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 method of enzymatically forming an N-,2-O-sulfated heparan sulfate (N,2O-HS) product in the absence of 3′-phosphoadenosine 5′-phosphosulfate (PAPS), the method comprising the following steps:
a. forming a reaction mixture comprising:
i. a sulfo group donor, the sulfo group donor consisting of an aryl sulfate compound;
ii. N-sulfated heparosan; and
iii. a non-natural hexuronyl 2-O sulfotransferase enzyme (2OST), engineered to have sulfotransferase activity with an aryl sulfate compound as a sulfo group donor and N-sulfated heparosan as a sulfo group acceptor;
b. binding the aryl sulfate compound within the enzyme active site; and
c. catalyzing the transfer of the sulfo group from the aryl sulfate compound to N-sulfated heparosan, thereby forming the N,2O-HS product.
2. The method of claim 1 , wherein the amino acid sequence of the non-natural 2OST enzyme is mutated relative to the amino acid sequence of a natural 2OST enzyme within enzyme class EC 2.8.2.-, wherein:
a. the natural 2OST enzyme comprises the following conserved amino acid sequence motifs:
i. a conserved amino acid sequence motif having the amino acid sequence, SEQ ID NO: 244;
ii. a conserved amino acid sequence motif having the amino acid sequence, SEQ ID NO: 245;
iii. a conserved amino acid sequence motif having the amino acid sequence, SEO ID NO: 246;
iv. a conserved amino acid sequence motif having the amino acid sequence, 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 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.
3. The method of claim 2 , wherein within the amino acid sequence of the non-natural 2OST enzyme, the mutated amino acid sequence SEQ ID NO: 248 is selected, and amino acid sequence SEQ ID NO: 245 is mutated to SEQ ID NO: 251.
4. The method of claim 2 , wherein within the amino acid sequence of the non-natural 2OST enzyme, the mutated 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.
5. The method of claim 2 , 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.
6. The method of claim 1 , wherein the aryl sulfate compound is selected from the group consisting of p-nitrophenyl sulfate (PNS) and 4-nitrocatechol sulfate (NCS).
7. The method of claim 1 , wherein the reaction mixture further comprises a glucuronyl C 5 -epimerase.
8. The method of claim 7 , wherein the N,2O-HS product comprises at least one polysaccharide having a C 5 -epimerized hexuronic acid residue and comprising a sequence motif having the structure of Formula VII, below: