Engineered aryl sulfate-dependent enzymes
The present invention provides several non-naturally occurring sulfotransferase enzymes that have been engineered to react with aryl sulfate compounds as sulfo group donors, instead of the natural substrate 3′-phosphoadenosine 5′-phosphosulfate (PAPS), and with heparosan-based polysaccharides, particularly heparan sulfate, as sulfo group acceptors. Each of the engineered sulfotransferase enzymes have a biological activity characterized by the position within the heparosan-based polysaccharide that receives the sulfo group, including glucosaminyl N-sulfotransferase activity, hexuronyl 2-O sulfotransferase activity, glucosaminyl 6-O sulfotransferase activity, or glucosaminyl 3-O sulfotransferase activity. Methods of using the engineered sulfotransferases to produce sulfated heparosan-based polysaccharides, including polysaccharides having anticoagulant activity, are also provided.
1. A non-natural hexuronyl 2-O sulfotransferase (2OST) enzyme having an amino acid sequence comprising multiple mutations relative to conserved amino acid residues and sequence motifs found in natural 2OST enzymes within enzyme class EC 2.8.2.-, wherein:
(a) the natural 2OST enzymes comprise the following conserved amino acid sequence motifs:
(i) a conserved amino acid sequence motif having the amino acid sequence of SEQ ID NO: 244;
(ii) a conserved amino acid sequence motif having the amino acid sequence of SEQ ID NO: 245;
(iii) a conserved amino acid sequence motif having the amino acid sequence of SEQ ID NO: 246;
(iv) a conserved amino acid sequence motif having the amino acid sequence of SEQ ID NO: 247; and
(b) within the amino acid sequence of the non-natural 2OST enzyme,
(i) amino acid sequence SEQ ID NO: 244 is mutated to an amino acid sequence selected from the group consisting of SEQ ID NO: 248 and SEQ ID NO: 249, and
(ii) amino acid sequence SEQ ID NO: 247 is mutated to SEQ ID NO: 250; and
(c) the non-natural 2OST enzyme has sulfotransferase activity in the absence of 3′-phosphoadenosine 5′-phosphosulfate, comprising a transfer of a sulfo group from an aryl sulfate compound to N-sulfated heparosan to form an N-,2-O-sulfated heparan sulfate product.
2. The non-natural 2OST enzyme of claim 1 , wherein within the amino acid sequence of the non-natural 2OST enzyme, amino acid sequence SEQ ID NO: 248 is selected, and amino acid sequence SEQ ID NO: 245 is mutated to SEQ ID NO: 251.
3. The non-natural 2OST enzyme of claim 1 , wherein within the amino acid sequence of the non-natural 2OST enzyme, the amino acid sequence SEQ ID NO: 249 is selected, amino acid sequence SEQ ID NO: 245 is mutated to SEQ ID NO: 252, and amino acid sequence SEQ ID NO: 246 is mutated to SEQ ID NO: 253.
4. The non-natural 2OST enzyme of claim 1 , wherein the non-natural 2OST enzyme comprises an amino acid sequence selected from the group consisting of the amino acid sequences SEQ ID NO: 63, SEQ ID NO: 65, SEQ ID NO: 68, and SEQ ID NO: 69.
5. The non-natural 2OST enzyme of claim 1 , wherein the amino acid sequence of the non-natural 2OST enzyme has at least 80% sequence identity with the amino acid sequence of a natural 2OST enzyme, the natural 2OST enzyme having amino acid sequence SEQ ID NO: 179.
6. The non-natural 2OST enzyme of claim 1 , wherein the aryl sulfate compound is selected from the group consisting of p-nitrophenyl sulfate and 4-nitrocatechol sulfate.
7. A non-natural hexuronyl 2-O sulfotransferase (2OST) enzyme engineered to have sulfotransferase activity in the absence of 3′-phosphoadenosine 5′-phosphosulfate (PAPS), the sulfotransferase activity comprising a transfer of a sulfo group from an aryl sulfate compound to a heparosan-based polysaccharide to form a 2-O-sulfated heparan sulfate product, wherein the heparan sulfate comprises N-sulfated heparan sulfate, and the 2-O -sulfated heparan sulfate product comprises N-,2-O-sulfated heparan sulfate,
wherein the amino acid sequence of the non-natural 2OST enzyme comprises at least one amino acid sequence motif selected from the group consisting of SEQ ID NO: 248, SEQ ID NO: 249, and SEQ ID NO:250.
8. The non-natural 2OST enzyme of claim 7 , wherein the non-natural 2OST enzyme has an amino acid sequence comprising multiple mutations relative to conserved amino acid residues found in natural 2OST enzymes within enzyme class EC 2.8.2.-, wherein:
natural 2OST enzymes have sulfotransferase activity with heparan sulfate and a sulfo group donor, the sulfo group donor consisting of PAPS, to form a 2-O-sulfated heparan sulfate product; and
the amino acid sequence of the non-natural 2OST enzyme has at least 80% sequence identity with the amino acid sequence of a natural 2OST enzyme, the natural 2OST enzyme having amino acid sequence SEQ ID NO: 179.
9. The non-natural 2OST enzyme of claim 8 , wherein
the amino acid sequence of the non-natural 2OST enzyme comprises an amino acid sequence motif having an amino acid sequence selected from the group consisting of SEQ ID NO: 248 and SEQ ID NO: 249.
10. The non-natural 2OST enzyme of claim 8 , wherein
the amino acid sequence of the non-natural 2OST enzyme comprises the amino acid sequence motif having the amino acid sequence of SEQ ID NO: 250.
11. The non-natural 2OST enzyme of claim 8 , wherein the aryl sulfate compound is selected from the group consisting of p-nitrophenyl sulfate and 4-nitrocatechol sulfate.
12. The non-natural 2OST enzyme of claim 7 , wherein the amino acid sequence of the non-natural 2OST enzyme comprises an amino acid sequence motif having an amino acid sequence selected from the group consisting of SEQ ID NO: 248 and SEQ ID NO: 249.
13. The non-natural 2OST enzyme of claim 7 , wherein the amino acid sequence of the non-natural 2OST enzyme comprises the amino acid sequence motif having the amino acid sequence of SEQ ID NO: 250.
14. The non-natural 2OST enzyme of claim 7 , wherein the aryl sulfate compound is selected from the group consisting of p-nitrophenyl sulfate and 4-nitrocatechol sulfate.