 2-O-(alpha-D-glucopyranosyl)glycerol recombinant enzyme, pH 6.5, 37°C Marinobacter adhaerens
39
-
 glycerol pH 6.5, 37°C Marinobacter adhaerens
39
-
 glycerol recombinant enzyme, pH 6.5, 37°C Marinobacter adhaerens

pH Optimum

pH Optimum Minimum pH Optimum Maximum Comment Organism
6.5
-
-
 Marinobacter adhaerens

pH Range

pH Minimum pH Maximum Comment Organism
4.5 7.5 over 20% of maximal activity within this range, profile overview Marinobacter adhaerens

General Information

General Information Comment Organism
evolution the enzyme belongs to the glycoside hydrolase GH13_18 family, simplified phylogenetic tree Marinobacter adhaerens
metabolism the enzyme specificity provides additional insights into bacterial metabolic routes, describing a phosphorolytic route for glucosylglycerol in a glucosylglycerol-producing organism Marinobacter adhaerens
additional information homology structure modeling using the crystal structure from the Bifidobacterium adolescentis sucrose phosphorylase (PDB IDs 1R7A, 2GDU, 2GDV) as template, docking study and enzyme-substrate interactions analysis, overview. Tyr194 and Gln336 each seemingly form a hydrogen bond with a hydroxyl group of glycerol, while Ala333 is situated at a suitable location for hydrophobic interaction with atom C1' Marinobacter adhaerens

kcat/KM [mM/s]

kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
1.4
-
 2-O-(alpha-D-glucopyranosyl)glycerol recombinant enzyme, pH 6.5, 37°C Marinobacter adhaerens
1.4
-
 phosphate pH 6.5, 37°C Marinobacter adhaerens
3.59
-
 2-O-(alpha-D-glucopyranosyl)glycerol pH 6.5, 37°C Marinobacter adhaerens
3.6
-
 2-O-(alpha-D-glucopyranosyl)glycerol recombinant enzyme, pH 6.5, 37°C Marinobacter adhaerens
5.57
-
 glycerol pH 6.5, 37°C Marinobacter adhaerens
5.6
-
 glycerol recombinant enzyme, pH 6.5, 37°C Marinobacter adhaerens
14.4
-
 alpha-D-glucose 1-phosphate recombinant enzyme, pH 6.5, 37°C Marinobacter adhaerens
14.4
-
 alpha-D-glucose 1-phosphate pH 6.5, 37°C Marinobacter adhaerens