Engineered aryl sulfate-dependent enzymes

A method of enzymatically forming an N-sulfated heparosan 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-deacetylated heparosan;
• and
• iii. a non-natural N-sulfotransferase enzyme (NST), engineered to have sulfotransferase activity with an aryl sulfate compound as a sulfo group donor and N-deacetylated heparosan as a sulfo group acceptor, wherein the non-natural NST enzyme comprises an amino acid sequence selected from the group consisting of SEQ ID NO:
• 5, SEQ ID NO:
• 7, SEQ ID NO:
• 9, SEQ ID NO:
• 11, SEQ ID NO:
• 13, SEQ ID NO:
• 15, SEQ ID NO:
• 18, SEQ ID NO:
• 19, SEQ ID NO:
• 20, SEQ ID NO:
• 21, SEQ ID NO:
• 22, SEQ ID NO:
• 23, SEQ ID NO:
• 24, and SEQ ID NO:
• 25;
• 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-deacetylated heparosan, thereby forming the N-sulfated heparosan product.