Engineered aryl sulfate-dependent enzymes

A method of enzymatically forming a 6-O-sulfated heparan sulfate 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) heparan sulfate, wherein the heparan sulfate comprises at least one polysaccharide selected from the group consisting of N-sulfated heparan sulfate (NS-HS), N-,2-O-sulfated heparan sulfate (N,2O-HS), 2-O sulfated heparan sulfate (2O-HS), and combinations thereof, and
• (iii) a non-natural glucosaminyl 6-O sulfotransferase enzyme (6OST), engineered to have sulfotransferase activity with an aryl sulfate compound as a sulfo group donor and heparan sulfate as a sulfo group acceptor,
• wherein the non-natural 6OST enzyme is a mutant of a natural 6OST enzyme within enzyme class EC 2.8.2-, the natural 6OST enzyme having sulfotransferase activity with heparan sulfate and a sulfo group donor, the sulfo group donor consisting of PAPS, to form a 6-O-sulfated heparan sulfate product,
• wherein the natural 6OST enzyme comprises a conserved amino acid sequence motif having the amino acid sequence, SEQ ID NO:
• 301, and within the amino acid sequence of the non-natural 6OST enzyme, amino acid sequence SEQ ID NO:
• 301 is mutated to an amino acid sequence motif selected from the group consisting of SEQ ID NO:
• 302, SEQ ID NO:
• 303, SEQ ID NO:
• 304, and SEQ ID NO:
• 305,
• 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 an amino acid sequence motif selected from the group consisting of SEQ ID NO:
• 306, SEQ ID NO:
• 307, SEQ ID NO:
• 308, and SEQ ID NO:
• 316, and
• wherein the natural 6OST enzyme comprises a conserved amino acid sequence motif having the amino acid sequence X13-T-R-A, wherein X13 is any amino acid, and the non-natural 6OST enzyme comprises an amino acid sequence motif selected from the group consisting of X13-T-R-A, wherein X13 is any amino acid, and a mutation to X13-T-X14-A, wherein X13 is any amino acid and X14 is alanine or glutamic acid;
• (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.