Active-site engineering of nucleotidylyltransferases and general enzymatic methods for the synthesis of natural and "unnatural" udp- and tdp-nucleotide sugars
AssigneeWisconsin Alumni Research Foundation
InventorsJon THORSON, Dimitar Nikilov
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Abstract
The present invention provides mutant nucleotidylyl-transferases, such as Ep, having altered substrate specificity; methods for their production; and methods of producing nucleotide sugars, which utilize these nucleotidylyl-transferases. The present invention also provides methods of synthesizing desired nucleotide sugars using natural and/or modified Ep or other nucleotidyltransferases; and nucleotide sugars sythesized by the present methods. The present invention further provides new glycosyl phosphates, and methods for making them.
Claims18
A nucleotide sugar produced by a method comprising combining NTP and α-D-hexopyranosyl phosphate in the presence of at least one mutated nucleotidylyltransferase.
A nucleotide sugar produced by a method comprising combining NTP and α-D-hexopyranosyl phosphate other than Glc1P in the presence of at least one nucleotidylyl-transferase.
A nucleotide sugar produced by a method comprising
- combining α-D-hexopyranosyl phosphate and NTP other than TTP, in the presence of at least one nucleotidylyltransferase.
A nucleotide sugar selected from the group consisting of
- Thymidine 5′-(α-D-glucopyranosyl diphosphate) (58);
- Uridine 5′-(α-D-glucopyranosyl diphosphate) (59);
- Thymidine 5′-(2-deoxy-α-D-glucopyranosyl diphosphate) (60);
- Uridine 5′-(2-deoxy-α-D-glucopyranosyl diphosphate) (61);
- Thymidine 5′-(3-deoxy-α-D-glucopyranosyl diphosphate) (62);
- Uridine 5′-(3-deoxy-α-D-glucopyranosyl diphosphate) (63);
- Thymidine 5′-(4-deoxy-α-D-glucopyranosyl diphosphate) (64);
- Uridine 5′-(4-deoxy-α-D-glucopyranosyl diphosphate) (65);
- Thymidine 5′-(6-deoxy-α-D-glucopyranosyl diphosphate) (66);
- Uridine 5′-(6-deoxy-α-D-glucopyranosyl diphosphate) (67);
- Thymidine 5′-(α-D-mannopyranosyl diphosphate) (68);
- Uridine 5′-(α-D-mannopyranosyl diphosphate) (69);
- Thymidine 5′-(α-D-galactopyranosyl diphosphate) (70);
- Uridine 5′-(α-D-galactopyranosyl diphosphate) (71);
- Thymidine 5′-(α-D-allopyranosyl diphosphate) (72);
- Uridine 5′-(α-D-allopyranosyl diphosphate) (73);
- Thymidine 5′-(α-D-altropyranosyl diphosphate) (74);
- Uridine 5′-(α-D-altropyranosyl diphosphate) (75);
- Thymidine 5′-(α-D-gulopyranosyl diphosphate) (76);
- Uridine 5′-(α-D-gulopyranosyl diphosphate) (77);
- Thymidine 5′-(α-D-idopyranosyl diphosphate) (78);
- Uridine 5′-(α-D-idopyranosyl diphosphate) (79);
- Thymidine 5′-(α-D-talopyranosyl diphosphate) (80);
- Uridine 5′-(α-D-talopyranosyl diphosphate) (81);
- Thymidine 5′-(6-amino-6-deoxy-α-D-glucopyranosyl diphosphate) (109);
- Uridine 5′-(6-amino-6-deoxy-α-D-glucopyranosyl diphosphate) (110);
- Thymidine 5′-(4-amino-4-deoxy-α-D-glucopyranosyl diphosphate) (111);
- Uridine 5′-(4-amino-4-deoxy-α-D-glucopyranosyl diphosphate) (112);
- Thymidine 5′-(3-amino-3-deoxy-α-D-glucopyranosyl diphosphate) (113);
- Uridine 5′-(3-amino-3-deoxy-α-D-glucopyranosyl diphosphate) (114);
- Thymidine 5′-(2-amino-2-deoxy-α-D-glucopyranosyl diphosphate) (115);
- Uridine 5′-(2-amino-2-deoxy-α-D-glucopyranosyl diphosphate) (116);
- Thymidine 5′-(6-acetamido-6-deoxy-α-D-glucopyranosyl diphosphate) (117);
- Uridine 5′-(6-acetamido-6-deoxy-α-D-glucopyranosyl diphosphate) (118);
- Thymidine 5′-(4-acetamido-4-deoxy-α-D-glucopyranosyl diphosphate) (119);
- Uridine 5′-(4-acetamido-4-deoxy-α-D-glucopyranosyl diphosphate) (120);
- Thymidine 5′-(3-acetamido-3-deoxy-α-D-glucopyranosyl diphosphate) (121);
- Uridine 5′-(3-acetamido-3-deoxy-α-D-glucopyranosyl diphosphate) (122);
- Thymidine 5′-(2-acetamido-2-deoxy-α-D-glucopyranosyl diphosphate) (123);
- Uridine 5′-(2-acetamido-2-deoxy-α-D-glucopyranosyl diphosphate) (124);
- Thymidine 5′-(4-amino-4,6-dideoxy-α-D-glucopyranosyl diphosphate) (125);
- Uridine 5′-(4-amino-4,6-dideoxy-α-D-glucopyranosyl diphosphate) (126);
Nucleotide sugars sythesized by a method comprising combining a nucleotidylyltransferase, α-D-glucopyranosyl phosphate, NTP and inorganic pyrophosphatase, and incubating.
Nucleotide sugars sythesized by a method comprising combining a nucleotidylyltransferase, α-D-glucopyranosyl phosphate, Mg+2, NTP and inorganic pyrophosphatase, and incubating;
- wherein the nucleotydylyltransferase is a natural nucleotidylyltransferase.
Nucleotide sugars sythesized by a method comprising combining a nucleotidylyltransferase, α-D-glucopyranosyl phosphate, Mg+2, NTP and inorganic pyrophosphatase, and incubating;
- wherein the nucleotidylyltransferase is a mutated nucleotidylyltransferase.
Nucleotide sugars synthesized by a method comprising combining NTP and α-D-hexopyranosyl phosphate in the presence of at least one mutated nucleotidylyltransferase, selected from the group consisting of
- Thymidine 5′-(6-amino-6-deoxy-α-D-glucopyranosyl diphosphate) (109);
- Uridine 5′-(6-amino-6-deoxy-α-D-glucopyranosyl diphosphate) (110);
- Thymidine 5′-(4-amino-4-deoxy-α-D-glucopyranosyl diphosphate) (111);
- Uridine 5′-(4-amino-4-deoxy-α-D-glucopyranosyl diphosphate) (112);
- Thymidine 5′-(3-amino-3-deoxy-α-D-glucopyranosyl diphosphate) (113);
- Uridine 5′-(3-amino-3-deoxy-α-D-glucopyranosyl diphosphate) (114);
- Thymidine 5′-(2-amino-2-deoxy-α-D-glucopyranosyl diphosphate) (115);
- Uridine 5′-(2-amino-2-deoxy-α-D-glucopyranosyl diphosphate) (116);
- Thymidine 5′-(6-acetamido-6-deoxy-α-D-glucopyranosyl diphosphate) (117);
- Uridine 5′-(6-acetamido-6-deoxy-α-D-glucopyranosyl diphosphate) (118);
- Thymidine 5′-(4-acetamido-4-deoxy-α-D-glucopyranosyl diphosphate) (119);
- Uridine 5′-(4-acetamido-4-deoxy-α-D-glucopyranosyl diphosphate) (120);
- Thymidine 5′-(3-acetamido-3-deoxy-α-D-glucopyranosyl diphosphate) (121);
- Uridine 5′-(3-acetamido-3-deoxy-α-D-glucopyranosyl diphosphate) (122);
- Thymidine 5′-(2-acetamido-2-deoxy-α-D-glucopyranosyl diphosphate) (123);
- Uridine 5′-(2-acetamido-2-deoxy-α-D-glucopyranosyl diphosphate) (124);
- Thymidine 5′-(4-amino-4,6-dideoxy-α-D-glucopyranosyl diphosphate) (125);
- and Uridine 5′-(4-amino-4,6-dideoxy-α-D-glucopyranosyl diphosphate) (126).
A α-D-hexopyranosyl phosphate comprising at least one phosphate selected from the group consisting of deoxy-α-D-glucopyranosyl, aminodeoxy-α-D-hexapyranosyl, and acetamidodeoxy-α-D-hexopyranosyl phosphates.
A nucleotide sugar library comprising two or more nuceleotide sugars produced by combining α-D-hexopyranosyl phosphate and NTP in the presence of at least one mutated nucleotidylyltransferase.
A nucleotide sugar library comprising two or more nucleotide sugars produced by combining α-D-hexopyranosyl phosphate other than Glc1P and NTP in the presence of at least one nucleotidylyltransferase.
A nucleotide sugar library comprising two or more nuceleotide sugars produced by combining α-D-hexopyranosyl phosphate and NTP other than TTP in the presence of at least one nucleotidylyltransferase.
A nucleotide sugar library comprising two or more nuceleotide sugars selected from the group consisting of
- Thymidine 5′-(α-D-glucopyranosyl diphosphate) (58);
- Uridine 5′-(α-D-glucopyranosyl diphosphate) (59);
- Thymidine 5′-(2-deoxy-α-D-glucopyranosyl diphosphate) (60);
- Uridine 5′-(2-deoxy-α-D-glucopyranosyl diphosphate) (61);
- Thymidine 5′-(3-deoxy-α-D-glucopyranosyl diphosphate) (62);
- Uridine 5′-(3-deoxy-α-D-glucopyranosyl diphosphate) (63);
- Thymidine 5′-(4-deoxy-α-D-glucopyranosyl diphosphate) (64);
- Uridine 5′-(4-deoxy-α-D-glucopyranosyl diphosphate) (65);
- Thymidine 5′-(6-deoxy-α-D-glucopyranosyl diphosphate) (66);
- Uridine 5′-(6-deoxy-α-D-glucopyranosyl diphosphate) (67);
- Thymidine 5′-(α-D-mannopyranosyl diphosphate) (68);
- Uridine 5′-(α-D-mannopyranosyl diphosphate) (69);
- Thymidine 5′-(α-D-galactopyranosyl diphosphate) (70);
- Uridine 5′-(α-D-galactopyranosyl diphosphate) (71);
- Thymidine 5′-(α-D-allopyranosyl diphosphate) (72);
- Uridine 5′-(α-D-allopyranosyl diphosphate) (73);
- Thymidine 5′-(α-D-altropyranosyl diphosphate) (74);
- Uridine 5′-(α-D-altropyranosyl diphosphate) (75);
- Thymidine 5′-(α-D-gulopyranosyl diphosphate) (76);
- Uridine 5′-(α-D-gulopyranosyl diphosphate) (77);
- Thymidine 5′-(α-D-idopyranosyl diphosphate) (78);
- Uridine 5′-(α-D-idopyranosyl diphosphate) (79);
- Thymidine 5′-(α-D-talopyranosyl diphosphate) (80);
- Uridine 5′-(α-D-talopyranosyl diphosphate) (81);
- Thymidine 5′-(6-amino-6-deoxy-α-D-glucopyranosyl diphosphate) (109);
- Uridine 5′-(6-amino-6-deoxy-α-D-glucopyranosyl diphosphate) (110);
- Thymidine 5′-(4-amino-4-deoxy-α-D-glucopyranosyl diphosphate) (111);
- Uridine 5′-(4-amino-4-deoxy-α-D-glucopyranosyl diphosphate) (112);
- Thymidine 5′-(3-amino-3-deoxy-α-D-glucopyranosyl diphosphate) (113);
- Uridine 5′-(3-amino-3-deoxy-α-D-glucopyranosyl diphosphate) (114);
- Thymidine 5′-(2-amino-2-deoxy-α-D-glucopyranosyl diphosphate) (115);
- Uridine 5′-(2-amino-2-deoxy-α-D-glucopyranosyl diphosphate) (116);
- Thymidine 5′-(6-acetamido-6-deoxy-α-D-glucopyranosyl diphosphate) (117);
- Uridine 5′-(6-acetamido-6-deoxy-α-D-glucopyranosyl diphosphate) (118);
- Thymidine 5′-(4-acetamido-4-deoxy-α-D-glucopyranosyl diphosphate) (119);
- Uridine 5′-(4-acetamido-4-deoxy-α-D-glucopyranosyl diphosphate) (120);
- Thymidine 5′-(3-acetamido-3-deoxy-α-D-glucopyranosyl diphosphate) (121);
- Uridine 5′-(3-acetamido-3-deoxy-α-D-glucopyranosyl diphosphate) (122);
- Thymidine 5′-(2-acetamido-2-deoxy-α-D-glucopyranosyl diphosphate) (123);
- Uridine 5′-(2-acetamido-2-deoxy-α-D-glucopyranosyl diphosphate) (124);
- Thymidine 5′-(4-amino-4,6-dideoxy-α-D-glucopyranosyl diphosphate) (125);
- Uridine 5′-(4-amino-4,6-dideoxy-α-D-glucopyranosyl diphosphate) (126);
Dependencies are inferred from the claim text.