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 a 6-O-sulfated heparan sulfate product, 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) heparan sulfate, wherein the heparan sulfate comprises N-,2-O-sulfated heparan sulfate (N,2O-HS); and
(iii) a non-natural glucosaminyl 6-O sulfotransferase enzyme (6OST), engineered to have sulfotransferase activity in the absence of 3′-phosphoadenosine 5′-phosphosulfate (PAPS), wherein the sulfotransferase activity comprises a transfer of the sulfo group from the aryl sulfate compound to heparan sulfate;
(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 the heparan sulfate, thereby forming the 6-O-sulfated heparan sulfate product.
2. The method of claim 1 , wherein the non-natural 6OST enzyme has an amino acid sequence comprising multiple mutations relative to conserved amino acid residues found in natural 6OST enzymes within enzyme class EC 2.8.2.-, wherein:
natural 6OST enzymes have sulfotransferase activity with heparan sulfate and a sulfo group donor, the sulfo group donor consisting of PAPS, to form the 6-O-sulfated heparan sulfate product; and
the amino acid sequence of the non-natural 6OST enzyme has at least 80% sequence identity with the amino acid sequence of a natural 6OST enzyme, the natural 6OST enzyme amino acid sequence selected from the group consisting of SEQ ID NO: 191, SEQ ID NO: 199, and SEQ ID NO: 201.
3. The method of claim 2 , wherein:
the natural 6OST enzyme comprises a conserved amino acid sequence motif having the amino acid sequence, SEQ ID NO: 254; and
within the amino acid sequence of the non-natural 6OST enzyme, amino acid sequence SEQ ID NO: 254 is mutated to SEQ ID NO: 257.
4. The method of claim 3 , wherein:
the natural 6OST enzyme comprises a conserved amino acid sequence motif having the amino acid sequence, SEQ ID NO: 256, and
within the amino acid sequence of the non-natural 6OST enzyme, amino acid sequence SEQ ID NO: 256 is mutated to SEQ ID NO: 260.
5. The method according to claim 2 , wherein the 6-O-sulfated heparan sulfate product comprises N-,2-O,6-O-sulfated heparan sulfate (N,2O,6O-HS).
6. The method according to claim 5 , wherein the N,2O,6O-HS product comprises at least one polysaccharide comprising a sequence motif having the structure of Formula X, below:
wherein X is either a sulfo group or an acetate group, and Y is either a sulfo group or a hydroxyl group.
7. The method of claim 2 , wherein the aryl sulfate compound is selected from the group consisting of p-nitrophenyl sulfate and 4-nitrocatechol sulfate.
8. The method of claim 2 , wherein the non-natural 6OST enzyme has at least 90% sequence identity with the amino acid sequence of the natural 6OST enzyme.
9. The method of claim 8 , wherein the natural 6OST enzyme amino acid sequence is SEQ ID NO: 191.