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-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.
2. The method according to claim 1 , wherein the N-deacetylated heparosan comprises N-acetyl glucosamine residues, and the N-acetyl glucosamine residues comprise less than 60% of the total glucosamine residues within the N-deacetylated heparosan.
3. The method according to claim 1 , wherein the N-deacetylated heparosan has a weight-average molecular weight in a range between and inclusive of 1,000 Da and 20,000 Da.
4. The method according to claim 3 , wherein the N-deacetylated heparosan has a weight-average molecular weight in a range between and inclusive of 9,000 Da and 12,500 Da.
5. A non-natural N-sulfotransferase enzyme (NST) enzyme engineered to have sulfotransferase activity with an aryl sulfate compound as a sulfo group donor and N-deacetylated heparosan as a sulfo group acceptor in the absence of 3′-phosphoadenosine 5′-phosphosulfate (PAPS), wherein the 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.
6. A nucleic acid molecule comprising a nucleotide sequence encoding for the amino acid sequence of the NST enzyme of claim 5 .
7. The nucleic acid molecule according to claim 6 , wherein the nucleic acid molecule is comprised within an expression vector.
8. The nucleic acid molecule according to claim 7 , wherein the expression vector further encodes for a fusion protein comprising the non-natural NST enzyme and one or more additional polypeptides.
9. The nucleic acid molecule according to claim 7 , wherein the expression vector is comprised within an isolated host cell, selected from the group consisting of a bacterial cell, a yeast cell, an insect cell, and a mammalian cell.