Activated Sugars
Kinase and nucleotidyltransferase enzymes for the production of activated sugars have been developed. These enzymes have improved stability for industrial application and relaxed specificity towards a variety of sugars. These enzymes are useful in, for example, the production of diverse NDP-sugars for glycosylation of aglycones of interest, production of oligosaccharides, production of other important glycosylated sugars, and in drug discovery applications.
1 . An isolated sugar-1-kinase, wherein the isolated sugar-1-kinase has sugar-1-kinase activity in a sugar-1-kinase assay and has a T 50 half-life at 30° C. of greater than 10 minutes.
2 . The isolated sugar-1-kinase of claim 1 , wherein the sugar-1-kinase assay is a 3,5-dinitrosalicylic acid (DNS) assay, a thin layer chromatography assay or a high-performance liquid chromatography assay.
3 . The isolated sugar-1-kinase of claim 1 , comprising at least 90% amino acid sequence identity to SEQ ID NO:12, SEQ ID NO:8, SEQ ID NO:9, or SEQ ID NO:10, wherein the isolated sugar-1-kinase has sugar-1-kinase activity in a 3,5-dinitrosalicylic acid (DNS) assay.
4 . The isolated sugar-1-kinase of claim 3 , comprising:
(a) SEQ ID NO:8 with the following mutations:
(i) N120S; D183E; T191S; Y376F; and T381S;
(ii) E71D and VI991;
(iii) D221G; or
(iv) a combination of one or more of the following mutations: N120S; D183E; T191S; Y376F; T381S; E71D; VI991; D221G; I341T; I341L, F375P F375M; F375Y; Y376K; Y376T; Y376P; and Y376F;
(b) SEQ ID NO:10 with the following mutations:
(i) N119H; K130N; S239G; F238Y; and I312L;
(ii) I312T and L332H;
(iii) Y341P and F342K;
(iv) Y341M and F342T;
(v) I312T; L332H; Y341P; and F342K; or
(vi) a combination of one or more of the following mutations: N119H; K130N; S239G; F238Y; I312L; I312T; L332H; Y341P; F342K; and Y341M; F342T; T168S; Y341P; Y341M; Y341F; F342K; F342T; F342P; F342Y;
(c) SEQ ID NO:9 with the following mutation: T177S; or
(d) SEQ ID NO:12 with a combination of one or more of the following mutations: D222G; I348T; I348L; F377P; F377M; F377Y; F378K; F378T; F378P; or F378Y.
5 . The isolated sugar-1-kinase of claim 3 , wherein the sugar-1-kinase comprises at least 90% amino acid sequence identity to SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16; or SEQ ID NO:18, wherein the isolated sugar-1-kinase has sugar-1-kinase activity in a sugar-1-kinase assay.
6 . A polynucleotide encoding the sugar-1-kinase of claim 3 .
7 . An expression vector or host cell that comprises the polynucleotide of claim 6 .
8 . An isolated nucleotidyltransferase comprising at least 90% amino acid sequence identity to SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, or SEQ ID NO:22, wherein the isolated nucleotidyltransferase has nucleotidyltransferase activity in a inorganic phosphate assay.
9 . The isolated nucleotidyltransferase of claim 8 , wherein the nucleotidyltransferase has a T 50 half-life at 30° C. of greater than 10 minutes.
10 . A polynucleotide encoding the nucleotidyltransferase of claim 8 .
11 . An expression vector or host cell that comprises the polynucleotide of claim 9 .
12 . A method of phosphorylating one or more sugars comprising contacting the sugars with the sugar-1-kinase of claim 1 , wherein phosphorylated sugar-1-phosphates are produced.
13 . The method of claim 12 , wherein the reaction temperature is greater than 30° C. and the conversion rate of sugar to sugar-1-phosphate is greater than 50%.
14 . The method of claim 12 , wherein the sugar is an L-sugar or a D-sugar.
15 . The method of claim 12 , wherein the sugar is D-galactose, L-galactose, L-glucose, D-glucose, D-glucoronate, L-rhamnose, D-arabinose, L-arabinose, L-xylose, D-xylose, L-ribose, D-ribose, D-fucose, D-fucose, L-fucose, L-xylose, L-lxyose, D-xylose, L-mannose, D-mannose, L-gulose, 6-azido-D-galactose, or a combination thereof.
16 . The method of claim 12 , further comprising contacting the sugar-1-phosphates with a nucleotidyltransferase to produce nucleoside-diphosphate (NDP) sugars.
17 . The method of claim 16 , wherein the nucleotidyltransferase and the sugar-1-kinase are contacted with the sugars at the same time or sequentially.
18 . A method of converting one or more sugar-1-phosphates to nucleoside-diphosphate (NDP) sugars comprising contacting the sugar-1-phosphates with the nucleotidyltransferases of claim 7 , wherein NDP sugars are produced.
19 . The method of claim 18 , wherein the reaction temperature is greater than 30° C. and the conversion rate of sugar-1-phosphates to NDP sugars is greater than 50%.
20 . The method of claim 18 , wherein the sugar-1-phosphate is an L-sugar-1-phosphate or a D-sugar-1-phosphate.
21 . The method of claim 18 , wherein the sugar-1-phosphate is D-galactose-1-phosphate, L-galactose-1-phosphate, L-glucose-1-phosphate, D-glucose-1-phosphate, D-glucoronate-1-phosphate, L-rhamnose-1-phosphate, D-arabinose-1-phosphate, L-arabinose-1-phosphate, L-xylose-1-phosphate, D-xylose-1-phosphate, L-ribose-1-phosphate, D-ribose-1-phosphate, D-fucose-1-phosphate, D-fucose-1-phosphate, L-fucose-1-phosphate, L-xylose-1-phosphate, L-lxyose-1-phosphate, D-xylose-1-phosphate, L-mannose-1-phosphate, D-mannose-1-phosphate, L-gulose-1-phosphate, 6-azido-D-galactose-1-phosphate, or a combination thereof.