Enzymatic Synthesis of Heparin - Sustainable Biotechnology: Sources of Renewable Energy - page 273

identified by Roberts' group to encode GlcNAc transferase activity, although the
purified protein was not obtained [48]. Our lab developed an effective approach to
express KfiA based on the published sequence [49]. The recombinant KfiA was har-
vested from bacterial culture at the yield of 10 mg/L. The substrate characterization
study concluded that KfiA has high specificity for the UDP-GlcNAc substrate. Also,
KfiA can efficiently transfer a GlcNAc group to an acceptor of various sizes, includ-
ing disaccharides. DeAngelis' group successfully identified and cloned heparosan
synthase pmHS1 [50] and pmHS2 [51] from P. multocida .UnlikeKfiA,pmHS1
and pmHS2 have both GlcNAc and GlcUA transferase activities although the sub-
strate specificities of pmHS1 and pmHS2 are believed to be distinct. The results
from these studies could provide a new approach for the synthesis of heparin/HS
backbone.
4.7 Alternative UDP Sugar Donor Substrates
UDP-sugar donors: UDP- N -acetylglucosamine (UDP-GlcNAc) and UDP-
glucuronic acid (UDP-GlcUA) are widely used in the synthesis of heparin/HS
oligosaccharide. The alternative UDP sugar donors can help us in the synthesis of
unnatural products with novel biological or chemical properties. Several unnatural
UDP-sugars have been synthesized and tested as substrates for GlcNAc or GlcUA
transferase. For example, DeAngelis and colleagues reported the enzyme-catalyzed
incorporation of unnatural UDP-sugar derivatives by pmHS1 and pmHS2 [58].
They found that pmHS1 required highly restricted donor structure, while pmHS2
was able to utilize several unnatural UDP-sugar analogs. For example, pmHS2 can
accept UDP sugars with acetamido-containing uronic acid as GlcUA donors and it
can tolerate glucosamine derivatives with longer acyl chain as GlcNAc donors. This
flexible specificity of pmHS2 could be used to prepare heparin/HS analogs with
novel structures. Recently, in order to study the specificity of different GlcUA trans-
ferases, Linhardt and colleagues have synthesized two UDP-GlcUA analogs: uridine
5 -diphosphoiduronic acid (UDP-IdoUA) and uridine 5 -diphosphohexenuronic
acid (UDP-HexUA) [59]. In this study, pmHS1 and pmHS2 were utilized as GlcUA
transferases. Unfortunately, their results demonstrated that UDP-HexUA failed to
serve as a substrate for pmHS1 and pmHS2. When UDP-IdoUA was used as the
substrate, sugar residues were transferred with low activity and only GlcUA was
incorporated into the products formed. According to the authors, this is either due to
the contamination of a small amount of UDP-GlcUA during the chemical synthesis
of UDP-IdoUA or UDP-IdoUA might be isomerized to UDP-GlcUA by the
GlcUA transferases via an unknown mechanism. These studies demonstrated the
potential application of unnatural UDP-sugars in the chemoenzymatic preparation
of synthetic HS/heparin.
In summary, our lab and other labs have demonstrated the potential of an
enzyme-based approach for the synthesis of HS. This approach is clearly capable of
preparing the HS with specific functions. The success of these efforts has improved