Transferases in Polymer Chemistry - Enzymatic Polymerisation - page 41

around 4 g HA per liter of the cultivated solution. At present, HA from various
sources, with different degrees of purity and molecular weights, is available for
medical applications.
HA

is synthesized by hyaluronan synthase (systematic name: alternating

UDP- α - N -acetyl- D -glucosamine: β - D -glucuronosyl-(1 → 3)-[nascent hyaluronan]

4- N -acetyl- β - D -glucosaminyltransferase and UDP- α - D -glucuronate: N -acetyl-

β - D -glucuronosyltransferase; EC

2.4.1.212) [ 223 - 226 ] . Hyaluronan synthase (HAS) is a single protein glycosyl-
transferase that is able to transfer two different monosaccharides, whereas most
glycosyltransferases catalyze one glycosidic transfer reaction exclusively.
Markovitz et al. successfully characterized the HAS activity from Streptococcus
pyogenes and discovered the enzyme's membrane localization and its requirements
for sugar nucleotide precursors and Mg 2 + [ 227 ]. DeAngelis et al. were the first to
succeed in the molecular cloning and characterization of the Group A Streptococcal
gene encoding the protein HasA, known to be in an operon required for bacterial HA
synthesis [ 228 , 229 ] . Following this, sequences of the genes encoding other HAS
proteins were identified using molecular biological techniques [ 220 , 224 , 225 , 230 -
243 ] . However, still little is known about the structure and mechanism of HAS.
The in vitro synthesis of HA oligomers and polymers using HAS and UDP-
sugars was reported by the group of Paul DeAngelis. The monosaccharide units
from UDP-GlcNAc and UDP-GlcA are transferred sequentially in an alternating
fashion to produce the disaccharide repeats of the heteropolysaccharide. Recombi-
nant derivatives of one HAS, PmHAS from the gram-negative bacterium Pasteurella
multocida type A [ 233 ] , have proved to be very useful for chemoenzymatic synthe-
ses of both oligosaccharides [ 244 ] and polysaccharides [ 245 , 246 ] .
In 2004, the PmHAS was employed in synchronized, stoichiometrically con-
trolled polymerization reactions in vitro to produce monodisperse HA polysaccha-
ride preparations [ 246 ]. Reaction synchronization is achieved by providing the HAS
with an oligosaccharide acceptor to bypass the slow polymer initiation step in vitro.
All HA chains are elongated in parallel and thus reach the same length, yielding a
population of narrow size distribution. The synthase adds all available UDP-sugar
precursors to the nonreducing termini of acceptors in a non-processive fashion, as
in the following equation:

- D -glucosaminyl-(1 → 4)-[nascent hyaluronan] 3- β

n ( UDP − GlcA )+ n ( UDP − GlcNAc )+ z ( GlcA − GlcNAc ) x → 2n ( UDP )

+ z ( GlcA − GlcNAc ) x +( n / z ) .

Therefore, size control is possible. For example, if there are many termini (i.e., z
is large), then a limited amount of UDP-sugars will be distributed among many
molecules and thus result in many short polymer chain extensions. Conversely, if
there are few termini (i.e., z is small), then the limited amount of UDP-sugars will be
distributed among few molecules and thus result in long polymer chain extensions.
With this, it became possible to synthesize highly defined HA polymer standards
that can be used for the characterization of polysaccharides by, for instance, size ex-
clusion chromatography equipped with a multi-angle light scattering detector [ 247 ] .