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Literature summary extracted from

  • Lu, W.; Leimkuhler, C.; Oberthür, M.; Kahne, D.; Walsh, C.T.
    AknK is an L-2-deoxyfucosyltransferase in the biosynthesis of the anthracycline aclacinomycin A (2004), Biochemistry, 43, 4548-4558.
    View publication on PubMed

Application

EC Number Application Comment Organism
2.4.1.B46 synthesis AknK may be a useful enzyme for the chemoenzymatic synthesis of anthracycline variants Streptomyces galilaeus

Cloned(Commentary)

EC Number Cloned (Comment) Organism
2.4.1.B46 subcloned into a pET-22b expression vector and expressed recombinantly in Escherichia coli as a C-terminal His6-tagged protein Streptomyces galilaeus
2.4.1.327 expressed in Escherichia coli BL21(DE3) cells Streptomyces galilaeus

KM Value [mM]

EC Number KM Value [mM] KM Value Maximum [mM] Substrate Comment Organism Structure
2.4.1.B46 0.104
-
 4-O-(alpha-L-rhodosaminyl)aklavinone pH 7.5, 25°C, cosubstrate: TDP-L-daunosamine Streptomyces galilaeus
2.4.1.B46 0.109
-
 4-O-(alpha-L-rhodosaminyl)aklavinone pH 7.5, 25°C, cosubstrate: dTDP-2-deoxy-alpha-L-fucose Streptomyces galilaeus
2.4.1.B46 0.138
-
 idarubicin pH 7.5, 25°C, cosubstrate: dTDP-alpha-L-2-deoxyfucose Streptomyces galilaeus
2.4.1.B46 0.149
-
 dTDP-2-deoxy-beta-L-fucose pH 7.5, 25°C, cosubstrate: 4-O-(alpha-L-rhodosaminyl)aklavinone Streptomyces galilaeus
2.4.1.B46 0.531
-
 dTDP-2-deoxy-beta-L-fucose pH 7.5, 25°C, cosubstrate: idarubicin Streptomyces galilaeus
2.4.1.B46 0.94
-
 dTDP-L-daunosamine pH 7.5, 25°C, cosubstrate: rhodosaminyl aklavinone Streptomyces galilaeus
2.4.1.327 0.1044
-
 rhodosaminyl aklavinone with TDP-L-daunosamine as cosubstrate, in 75 mM Tris (pH 7.5), 12 mM MgCl2, at 25°C Streptomyces galilaeus
2.4.1.327 0.1091
-
 rhodosaminyl aklavinone with TDP-L-2-deoxyfucose as cosubstrate, in 75 mM Tris (pH 7.5), 12 mM MgCl2, at 25°C Streptomyces galilaeus
2.4.1.327 0.138
-
 idarubicin in 75 mM Tris (pH 7.5), 12 mM MgCl2, at 25°C Streptomyces galilaeus
2.4.1.327 0.1489
-
 dTDP-L-2-deoxyfucose with rhodosaminyl aklavinone as cosubstrate, in 75 mM Tris (pH 7.5), 12 mM MgCl2, at 25°C Streptomyces galilaeus
2.4.1.327 0.531
-
 dTDP-L-2-deoxyfucose with idarubicin as cosubstrate, in 75 mM Tris (pH 7.5), 12 mM MgCl2, at 25°C Streptomyces galilaeus
2.4.1.327 0.94
-
 dTDP-L-daunosamine in 75 mM Tris (pH 7.5), 12 mM MgCl2, at 25°C Streptomyces galilaeus

Molecular Weight [Da]

EC Number Molecular Weight [Da] Molecular Weight Maximum [Da] Comment Organism
2.4.1.B46 65000
-
 x * 65000, SDS-PAGE Streptomyces galilaeus
2.4.1.327 50685
-
 x * 50685, calculated from amino acid sequence Streptomyces galilaeus
2.4.1.327 50699
-
 x * 50699, MALDI-TOF mass spectrometry Streptomyces galilaeus
2.4.1.327 65000
-
 x * 65000, His6-tagged enzyme, SDS-PAGE Streptomyces galilaeus

Natural Substrates/ Products (Substrates)

EC Number Natural Substrates Organism Comment (Nat. Sub.) Natural Products Comment (Nat. Pro.) Rev. Reac.
2.4.1.B46 dTDP-2-deoxy-beta-L-fucose + 4-O-(alpha-L-rhodosaminyl)aklavinone Streptomyces galilaeus
-
 dTDP + 4-O-[(4-O-2-deoxy-alpha-L-fucosyl)-alpha-L-rhodosaminyl]aklavinone
-
 ?
2.4.1.327 dTDP-2-deoxy-beta-L-fucose + aclacinomycin T Streptomyces galilaeus
-
 dTDP + aclacinomycin S
-
 ?
2.4.1.327 dTDP-2-deoxy-beta-L-fucose + aclacinomycin T Streptomyces galilaeus ATCC 31615
-
 dTDP + aclacinomycin S
-
 ?

Organism

EC Number Organism UniProt Comment Textmining
2.4.1.B46 Streptomyces galilaeus Q9L555
-
-
2.4.1.327 Streptomyces galilaeus
-
-
-
2.4.1.327 Streptomyces galilaeus ATCC 31615
-
-
-

Purification (Commentary)

EC Number Purification (Comment) Organism
2.4.1.B46
-
 Streptomyces galilaeus
2.4.1.327 Ni-NTA resin column chromatography Streptomyces galilaeus

Substrates and Products (Substrate)

EC Number Substrates Comment Substrates Organism Products Comment (Products) Rev. Reac.
2.4.1.B46 dTDP-2-deoxy-alpha-L-fucose + 4-O-[(4-O-2-deoxy-alpha-L-fucosyl)-alpha-L-rhodosaminyl]aklavinone AknK also catalyzes a tandem addition of a second L-2-deoxyfucosyl moiety, albeit with reduced activity, to the natural disaccharide chain to produce L-deoxyfucosyl-L-deoxyfucosyl-L-rhodosaminyl aklavinone, a variant of the natural aclacinomycin A. The second transfer reaction is 3 orders of magnitude slower than the first sugar transfer reaction catalyzed by AknK Streptomyces galilaeus dTDP + 4-O-[[4-O-2-deoxy-alpha-L-fucosyl-(4-O-alpha-L-2-deoxyfucosyl)]alpha-L-rhodosaminyl]aklavinone
-
 ?
2.4.1.B46 dTDP-2-deoxy-alpha-L-fucose + idarubicin
-
 Streptomyces galilaeus dTDP + 4'-O-(2-deoxy-alpha-L-fucosyl)idarubicin
-
 ?
2.4.1.B46 dTDP-2-deoxy-beta-L-fucose + 4-O-(alpha-L-rhodosaminyl)aklavinone
-
 Streptomyces galilaeus dTDP + 4-O-[(4-O-2-deoxy-alpha-L-fucosyl)-alpha-L-rhodosaminyl]aklavinone
-
 ?
2.4.1.B46 dTDP-L-daunosamine + 4-O-(alpha-L-rhodosaminyl)aklavinone the enzyme accepts L-daunosamine as an alternative sugar substrate, albeit with a 270fold decrease in catalytic efficiency (kcat/Km) Streptomyces galilaeus dTDP + 4-O-[(4-O-alpha-L-daunosaminyl)-alpha-L-rhodosaminyl]aklavinone
-
 ?
2.4.1.B46 additional information AknK also accepts an alternate dTDP-L-sugar, dTDP-L-daunosamine, and other monoglycosylated anthracyclines, including daunomycin, adriamycin, and idarubicin, to build alternate disaccharides on variant anthracycline backbones Streptomyces galilaeus ?
-
 ?
2.4.1.327 dTDP-2-deoxy-beta-L-fucose + aclacinomycin T
-
 Streptomyces galilaeus dTDP + aclacinomycin S
-
 ?
2.4.1.327 dTDP-2-deoxy-beta-L-fucose + aclacinomycin T
-
 Streptomyces galilaeus ATCC 31615 dTDP + aclacinomycin S
-
 ?
2.4.1.327 dTDP-L-2-deoxyfucose + aclacinomycin S
-
 Streptomyces galilaeus dTDP + 2-deoxy-alpha-D-fucosyl-aclacinomyin S
-
 ?
2.4.1.327 dTDP-L-2-deoxyfucose + aclacinomycin S
-
 Streptomyces galilaeus ATCC 31615 dTDP + 2-deoxy-alpha-D-fucosyl-aclacinomyin S
-
 ?
2.4.1.327 dTDP-L-2-deoxyfucose + aclacinomycin T
-
 Streptomyces galilaeus dTDP + aclacinomycin S
-
 ?
2.4.1.327 dTDP-L-2-deoxyfucose + aclacinomycin T
-
 Streptomyces galilaeus ATCC 31615 dTDP + aclacinomycin S
-
 ?
2.4.1.327 idarubicin + dTDP-L-2-deoxyfucose
-
 Streptomyces galilaeus dTDP + 4'-O-(alpha-L-fucosyl)idarubicin
-
 ?
2.4.1.327 idarubicin + dTDP-L-2-deoxyfucose
-
 Streptomyces galilaeus ATCC 31615 dTDP + 4'-O-(alpha-L-fucosyl)idarubicin
-
 ?
2.4.1.327 rhodosaminyl aklavinone + dTDP-L-daunosamine
-
 Streptomyces galilaeus dTDP + daunosaminyl-rhodosaminyl-aklavinone
-
 ?
2.4.1.327 rhodosaminyl aklavinone + dTDP-L-daunosamine
-
 Streptomyces galilaeus ATCC 31615 dTDP + daunosaminyl-rhodosaminyl-aklavinone
-
 ?

Subunits

EC Number Subunits Comment Organism
2.4.1.B46 ? x * 65000, SDS-PAGE Streptomyces galilaeus
2.4.1.327 ? x * 50685, calculated from amino acid sequence Streptomyces galilaeus
2.4.1.327 ? x * 50699, MALDI-TOF mass spectrometry Streptomyces galilaeus
2.4.1.327 ? x * 65000, His6-tagged enzyme, SDS-PAGE Streptomyces galilaeus

Synonyms

EC Number Synonyms Comment Organism
2.4.1.B46 AknK
-
 Streptomyces galilaeus
2.4.1.327 AknK
-
 Streptomyces galilaeus
2.4.1.327 L-2-deoxyfucosyltransferase
-
 Streptomyces galilaeus

Temperature Optimum [°C]

EC Number Temperature Optimum [°C] Temperature Optimum Maximum [°C] Comment Organism
2.4.1.B46 25
-
 assay at Streptomyces galilaeus

Turnover Number [1/s]

EC Number Turnover Number Minimum [1/s] Turnover Number Maximum [1/s] Substrate Comment Organism Structure
2.4.1.327 0.035
-
 rhodosaminyl aklavinone with TDP-L-daunosamine as cosubstrate, in 75 mM Tris (pH 7.5), 12 mM MgCl2, at 25°C Streptomyces galilaeus
2.4.1.327 0.09
-
 idarubicin in 75 mM Tris (pH 7.5), 12 mM MgCl2, at 25°C Streptomyces galilaeus
2.4.1.327 1.09
-
 rhodosaminyl aklavinone with TDP-L-2-deoxyfucose as cosubstrate, in 75 mM Tris (pH 7.5), 12 mM MgCl2, at 25°C Streptomyces galilaeus

pH Optimum

EC Number pH Optimum Minimum pH Optimum Maximum Comment Organism
2.4.1.B46 7.5
-
 assay at Streptomyces galilaeus

kcat/KM [mM/s]

EC Number kcat/KM Value [1/mMs-1] kcat/KM Value Maximum [1/mMs-1] Substrate Comment Organism Structure
2.4.1.B46 0.037
-
 dTDP-L-daunosamine pH 7.5, 25°C, cosubstrate: 4-O-(alpha-L-rhodosaminyl)aklavinone Streptomyces galilaeus
2.4.1.B46 0.17
-
 dTDP-2-deoxy-alpha-L-fucose pH 7.5, 25°C, cosubstrate: idarubicin Streptomyces galilaeus
2.4.1.B46 0.33
-
 4-O-(alpha-L-rhodosaminyl)aklavinone pH 7.5, 25°C, cosubstrate: TDP-L-daunosamine Streptomyces galilaeus
2.4.1.B46 0.65
-
 idarubicin pH 7.5, 25°C, cosubstrate: dTDP-2-deoxy-alpha-L-fucose Streptomyces galilaeus
2.4.1.B46 5.7
-
 4-O-(alpha-L-rhodosaminyl)aklavinone pH 7.5, 25°C, cosubstrate: dTDP-2-deoxy-alpha-L-fucose Streptomyces galilaeus
2.4.1.B46 7.3
-
 dTDP-2-deoxy-alpha-L-fucose pH 7.5, 25°C, cosubstrate: 4-O-(alpha-L-rhodosaminyl)aklavinone Streptomyces galilaeus