Vol.1, No.4, 97-111 (2013) Advances in Enzyme Research
Structural and functional evidence for two separate
oligosaccharide binding sites of Pasteurella
multocida hyaluronan synthase
Floor K. Kooy1,2, Hendrik H. Beeftink2*, Michel H. M. Eppink2, Johannes Tramper2,
Gerrit Eggink1,2, Carmen G. Boeriu1
1Food and Biobased Research, Wageningen University and Research Center, Wageningen, The Netherlands
2Bioprocess Engineering, Wageningen University and Research Center, Wageningen, The Netherlands;
*Corresponding Author: rik.beeftink@wur.nl
Received 28 June 2013; revised 12 August 2013; accepted 24 August 2013
Copyright © 2013 Floor K. Kooy et al. This is an open access article distributed under the Creative Commons Attribution License,
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Pasteurella multocida hyaluronan synthase
(PmHAS) is a bi-functional glycosyltransferase,
containing a β1,3-glucuronyltransferase and
β1,4-N-acetylglucosaminetransferase domain.
PmHAS catalyzes the elongation of hyaluronan
(HA) through the sequential addition of single
monosaccharides to the non-reducing end of
the hyaluronan chain. Research is focused on
the relation between the length of the HA oligo-
saccharide and the single-step elongation ki-
netics from HA4 up to HA9. It was found that the
turnover number kcat increased with length to
maximum values of 11 and 14 s1 for NAc- and
UA-transfer, respectively. Interestingly, the spe-
cificity constant kcat/KM increased with polymer
length from HA5 to HA7 to a value of 44 mM1·s1,
indicating an oligosaccharide binding site with
increasing specificity towards a heptasaccha-
ride at the UA domain. The value of kcat/KM re-
mained moderately constant around 8 mM1·s1
for HA4, HA6, and HA8, indicating a binding site
with significantly lower binding specificity at the
NAc domain than at the UA domain. These find-
ings are further corroborated by a structural
homology model of PmHAS, revealing two dis-
tinct sites for binding of oligosaccharides of
different sizes, one in each transferase domain.
Structural alignment studies between PmHAS
and glycosyltransferases of the GT-A fold
showed significant similarity in the binding of
the UDP-sugars and the orientation of the ac-
ceptor substrate. These similarities in substrate
orientation in the active site and in essential
amino acid residues involved in substrate bind-
ing were utilized to localize the two HA oligo-
saccharide binding sites.
Keywords: Pasteurella; Hyaluronan; Binding Site;
Polymerization; Co-Polymers
Enzymatic production of glycosaminoglycans has in-
creasingly attracted attention over the last two decades as
these polysaccharides are applied multifold in pharmacy
and cosmetics. For organ integrity and functioning, gly-
cosaminoglycans are essential since they activate signal-
ing pathways that control cell proliferation, differentia-
tion, adhesion, and migration [1,2]. The controlled pro-
duction of oligosaccharides with a defined chain length
and sulfate groups would constitute a breakthrough in
medical sciences with potential applications in, for ex-
ample, anti-cancer therapeutics [3] and disrupting viral
invasion and pathogenesis [1]. In nature, glycosami-
noglycans are produced by glycosyltransferases that
catalyze the transfer of an activated donor sugar to an
oligosaccharide acceptor.
One particular glycosaminoglycan is hyaluronan (HA),
an alternating copolymer of β3-N-acetylglycosamine
(GlcNAc) and β4-glucuronic acid (GlcUA). Following
the initial HA isolation from animal tissues [4-7], it was
ascertained that HA was also produced by a small num-
ber of microbial pathogens [8-10], employing HA as a
cloak in order to disguise themselves from the mammal-
ian immune system. Numerous cultivation procedures
have been developed to produce HA utilizing either these
pathogenic microorganisms [8,11,12] or safe recombi-
nant hosts [13-18], containing the hyaluronan synthases
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F. K. Kooy et al. / Advances in Enzyme Research 1 (2013) 97-111
(HAS) that synthesize HA. The quality of the product is
crucial since applications of HA depend on the oligo- or
polysaccharide length; consequently, the production of
HA with defined lengths is required. Innovative produc-
tion techniques aspire for high molecular weight HA
with a moderate length distribution, or polydispersity, by
inflicting stress through culture conditions [12,19-21] or
by avoiding HA degradation through hyaluronidase [13,
Recently, it was demonstrated that, for Streptococcus
zooepidemicus, overexpression of genes involved in
UDP-GlcNAc biosynthesis increased the molecular
weight of the HA products [22], indicating that the chain
length is controlled by the availability of the substrates.
This is corroborated by kinetic data from HAS enzymes
of different sources, demonstrating a significantly larger
KMNAc value than KMUA value [23-26]. Furthermore, the
hyaluronan synthase in Pasteurella multocida (PmHAS)
possesses the ability to elongate HA oligosaccharides
[27], which offers an additional opportunity to optimize
product length and polydispersity. The addition of HA
oligosaccharides to the reaction in the presence of both
UDP-sugars exacerbates the polymerization rate of
PmHAS and diminishes the polydispersity of the HA
products compared to reactions initiated with only the
UDP-sugars [28]. Although these findings have resulted
in increased molecular weight products with minimal
polydispersity, the kinetic elongation mechanism of HAS
enzymes supporting these results remains unknown.
The following is an investigation of single-step elon-
gation kinetics of various HA oligosaccharide chain
lengths (HA4 up to HA9). Several single-substrate models
were evaluated and kinetic parameters were determined
for each individual oligosaccharide. Binding affinities for
the UA-transferase site were ascertained to be considera-
bly higher than those for the NAc-transferase site and
two physically distinct binding sites were indicated and
supported by a structural homology model for PmHAS,
based on the crystal structure of chondroitin polymerase
K4CP [29]. Chondroitin polymerase and PmHAS have a
prominent sequence identity and sequence homology
(62% and 79%, respectively), resulting in a reliable
structural model for PmHAS. With the support of struc-
tural alignment studies, similarities in the active sites of
PmHAS and other glycosyltransferases have been ascer-
tained such as the location of acceptor binding sites and
several conserved amino acids involved in binding the
substrates. Conserved regions have been reported before
for UDP-sugar binding sites, whereas, in this study, we
have also determined structural similarities for the ac-
ceptor binding site. To summarize, this study presents
evidence for two distinct oligosaccharides binding sites
within PmHAS which affects the polydispersity of the
HA products.
2.1. Characterization of PmHAS
All reagents were purchased from either Fisher or
Sigma-Aldrich unless stated otherwise. Purified PmHAS
was provided by Merck & Co. (formerly Organon N.V.).
PmHAS represents the soluble PmHAS1-703 enzyme, as
described by Jing and DeAngelis[30], cloned and ex-
pressed in a pET101/D-TOPO expression vector (Invi-
trogen) with an additional V5 epitope and polyhistidine
(6x His) region at the C-terminal end of the enzyme.
PmHAS was purified from the crude extract employing
affinity chromatography on Ni-NTA columns (Qiagen).
A coupled-enzyme assay, similar to assays created for
other glycosyltransferases[23,31,32], was developed to
measure PmHAS activity. The coupled-enzyme assay
directly links the increase in the UDP by-product of the
PmHAS elongation to the decrease of NADH that was
spectrophotometrically measured at 340 nm. PmHAS
activity was measured varying one of the reaction condi-
tions, while keeping the others constant. The standard
reaction buffer incorporated 5 mM MgCl2, 112.5 mM
KCl, 1 M ethylene glycol, and 50 mMTris·HCl (pH 8.0)
and the assay components 60 U PK/ml, 75 U LDH/ml, 2
mM PEP, and 0.4 mM NADH. Bis-Tris·HCl was used
for experiments below pH 7, and Tris·HCl for the ex-
periments above or at pH 7. The following reaction con-
ditions were varied and measured with the coupled-en-
zyme assay: pH 5.6 - 9; temperature 20˚C - 40˚C; 5 mM
of either MgCl2, MnCl2, CoCl2, NiCl2, or CaCl2; MgCl2 5
- 50 mM; viscous buffer either 1 M trehalose, 1 M su-
crose, or 0.1 - 2 M ethylene glycol. Concentrations of
PmHAS, sugar nucleotides, and HA4 were kept constant
at 50 μg/ml, 5.5 mM and 0.1 mM, respectively. In ex-
periment with varying MgCl2 concentrations, substrate
concentrations were 1mM for both UDP-sugars and
0.1mM for HA4.
The reactions were measured at 35˚C for 20 min in
96-well150 μl UV star microplates (Greiner Bio-One,
Germany) and a temperature-controlled Safire spectro-
photometer (Tecan, Switzerland). Following measure-
ment of the absorbance reduction, reactions were discon-
tinued by 15 min of heating at 95˚C and then placed in
the freezer (20˚C) until analysis through gel electro-
phoresis. PmHAS activity was also examined for 1 and 5
hours of reaction at KCl concentrations ranging from 0 to
200 mM; because KCl is needed for PK activity, this ex-
periment was only analyzed through gel electrophoresis.
2.2. HA Product Analysis by Gel
Reaction mixtures were analyzed on 20% TBE poly-
acrylamide gels (Invitrogen) by gel electrophoresis and
Copyright © 2013 SciRes. OPEN ACCESS
F. K. Kooy et al. / Advances in Enzyme Research 1 (2013) 97-111 99
stained by Stains-All [33]. On the gels, Generuler DNA
ladder Ultra low range (Invitrogen) was employed as a
2.3. PmHAS Activity in Single-Step
Initial rates were evaluated at 35˚C for each one-step
elongation from HA4 up to HA
9 with the coupled-en-
zyme assay with 60 u PK/ml, 75 u LDH/ml, 2 mM
phosphoenolpyruvate, 0.4 mM NADH, 15 mM MgCl2,
112.5 mM KCl, 1 M ethylene glycol, 50 mMTris·HCl at
pH 8.0. Reaction mixtures contained a single purified
HA oligomer (obtained from Hyalose, L.L.C, USA) and
one tyoe of monomer. Single-step elongations of even-
numbered oligosaccharides as a substrateproceeded at
saturating UDP-GlcNAc concentrations of 40 mM in the
absence of UDP-GlcUA, while elongationsof odd-num-
bered oligomers were performed at saturating UDP-
GlcUA concentrations of 1 mM in the absence of UDP-
GlcNAc. This setup ascertained reactions to be sin-
gle-step elongations. After 5 min of incubation, the reac-
tion was initiated by addition of 5 μg/ml PmHAS and
various oligosaccharide concentrations (0.1, 0.5, 1, 2, 4,
or 6 mM). Reactions were discontinued by 15 min heat-
ing at 95˚C; enzymes were then removed with a Micro-
con YM-30 centrifugal filter unit (Millipore). Samples
were desalted with Dowex AG 50W-X8 (Bio-rad Labo-
ratories) before measuring product formation by Matrix
Assisted Laser Desorption/Ionization Time of Flight
Mass Spectrometry (MALDI-TOF MS).
For the MALDI-TOF MS analysis, an Ultraflex work-
station (BrukerDaltonics, Germany) with a 337 nm laser
was employed. The mass spectrometer was operated in
positive mode and calibrated with a mixture of malto-
dextrins (mass range 250 - 2500 Da). The laser irradiance
was adjusted between 29% and 32% of the full laser
power and, following a delayed extraction period of 200
ns. Ions were accelerated by a 25 kV voltage and de-
tected in the reflector mode. For data collection, 200
shots were used. Samples were diluted 10 times in a ma-
trix solution prepared with solution of 10 mg of 2,5-di-
hydroxybenzoic acid (DHB) in water. ; for analysis, 2 ml
of the mixture was transferred to a MALDI sample plate
and dried under a stream of warm air.
2.4. Analysis of Kinetic Data
Three one-substrate equations were matched to the
kinetic datato find the best fit. The Michaelis Menten,
Hill, and substrate inhibition equations [34,35] were fit-
ted in Excel with unweighted nonlinear regression:
max Mich aelis
 (3)
with v indicating the reaction rate by volume, vmax its
(virtual) maximum and equal to kcatE, kcat the turnover
number, E the enzyme concentration, HA the oligosac-
charide concentration, KM the saturation constant, KI the
inhibition constant. The uncertainties of the fits, standard
deviations of the parameters and the correlation matrices
were determined with the Excel SolverAid macro [36].
Goodness of fit for the three models was evaluated from
graphical plots, such as residual and normal probability
plots, and by analysis of the following goodness-of-fit
estimators: stability of the model; the corrected Akaike
criterion; Sy.x; the correlation between the estimated pa-
rameters kcat, KM, n or Ki; and the standard deviation of
these estimates [37,38].
2.5. Competition Studies
Competition at the oligosaccharide site was evaluated
by measuring the activity of, for example, the elongation
of an even-numbered oligosaccharide by UDP-GlcNAc
as a sugar donor in the presence of an odd-numbered HA
that cannot be extended by this donor sugar [39]. Activ-
ity was measured by the coupled-enzyme assay under
identical reaction conditions as used for kinetic studies
but also containing 10 mM UDP-GlcUA or 20 mM UDP-
GlcNAc. Reactions of HA4, HA5 and HA6 were indi-
vidually monitored in the presence of a competing oli-
gosaccharide with a molar ratio between the reacting and
competing oligosaccharides of 1:1 or 1:10. Reactions of
0.3 mM reacting oligosaccharide with the omission of
the competing oligosaccharide were taken as a reference.
2.6. Structure Homology Modeling
Model building and energy minimization of PmHAS
was performed with Modeler using the Accelrys Discov-
ery Studio 2.1 software package with K4CP as a template
structure. The protein model was validated with Pro-
files-3D (Accelrys Discovery Studio 2.1), the stereo-
chemical quality of the homology model was verified by
PROCHECK [40], and the protein folding was assessed
with PROSAII [41]. Docking studies of HA oligosaccha-
rides were performed with the program AutodockVina
[42]. Structural alignment was performed utilizing Dali-
Lite [43]. Structural alignment of enzymes was evaluated
by their root mean square deviation (RSMD) and Z-
scores. Low RSMD values (below 4.0 Å) and elevated
Z-scores (above 2) are an indication of a favorable
structural superimposition and may indicate a conserved
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F. K. Kooy et al. / Advances in Enzyme Research 1 (2013) 97-111
fold structure. All structural images were generated with
PyMOL version 0.99 (Delano Scientific LLC, San Carlos,
California, USA).
2.7. Polymerization Reactions with
Purified HA4 oligosaccharides were labeled with the
fluorophoreanthranillic acid at the reducing end of the
chain. Labeling and purification of HA4-fluor was ac-
complished as described before [33]. All reactions con-
tained 2 mM UDP-GlcUA, 40 mM UDP-GlcNAc, 15
mM MgCl2, 1 M ethylene glycol, and 50 mMTris·HCl at
pH 8.0. The HA4-fluor concentration was maintained at
saturating values (2.5 mM) or at subsaturating values
(0.1 mM). Reactions were initiated by adding either 15
or 30 μg/ml PmHAS and performed in 7.5 μl of reaction
volume in PCR eppendorf tubes. The reaction progress
was analyzed for 130 min at 30˚C; every 5 or 10 min, the
reaction of 1 sample tube was discontinued by freezing
in liquid nitrogen and maintaining it at 20˚C. Following
the experiments, all samples were heated for 15 min at
95˚C and analyzed on 20% TBE polyacrylamide gels; gel
images were processed as described elsewhere [33].
3.1. Kinetic Characteristics
A combination of kinetic characterization and struc-
tural modeling was employed in order to study the po-
lymerization of hyaluronan by Pasteurella multocida
hyaluronan synthase (PmHAS), focusing on the influ-
ence of the oligosaccharide length on the turnover num-
ber (kcat) and the specificity constant (kcat/KM). Kinetic
results are first introduced that characterize the optimal
conditions for PmHAS activity. To accomplish this, sin-
gle-step elongation kinetics were investigated, including
the influence of competing oligosaccharides on the po-
lymerization rate. Subsequently, a structural homology
model of PmHAS is considered, and structural relations
to other glycosyltransferases are then submitted. Utiliz-
ing the determined kinetic parameters, we demonstrate
that - due to two oligosaccharide binding sites - product
polydispersity increases at sub-saturating HA concentra-
3.1.1. Characterization of PmHAS
The polymerization activity of PmHAS was measured
to study optimal conditions for elongation with HA4 as
template and equimolar amounts of both UDP sugar sub-
strates. The reaction conditions that were varied included:
pH, temperature, the nature of the divalent ions and the
stabilizing buffer components. PmHAS activity has a pH
optimum between 7.5 and 9 (Figure 1(A)), with highest
activity at pH 8. Activity increased with increasing tem-
perature (Figure 1(B)) to 35˚C, after which activity de-
creases, probably due to enzyme inactivation. At optimal
pH and temperature, elevated molecular weight products
were obtained with a narrow product range (Figures 1(A)
and (B)). The nature of the divalent metal ions signify-
cantly affected the PmHAS activity; most efficient was
Mg2+, whereas the activity decreased to 70% with Mn2+
or Co2+, to 28% for Ni2+ and to 22% for Ca2+ (Figure
1(C)). PmHAS activity was optimal at 15 mM of MgCl2.
Bufferviscogens, such as ethylene glycol or trehalose,
not only influenced PmHAS activity, but also affected
product polydispersity (Figure 1(D)). Significantly lower
polydispersity and a high activity were found at 1 M of
ethylene glycol. Varying the ethylene glycol concentra-
tion from 0.1 up to 2 M resulted in minor changes in
PmHAS activity; activity was highest at 1 M ethylene
glycol (not shown). The following conditions were
therefore selected for kinetic experiments: pH 8.0, 35˚C,
15 mM Mg2+ and 1 M ethylene glycol.
PmHAS activity in cell membrane preparations has
been previously described with a maximum between
pH 6.8 and 7.6 and a 2 - 3 fold higher activity withMn2+
than with Mg2+ [44]. Differences in optimal conditions
are possibly a result of the use of isolated PmHAS in our
studies. Similar pH and temperature optima were re-
ported for hyaluronan synthases from Streptococcus
equisimilis (SeHAS) and Xenopuslaevis (XlHAS) [24,
45]. The metal ion preference of PmHAS is also compa-
rable with that observed for XlHAS. The most effective
divalent metal ion for XlHAS was Mg2+ with a 4- to
10-fold reduction with Mn2+, Ni2+ or Co2+ [24]. Al-
though the metal ion preference was not reported for
other HAS enzymes, the corresponding activity assays
included 15 - 20 mM of MgCl2 [25,26,46,47], suggesting
that Mg2+ is the preferred ion for HAS enzymes in gen-
eral. In addition, viscous compounds increased SeHAS
activity as well as PmHAS activity when employing eth-
ylene glycol and sucrose below 0.5 M; however, when
increasing the concentration of these viscogens, SeHAS
activity exhibited inhibition [45].
3.1.2. Influence of Oligosaccharide Length on
kcat and KM
HAn oligosaccharides, (with n ranging from 4 to 9),
were individually elongated in single-step reactions with
the corresponding sugar nucleotide in excess (UDP-
GlcNAc for even-numbered and UDP-GlcUA for odd-
numbered oligosaccharides). The non-reducing end,
where elongation occurs, contains a GlcUA sugar for the
even-numbered oligosaccharides, and, mutatis mutandis,
a GlcNAc sugar for the odd-numbered oligosaccharides.
The reaction progress was analyzed employing the cou-
led enzyme assay described in the Experimental section. p
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F. K. Kooy et al. / Advances in Enzyme Research 1 (2013) 97-111
Copyright © 2013 SciRes.
Figure 1. Specific activity of PmHAS under different reaction conditions. (A) pH dependency of PmHAS ac-
tivity; reaction products at pH 5.6, 7, 8 and 9 were analyzed on gel; most abundant and longest products were
observed at p H8. M indicates the Generuler DNA ladder; bars demonstrate the corresponding duplicates. (B)
Temperature dependency of PmHAS activity; reactions products at 20˚C, 25˚C, 35˚C and 40˚C were analyzed
on gel. Product size and amount concur with activity measurements; (C) Effect of divalent ions on PmHAS
activity. Best results were obtained with 15 mM Mg2+. (D) Effect of viscogens on PmHAS activity; sucrose
(S), trehalose (T) and ethylene glycol (E) were compared to a viscogen-less buffer as negative control (N.C.).
Gel analysis of reaction products show ethylene glycol to stimulate low polydispersity.
MALDITOF-MS confirmed the formation of the ex-
pected products HA(n + 1) in all reactions (data not shown).
Since the corresponding UDP-sugar is in excess, the
elongation can be considered as a one-substrate reaction
[34]. Three models for one substrate kinetics were used
for fitting the data by nonlinear regression: the Micha-
elis-Menten equation, the substrate-inhibition equation
and the Hill equation (see the Experimental section).
These models were selected to determine if PmHAS
elongates HA through classical Michaelis kinetics or if
the elongation was regulated by other mechanisms, such
as cooperativity or substrate inhibition.
Goodness-of-fit was judged statistically by examina-
tion of residual and normal probability plots, by evalua-
tion of standard deviations of the estimated parameters
kcat and KM, and by statistical tests such as the corrected
Akaike criterion. These statistical tests demonstrated that
all three models fitted well (Ta bl e 1). However, the Hill
equation was found to reduce to the Michaelis Menten
equation, since the estimated Hill number n equaled
unity for every reaction. In addition, the substrate inhibi-
tion model did not allow accurate estimation of kcat and
KM. The substrate inhibition model resulted in substantial
standard deviations for these parameters, and a very
strong correlation between kcat and KM varying from 0.90
to 0.99, which was not observed for the other models. In
summary, the Michaelis Menten equation was superior
for the present concentration range, although substrate
inhibition may possibly become relevant at elevated oli-
gosaccharide concentrations.
Experimental data and their Michaelis Menten fit are
depicted in Figure 2; the resulting values for kcat and KM
are given in Ta ble 2. To the best of our knowledge, it is
the first time that these kinetic parameters are reported
for individual oligosaccharides for any of the HAS en-
zymes. Globally, the value of the turnover number kcat is
seen to increase with oligosaccharide length; this in-
crease levels off at higher lengths. In addition, even-
numbered HA polymers seem to feature a lower kcat than
the next larger odd-numbered ones. Figure 3 illustrates
this behavior and shows a general increase with length as
well as (considering the relatively small standard devia-
tions) the difference in kcat values between odd-numbered
and even-numbered oligosaccharides. Since no literature
values for kcat are available as a reference, we use spe-
cific activity values for comparison. The UA-transferase
specific activity of approximately 450 μmol/mg·hr ob-
served for 1 mM HA7 or HA9 at 1 mM UDP-GlcUA
(Figure 2) are virtually equal to the reported value of
484 μmol/mg·hr for HA21 [39]. This indicates that the
maximal rate at the UA-transferase domain is attained
utilizing a heptasaccharide or longer. The NAc-trans-
ferase specific activities with values of 175 μmol/mg·hr
observed in our study for 1 mM HA8 at 1 mM
UDP-GlcNAc is seven times higher than reported [39].
Maximal NAc-transferase rates were only reached in our
studies at elevated UDP-GlcNAc concentration of 40
M; which is considerably higher than the value of m
F. K. Kooy et al. / Advances in Enzyme Research 1 (2013) 97-111
Figure 2. Effect of HA concentration and length on the specific activity of PmHAs. Elongation
rates for three even-numbered oligosaccharides, HA4 (), HA6 (), and HA8 (), and three
odd-numbered oligosaccharides, HA5 (), HA7 (), and HA9 (), were measured at varying
oligosaccharide concentrations from 0.1 to 6mM and the corresponding UDP-sugar in excess. The
lines, solid ones for HA4, HA8, HA5 and HA9 and dashed ones for HA6 and HA7, are Michaelis
Menten fits.
Ta bl e 1. Model selection on basis of goodness of fit for three
kinetic models: 1MichaelisMenten, Eq.1; 2 Hill, Eq.2; 3 sub-
strate inhibition, Eq.3 for single-step elongation of HA by
substrate eq. SSr AICcDAICc Parameter
correlation Sy.x
HA4 1 1607 67.8 0.0 0.81 12.7
2 1469 71.4 3.6 0.41 - 0.7712.8
3 1186 68.8 1.0 0.91 - 0.9711.5
1 9228 88.7 0.0 0.86 30.4
2 8713 92.8 4.1 0.66 - 0.8831.1
3 8369 92.3 3.6 0.94 - 0.9830.5
1 7511 85.7 0.0 0.91 26.7
2 6590 89.4 3.7 0.94 - 0.9927.1
3 7151 90.4 4.7 0.92 - 0.9728.2
1 6956 85.3 6.3 0.85 26.4
2 4429 84.6 5.6 0.34 - 0.7422.2HA5
3 2777 79.0 0.0 0.97 - 0.9917.6
1 18732 97.2 0.0 0.76 43.3
2 18609 101.94.7 0.39 - 0.7745.5
3 18732 102.04.8 0.79 - 0.9345.6
1 12289 81.1 0.0 0.82 39.2
2 9386 84.4 3.3 0.29 - 0.7036.6
3 10652 85.7 4.6 0.86 - 0.9539.0
Table 2. Parameter estimates and uncertainties for Michaelis
Menten kinetic constants for elongation reactions of various
lengths of hyaluronic acid.
Reaction substratekcat (s1) KM (mM) kcat/KM (mM1·s1)
HA4 4.6 ± 0.2 0.5 ± 0.1 8.7 ± 0.2
HA6 9.9 ± 0.7 0.9 ± 0.2 10.7 ± 0.2
HA8 10.8 ± 0.8 1.6 ± 0.3 6.9 ± 0.2
HA5 8.7 ± 0.5 0.8 ± 0.17 10.8 ± 0.2
HA7 14.0 ± 0.6 0.3 ± 0.07 44.4 ± 0.1
HA9 13.8 ± 0.7 0.4 ± 0.08 34.1 ± 0.2
polymer length (-)
kcat (s-1)
Figure 3. Relation between turnover number kcat and oligomer
length for odd-numbered and even-numbered oligomers. Lines
are for visual guidance only.
1 mM UDP-GlcNAc reported in literature [39].
Interestingly, there is a significant difference between
KM’s of the even-numbered and odd-numbered oligosac-
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F. K. Kooy et al. / Advances in Enzyme Research 1 (2013) 97-111 103
charides. The KM’s of the odd-numbered oligosaccha-
rides are decreasing with the increase of the chain length,
while, on the contrary, those for the even-numbered in-
crease. Understanding these trends is accomplished by
assessing the differences in the specificity constant
kcat/KM between even-numbered and odd-numbered oli-
gosaccharides. The specificity constant kcat/KM contains
the affinity 1/KM of the enzyme towards substrates [34,48]
and is seen to increases (Table 2) from 10.8 to 44.4
mM1·s1 for HA5 and HA7; after that, a slight decrease
is observed to 34.1 mM1·s1for HA9. This suggests that
the two sugar residues at the reducing end of HA7
(GlcUA2-GlcNAc1), compared to HA5, enhance the
binding to the oligosaccharide site by interacting more
strongly. The specificity constant of HA9 does not in-
crease further; apparently, it does not develop additional
interactions with the binding site. By investigating initial
polymerization rates of HA acceptor analogs up to hex-
asaccharides, Williams et al. [39] demonstrated that the
minimal oligosaccharide length for high-efficiency elon-
gation is comprised of, at the least, a trisaccharide with
two glucuronic acid residues. This indicates that the oli-
gosaccharide binding site at the UA-transferase site has
the capacity to bind a minimum of three saccharide resi-
dues, as suggested by Williams [39], and a maximum of
seven saccharide residues, as proposed in this study.
Contradictorily, the kcat/KM value for even-numbered
oligosaccharides is moderately constant ranging from 6.9
to 10.7 mM1·s1. The variance in binding specificity of
PmHAS for odd and even-numbered oligosaccharides
would be difficult to understand if there was only one
oligosaccharide binding site in PmHAS at which both
elongations (NAc and UA) would occur. These differ-
ences can only be explicated by two separate oligosac-
charide binding sites for odd and even-numbered oligo-
saccharides. A long oligosaccharide binding site at the
UA-transferase site could explain the increased specific-
ity constant for HA7, whereas the moderately constant
specificity constant of HA4, HA6 and HA8 can be ex-
plained by a short oligosaccharide binding site interact-
ing with the first four sugar residues of HA4, HA6 and
HA8 at the NAc-transferase site.
3.1.3. Competition Studies
To further examine the difference in binding specificity
for odd-numbered and even-numbered oligosaccharides,
we conducted competition studies to investigate 1) the
influence of HA5 as an inhibitor of HA4 elongation with
UDP-GlcNAc, 2) the influence of HA4 as an inhibitor of
HA5 elongation with UDP-GlcUA, and 3) the competi-
tion between HA4and HA6 for NAc-transferase activity.
For each individual step elongation, enzyme activity in
the absence of the competing oligosaccharide was meas-
ured as a reference. Two reactions were conducted with a
molar ratio between the reacting and competing oligo-
saccharide of 1:1 or 1:10 (Figure 4). Theoretically, if
only a single oligosaccharide binding site was evident,
the reacting and competing oligosaccharides would con-
currently bind, and are therefore compete for the site. If
two oligosaccharide binding sites were evident, a com-
peting oligosaccharide (HA4 or HA5) might still bind
non-productively at the elongation site, since these com-
petitors only differ from the reacting oligosaccharide in
the last sugar at the non-reducing end. In both cases, the
competing oligosaccharide binds at the reacting site, and
the measured activity should decrease compared to the
reference reaction.
The results in Figure 4(A) show that HA5 does not in-
fluence the reaction between HA4 with UDP-GlcNAc,
nor does HA4 create variation with the rates of the reac-
tion between HA5 with UDP-GlcUA. The absence of
competition confirms that there are two separate oligo-
saccharide binding sites within PmHAS, one for each
transferase activity, and that these binding sites are very
specific for their substrates. Previously, Williams et al.
[39] had also observed absence of competition for a par-
ticular class of longer oligosaccharides (HA14 and HA15).
Both HA4 and HA6 oligosaccharides can react with
UDP-GlcNAc at the same site, and this is confirmed by
the experimental outcomes. The reaction rate measured
in experiments with both substrates simultaneously
equals the sum of the individual elongation rates of HA4
and HA6 (Figure 4(B)). The specificity constants of HA4
and HA6 are generally the same, which is the result of
approximately twice as high KM and kcat values for HA6
compared to the KM and kcat values of HA4 (see Table 2).
In the reactions with 3 mM HA4, i.e. the equivalent of
KM of HA4, the kcat is almost attained for the HA4 reac-
tions, corresponding to a specific activity of 177 μmol/
mg·hr (see Figure 4 and the last two white bars in Fig-
ure 4(B)). The specific activity (see second to last white
bar in Figure 4(B)) increased, moreover, to 213 μmol/
mg·hr by the addition of 0.3 mM HA6. On the contrary,
the kcat of HA6 is not attained, since 3 mM HA6, the
equivalent of ~3*KM of HA6, is not sufficient to achieve
saturating concentrations (last two grey bars in Figure
4(B)). The results from the competition studies between
HA4 and HA6 thus demonstrate that elongation of these
two oligosaccharides is dependent on their concentration
levels and that, under the current conditions, the com-
bined rate is the sum of the individual rates.
3.2. Structural Characteristics
A three-dimensional model of PmHAS was con-
structed based on the recently obtained crystal structure
of K4CP chondroitin polymerase [29], which exhibits a
sequence identity of 62% and sequence homology of
79% compared to PmHAS. The Ramachandran plot ob-
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F. K. Kooy et al. / Advances in Enzyme Research 1 (2013) 97-111
Copyright © 2013 SciRes.
Figure 4. Influence of the competing oligosaccharide on the PmHAS
elongation reaction. (A) The light grey bars show the results of HA5
as competitor to the elongation reaction of HA4 with UDP-GlcNAc,
whereas the dark grey depicts the HA5 elongation reaction with
UDP-GlcUA and competing oligosaccharide HA4; (B) The results of
HA6 as “competing” oligosaccharide are shown in grey, and for HA4
in white.
tained from PROCHECK demonstrated that the stereo-
chemical quality of the model was very valuable. Only
two residues, Ala310 and Asn411, show slightly deviating
Phi and Psi angles that do not project consequences on
the overall structure; both of these residues are in flexible
loops. The structure of PmHAS residues 72 to 688 is
almost identical to the K4CP structure (cf. the overlay of
the two structures, Figure 5(A)); only the last 15 resi-
dues (689 - 703) could not be modeled. This structural
model thus allows localization of the two oligosaccharide
binding sites that were inferred from the kinetic studies.
PmHAS consists of three domains (Figure 5(B)): an
N-terminal domain (residues 72 to 134); the NAc-trans-
ferase-domain (residues 135 to 426); and the UA-trans-
ferase domain (residues 427 - 688). The N-terminal do-
main consists of a random coil and two α-helices, and the
first 71 residues are missing as no alignment could be
discerned for this N-terminal region. The NAc-trans-
ferase domain of PmHAS contains 13 α-helices and 12
β-strands. The UA-transferase domain consists of 10
α-helices and 12 β-strands as with the structure of K4CP.
Both the NAc-transferase and the UA-transferase do-
mains of PmHAS adopt the GT-A fold, which consists of
an α/β/α sandwich and is one of the fold types in glycol-
syltransferases [49] (Figure 5). The orientation of the
two active sites and the substantial distance of over 60 Å
make a single oligosaccharide binding site within
PmHAS unlikely (Figure 5).
The PmHAS model was further analyzed for structural
alignment with GT-A folded glycosyltransferases to
identify conserved regions in structure and sequence.
Conserved regions, such as amino acid residues involved
in binding substrates, are frequently important in enzyme
functionality. Below, similarities in substrate binding and
substrate orientation are employed to ascertain binding
sites of HA oligosaccharides in PmHAS. Various GT-A
folded glycosyltransferases were structurally aligned
with PmHAS (abbreviations in parentheses): UDP-
GalNAc:polypeptide α-N-acetylgalactosaminyl-transferase
T2 (hT2, [50]), β1,3-glucuronosyltransferase (GlcAT1,
[51]), β1,3-glucuronosyltransferase (GlcAT-P, [52]), β1,
4-galactosyltransferase (β4Gal-T1, [53]), α1,4-N-ace-
tylhexosaminlytransferase (EXTL2, [54]), α1,3-galac-
tosyltransferase (α3GT, [55]), blood group A α1,3-N-
galactosaminyltransferase (hGTA, [56]), α1,4-galacto-
syltransferase (LgtC, [57]).
3.2.1. UDP-Sugar Binding Site
Although the structurally aligned enzymes represent a
broad spectrum of glycosyltransferase reactions, regions
that bind the UDP-sugars are conserved, resulting in
strong similarities such as the orientation of the UDP-
sugar in the site (Figure 6). The DXD motif [58,59] is
extremely conserved within GT-A folded glycosyltrans-
ferases and forms a complex with divalent ions, such as
Mn2+ and Mg2+, which are crucial for UDP-sugar binding.
The DXD motif in PmHAS is defined as Asp247, Cys248
and Asp249 within the NAc-transferase site and Asp527,
Ser528, and Asp529 within the UA-transferase site. Muta-
tion studies within PmHAS have exhibited that varying
any of these Asp residues deactivates the transferase site
containing that DXD motif [60]. In addition, the DGS
motif in PmHAS contains an Asp residue that is con-
erved in certain aligned glycosyltransferases as well. s
F. K. Kooy et al. / Advances in Enzyme Research 1 (2013) 97-111 105
(A) (B)
Figure 5. Structural homology model of PmHAS. (A) Superimposition of the crystal structure of K4CP
chondroitin polymerase (green) and the PmHAS homology model (blue) with RMSD value and Z-score of
0.5Å and 53.4, respectively. Substrates UDP (orange) and UDP-GalNAc (yellow), depicted as sticks, and
Mn2+, as red dots, were originally present in the crystal structure. Secondary structural elements are indi-
cated by ribbons for α-helices, and arrows for β-strands. (B) Overall structure of PmHAS with UDP-GlcUA
(green) and UDP-GlcNAc (yellow) in sticks and Mn2+ as red dots. The structure is divided in three domains:
the N-terminal region (residues 72 - 134) in green, the UA-transferase (427 - 688) in orange and the NAc-
transferase (135 - 426) in blue.
This Asp residue interacts with the N3 of the uracyl
group within the UDP moiety. Mutation of Asp196 or
Asp477 in PmHAS resulted in loss of NAc- or UA-trans-
ferase activity [30], respectively, which emphasizes the
importance of these residues.
3.2.2. Acceptor Binding Site
Since PmHAS contains β1,3-glucuronyltransferase
and β1,4-N-acetylglucosaminyltransferase domains, the
aligned glycosyltransferases were selected on the basis of
the ability to elongate the acceptor at the 3OH or 4OH
group with the donor sugar. The structural alignments
indicate that the acceptor has distinct orientations to-
wards the UDP-sugar, depending on the type of linkage
formed following the sugar moiety transfer (Figure 6).
The α-configuration of the C1 in the sugar moiety at-
tached to UDP is preserved in the product after the con-
veyance by retaining glycosyltransferases, whereas in-
verting glycosyltransferases convert this into a β-linked
product. Consequently, the attacking OH group in the
acceptor in retaining enzymes is arranged sequentially to
the C1 of the sugar moiety, ready to form the α-linkage
(Figures 6(B) and (D)). Similarly, the attacking OH
group in inverting glycosyltransferases is positioned op-
posite to the α-linked UDP (Figures 6(A) and (C)). This
is emphasized by the reported hydrogen bonds in retain-
ing glycosyltransferases between the attacking OH group
in the acceptor and an oxygen atom of the β-phosphate
group in UDP [55-57,64]. These hydrogen bonds were
not evident in inverting enzymes, where the distance
between UDP and the acceptor is too extensive.
In addition to the similarities in acceptor orientation,
aromatic hydrophobic residues Phe, Trp, and Tyr are lo-
cated near every catalytic center. Their roles in the ac-
ceptor binding site appear to depend on their location and
orientation within the active site. Mutation studies in the
aligned glycosyltransferases illustrate that aromatic hy-
drophobic residues exhibit specific affinity towards the
acceptor [65,66], perform as a stabilizer of the transition
state [66], or assist in the orientation of the acceptor into
the correct position [53].
3.2.3. Oligosaccharide Positioning
Structural similarities among PmHAS and other gly-
cosyltransferases demonstrated that the HA oligosaccha-
ride should be positioned underneath the C1 of the sugar
moiety to form β-linkages, since PmHAS has two in-
verting transferase domains. AutodockVina [42] was
employed to model HA6 and HA7 into the NAc-trans-
ferase and UA-transferase sites (Figure 7), respectively.
The orientations of HA6 and HA7 (Figures 7(A) and (B))
concur with the results from Figure 6. Additionally, the
amino acids that are likely to interact with the substrates
are depicted as sticks (Figure 7).
Kinetic results indicated that there is a difference in
binding specificity between the NAc-transferase and
UA-transferase sites and that in the UA-transferase site
he specificity constant strongly increases upon elongation t
Copyright © 2013 SciRes. OPEN ACCESS
F. K. Kooy et al. / Advances in Enzyme Research 1 (2013) 97-111
Figure 6. Substrate orientation in active sites of structurally aligned glycosyltransferases of
the GT-A fold. Crystal structures of enzymes with both donor and acceptor substrates were
superimposed to investigate the orientation of the attacking OH group of the acceptor toward
the UDP-sugar. Grey arrows point out the direction of the reacting OH group to the C1 of the
sugar moiety. The DXD motif, the proposed catalytic residue and other conserved residues are
indicated. (A) inverting β1,3-transferases GlcAT-1 in dark blue (1fgg [51] and 1kws [61],
Galβ1-3Gal and UDP-GlcUA in green) and GlcAT-P in blue (1v84 [52], N-acetyllactosamine
and UDP in orange); (B) retaining α1,3-transferases α3GT in dark blue (1o7q [55] and 1g93
[62], N-acetyllactosamine and UDP-Gal in purple) and hGTA in blue (1lzi [56], H-antigen and
UDP in pink); (C) inverting β1,4-transferase β4Gal-T1 in dark blue (1tvy [63], UDP-Gal in
green) with three acceptors (chitotriose in orange, 2ah9 [53]; trisaccharideGlcNAcβ1,2-
Manα1,6-Manβ-OR (1,2-1,6-arm) in green, 2aec [53]; trisaccha-rideGlcNAcβ1,4-Manα1,
3-Manβ-OR (1,4-1,3-arm) in orange, 2agd); (D) retaining α1,4- transferases EXTL2 in dark
blue (1on8 and 1on6 [54], GlcUAβ1-3Galβ1-O-naphthale-nemethanol and UDP-GalNAc in
purple), and LgtC in blue (1ga8 [57], 4’-deoxylactose and UDP-2-deoxy-2fluoro-galactose in
from HA5 to HA7. Both results can be explained by the
structural model. First, the channel-shaped oligosac-
charide binding site in the UA-transferase site is longer
than in the NAc-transferase site (Figure 7(C)). At the
NAc-transferase site, only the first four sugar residues at
the non-reducing end of HA6 are in direct contact with
amino acid residues in the oligosaccharide site; other
sugar residues have considerable additional degrees of
freedom without amino acid interactions. In addition, all
sugar residues of HA7 at the UA-transferase site are en-
compassed by the oligosaccharide binding site, resulting
in a higher specificity constant than for HA5.
3.3. Polydispersity
Present kinetic parameters were utilized to monitor the
effect of HA4 concentration on the polydispersity of the
HA products over time. Two situations were selected,
specifically, a polymerization reaction with the concen-
tration of the oligosaccharide acceptor at (a) unsaturated
levels (KM/5) and at (b) saturated levels (5·KM). For both
reactions, the UDP-sugars were at saturated concentra-
tions of 2 mM UDP-GlcUA and 40 mM UDP-GlcNAc,
respectively. To visualize all products, HA4 labeled with
anthranillic acid (HA4-fluor) was used for polymerization.
Since in a previous study [33], we established that HA4-
Copyright © 2013 SciRes. OPEN ACCESS
F. K. Kooy et al. / Advances in Enzyme Research 1 (2013) 97-111 107
Figure 7. Docking of HA oligosaccharides in the PmHAS active sites. On top are the results shown for the UA-transferase site with a
heptasaccharide (HA7) modeled into the active site and, vice versa, the results for the NAc-transferase with a hexasaccharide (HA6)
below. The orientation of the acceptors towards the UDP-sugars is demonstrated in (A) and (B), and an overview of the PmHAS
struc- ture with the two acceptor binding sites is given in (C). The surface of PmHAS is made transparent to show the substrate ori-
entation and the amino acid residues that are most likely involved in substrate binding (shown in sticks).
fluor and HA4 are kinetically similar, we assumed that
HA4-fluor and HA4 have comparable KM values.
In Figure 8, the polydispersity is seen to increase over
time for both reactions with significantly more HA
products in reactions with unsaturated HA4-fluor con-
This result is in agreement with data of Mulders and
Beeftink, who theoretically demonstrated the size distri-
bution to be more prominent at higher reaction orders in
HA concentration [67]. The observed increase in
polydispersity over time is the effect of elongation at two
separate active sites. Oligosaccharide elongation in
PmHAS can only occur by the sequential binding, elon-
gation and release of the growing HA chain. Since the
two oligosaccharide binding sites in PmHAS are a con-
siderable distance from each other, the HA oligosaccha-
ride extended at one transferase site is not immediately
extended by the other. At unsaturated HA4-fluor concen-
trations, this effect is magnified because complex forma-
Figure 8. Influence of HA4-fluor concentration on product
polydispersity over time. Reactions were followed for 130 min
and analyzed on 20% TBE polyacrylamidegel. (a) Reactions
containing 0.1 mM HA4-fluor, 2 mM UDP-GlcUA, 40 mM
UDP-GlcNAc and 15 μg/ml PmHAS. (b) Reactions containing
2.5 mM HA4-fluor, 2 mM UDP-GlcUA, 40 mM UDP-GlcNAc
and 30 μg/ml PmHAS. M and M2 are other reactions with
HA4-fluor used as markers.
Copyright © 2013 SciRes. OPEN ACCESS
F. K. Kooy et al. / Advances in Enzyme Research 1 (2013) 97-111
tion between PmHAS and HA4-fluor is decelerated, re-
sulting in an increased polydispersity. Polydispersity of
HA products can be decreased by controlling the initial
concentrations of HA oligosaccharide and UDP-sugars.
Evidence is presented for two separate oligosaccharide
binding sites within PmHAS, one in each transferase
domain. Based on kinetic and structural analysis, we
demonstrate that the two transferase domains act as two
independent enzymes, where GlcNAc is transferred at
the NAc-transferase domain and GlcUA at the UA-
transferase domain. The transferase domains in PmHAS
indicate structural similarity with glycosyltransferases of
the GT-A fold. With structural alignment of these en-
zymes, we identified general characteristics of acceptor
binding sites in these enzymes that are useful in locating
oligosaccharide binding sites in other GT-A folded gly-
cosyltransferases. We propose two locations for these
oligosaccharide binding sites that are considerably dis-
tant from each other. As a result of this extensive dis-
tance, the number of products will increase over time in a
polymerization reaction unless single-step reactions are
imposed. The molecular weight and polydispersity of the
product can be controlled by the availability of the sub-
The authors thank Jan Springer and Aukje Zimmerman for the pro-
duction of PmHAS; Martinus van Boekel for helpful discussions on
statistical analysis; and Lambertus van den Broek for technical assis-
tance with MALDI-TOF MS experiments.
The project is a collaboration with Merck & Co. (formerly Organon
N.V.) and financially supported by the Netherlands Ministry of Eco-
nomic Affairs and B-Basic partners (www.b-basic.nl) through B-Basic,
a public-private NWO-ACTS program (ACTS = Advanced Chemical
Technologies for Sustainability).
[1] Gandhi, N.S. and Mancera, R.L. (2008) The structure of
glycosaminoglycans and their interactions with proteins.
Chemical Biology & Drug Design, 72, 455-482.
http://dx.doi.or g/ 10.1111/j.1747-0285.2008.00741.x
[2] Schaefer, L. and Schaefer, R.M. (2010) Proteoglycans:
From structural compounds to signaling molecules. Cell
and Tissue Research, 339, 237-246.
[3] Stern, R. (2008) Association between cancer and “acid
mucopolysaccharides”: An old concept comes of age,
finally. Seminars in Cancer Biology, 18, 238-243.
[4] Meyer, K. and Palmer, J.W. (1934) The polysaccharide of
the vitreous humor. Journal of Biological Chemistry, 107,
[5] Boas, N.F. (1949) Isolation of hyaluronic acid from the
cock’s comb. Journal of Biological Chemistry, 181, 573-
[6] Meyer, K. and Chaffee, E. (1941) The mucopolysac-
charides of skin. Journal of Biological Chemistry, 138,
[7] Chain, E. and Duthie, E.S. (1940) Identity of hyal-
uronidase and spreading factor. British Journal of
Experimental Pathology, 21, 324-338.
[8] Kendall, F.E., Heidelberger, M. and Dawson, M.H. (1937)
A serologically inactive polysaccharide elaborated by
mucoid strains of group A hemolytic streptococcus.
Journal of Biological Chemistry, 118, 61-69.
[9] Carter, G.R. and Annau, E. (1953) Isolation of capsular
polysaccharides for colonial variants of Pasteurella mul-
tocida. American Journal of Veterinary Research, 14,
[10] MacLennan, A.P. (1956) The production of capsules,
hyaluronic acid and hyaluronidase by 25 strains of group
C streptococci. Journal of General Microbiology, 15,
485-491. http://dx.doi.org/10.1099/00221287-15-3-485
[11] Thonard, J.C., Migliore, S.A. and Blustein, R. (1964)
Isolation of hyaluronic acid from broth cultures of strep-
tococci. Journal of Biological Chemistry, 239, 726-728.
[12] Armstrong, D.C. and Johns, M.R. (1997) Culture con-
ditions affect the molecular weight properties of
hyaluronic acid produced by Streptococcus zooepide-
micus. Applied & Environmental Microbiology, 63, 2759-
[13] Widner, B., et al. (2005) Hyaluronic acid production in
Bacillus subtilis. Applied & Environmental Microbiology,
71, 3747-3752.
[14] DeAngelis, P.L., Papaconstantinou, J. and Weigel, P.H.
(1993) Isolation of a Streptococcus pyogenes gene locus
that directs hyaluronan biosynthesis in acapsular mutants
and in heterologous bacteria. Journal of Biological Che-
mistry, 268, 14568-14571.
[15] Chien, L.-J. and Lee, C.-K. (2007) Hyaluronic acid
production by recombinant Lactococcus lactis. Applied
Microbiology and Biotechnology, 77, 339-346.
[16] Yu, H. and Stephanopoulos, G. (2008) Metabolic en-
gineering of Escherichia coli for biosynthesis of hy-
aluronic acid. Metabolic Engineering, 10, 24-32.
[17] Mao, Z. and Chen, R.R. (2007) Recombinant synthesis of
hyaluronan by agrobacterium sp. Biotechnology Progress,
23, 1038-1042.
[18] Mao, Z., Shin, H.-D. and Chen, R. (2009) A recombinant
E. coli bioprocess for hyaluronan synthesis. Applied
Microbiology and Biotechnology, 84, 63-69.
[19] Johns, M.R., Goh, L.-T. and Oeggerli, A. (1994) Effect of
pH, agitation and aeration on hyaluronic acid production
by Streptococcus zooepidemicus. Biotechnology Letters,
16, 507-512. http://dx.doi.org/10.1007/BF01023334
Copyright © 2013 SciRes. OPEN ACCESS
F. K. Kooy et al. / Advances in Enzyme Research 1 (2013) 97-111 109
[20] Huang, W.-C., Chen, S.-J. and Chen, T.-L. (2006) The
role of dissolved oxygen and function of agitation in
hyaluronic acid fermentation. Biochemical Engineering
Journal, 32, 239-243.
[21] Kim, J.-H., et al. (1996) Selection of a Streptococcus equi
mutant and optimization of culture conditions for the
production of high molecular weight hyaluronic acid.
Enzyme and Microbial Technology, 19, 440-445.
[22] Chen, W.Y., et al. (2009) Hyaluronan molecular weight is
controlled by UDP-N-acetylglucosamine concentration in
Streptococcus zooepidemicus. Journal of Biological Che-
mistry, 284, 18007-18014.
[23] Krupa, J.C., et al. (2007) Quantitative continuous assay
for hyaluronan synthase. Analytical Biochemistry, 361,
218-225. http://dx.doi.org/10.1016/j.ab.2006.11.011
[24] Pummill, P.E., Achyuthan, A.M. and DeAngelis, P.L.
(1998) Enzymological characterization of recombinant
Xenopus DG42, a vertebrate hyaluronan synthase. Jour-
nal of Biological Chemistry, 273, 4976-4981.
[25] Itano, N., et al. (1999) Three isoforms of mammalian
hyaluronan synthases have distinct enzymatic properties.
Journal of Biological Chemistry, 274, 25085-25092.
[26] Tlapak-Simmons, V.L., et al. (1999) Kinetic character-
ization of the recombinant hyaluronan synthases from
Streptococcus pyogenes and Streptococcus equisimilis.
Journal of Biological Chemistry, 274, 4246-4253.
[27] DeAngelis, P.L. (1999) Molecular directionality of poly-
saccharide polymerization by the Pasteurella multocida
hyaluronan synthase. Journal of Biological Chemistry,
274, 26557-26562.
[28] Jing, W. and DeAngelis, P.L. (2004) Synchronized che-
moenzymatic synthesis of monodisperse hyaluronan po-
lymers. Journal of Biological Chemistry, 279, 42345-
42349. http://dx.doi.org/10.1074/jbc.M402744200
[29] Osawa, T., et al. (2009) Crystal structure of chondroitin
polymerase from Escherichia coli K4. Biochemical and
Biophysical Research Communications, 378, 10-14.
[30] Jing, W. and DeAngelis, P.L. (2000) Dissection of the two
transferase activities of the Pasteurella multocida hy-
aluronan synthase: Two active sites exist in one po-
lypeptide. Glycobiology, 10, 883-889.
[31] Fitzgerald, D.K., et al. (1970) Enzymic assay for gala-
ctosyl transferase activity of lactose synthetase and [alpha]-
lactalbumin in purified and crude systems. Analytical
Biochemistry, 36, 43-61.
[32] Gosselin, S., et al. (1994) A continuous spectro-
photometric assay for glycosyltransferases. Analytical
Biochemistry, 220, 92-97.
[33] Kooy, F.K., et al. (2009) Quantification and character-
ization of enzymatically produced hyaluronan with
fluorophore-assisted carbohydrate electrophoresis. Analy-
tical Biochemistry, 384, 329-336.
[34] Cornish-Bowden, A. (1995) Fundamentals of enzyme
kinetics. Portland Press Ltd., London.
[35] Cook, P.F. and Cleland, W.W. (2007) Enzyme kinetics
and mechanism. Garland Science, London.
[36] De Levie, R. (2004) Macros for least-squares & for the
propagation of imprecision, in advanced excel for scien-
tific data analysis. Oxford University Press, New York.
[37] Van Boekel, M.A.J.S. (2010) Kinetic modeling of reac-
tions in foods. CRC Press, Boca Raton.
[38] Motulsky, H. and Christopoulos, A. (2003) Fitting models
to biological data using linear and nonlinear regression: A
practical guide to curve fitting. GraphPad Software Inc.,
San Diego.
[39] Williams, K.J., Halkes, K.M., Kamerling, J.P. and DeAn-
gelis, P.L. (2006) Critical elements of oligosaccharide
acceptor substrates for the Pasteurella multocida hyaluro-
nan synthase. Journal of Biological Chemistry, 281, 5391-
5397. http://dx.doi.org/10.1074/jbc.M510439200
[40] Laskowski, R.A., MacArthur, M.W., Moss, D.S. and Tho-
rnton, J.M. (1993) PROCHECK: A program to check the
stereochemical quality of protein structures. Journal of
Applied Crystalography, 26, 283-291.
[41] Sippl, M.J. (1993) Recognition of errors in three-dimen-
sional structures of proteins. Proteins: Structure, Func-
tion, and Genetics, 17, 355-362.
[42] Trott, O. and Olson, A.J. (2009) AutoDock Vina: Improving
the speed and accuracy of docking with a new scoring
function, efficient optimization, and multithreading. Jour-
nal of Computational Chemistry, 31, 455-461.
[43] Holm, L. and Sander, C. (1996) Mapping the protein uni-
verse. Science, 273, 595-602.
[44] DeAngelis, P.L. (1996) Enzymological characterization of
the Pasteurella multocida hyaluronic acid synthase. Bi o-
chemistry, 35, 9768-9771.
[45] Tlapak-Simmons, V.L., Baron, C.A. and Weigel, P.H. (2004)
Characterization of the purified hyaluronan synthase from
Streptococcus equisimilis. Biochemistry, 43, 9234-9242.
[46] Yoshida, M., Itano, N., Yamada, Y. and Kimata, K. (2000)
In Vitro synthesis of hyaluronan by a single protein
derived from mouse HAS1 Gene and characterization of
amino acid residues essential for the activity. Journal of
Biological Chemistry, 275, 497-506.
[47] Kumari, K. and Weigel, P.H. (1997) Molecular cloning,
expression, and characterization of the authentic hyalu-
ronan synthase from Group C Streptococcus equisimilis.
Copyright © 2013 SciRes. OPEN ACCESS
F. K. Kooy et al. / Advances in Enzyme Research 1 (2013) 97-111
Copyright © 2013 SciRes. OPEN ACCESS
Journal of Biological Chemistry, 272, 32539-32546.
[48] Eisenthal, R., Danson, M.J. and Hough, D.W. (2007) Ca-
talytic efficiency and kcat/KM: A useful comparator? Trends
in Biotechnology, 25, 247-249.
[49] Breton, C., Snajdrová, L., Jeanneau, C., Koca, J. and Im-
berty, A. (2006) Structures and mechanisms of glycosyl-
transferases. Glycobiology, 16, 29R-37R.
[50] Fritz, T.A., Raman, J. and Tabak, L.A. (2006) Dynamic
association between the catalytic and lectin domains of
human UDP-GalNAc: Polypeptide a-N-acetylgalactosami-
nyltransferase-2. Journal of Biological Chemistry, 281,
8613-8619. http://dx.doi.org/10.1074/jbc.M513590200
[51] Pedersen, L.C., Tsuchida, K., Kitagawa, H., Sugahara, K.,
Darden, T.A. and Negishi, M. (2000) Heparan/Chondroi-
tin sulfate biosynthesis. Structure and mechanism of human
glucuronyltransferase I. Journal of Biological Chemistry,
275, 34580-34585.
[52] Kakuda, S., Shiba, T., Ishiguro, M., Tagawa, H., Oka, S.,
Kajihara, Y., Kawasaki, T., Wakatsuki, S. and Kato, R.
(2004) Structural basis for acceptor substrate recognition
of a human glucuronyltransferase, GlcAT-P, an enzyme
critical in the biosynthesis of the carbohydrate epitope
HNK-1. Journal of Biological Chemistry, 279, 22693-
22703. http://dx.doi.org/10.1074/jbc.M400622200
[53] Ramasamy, V., Ramakrishnana, B., Boeggeman, E., Ratner,
D.M., Seeberger, P.H. and Qasba, P.K. (2005) Oligosac-
charide preferences of β1,4-galactosyltransferase-I: Crystal
structures of Met340His mutant of human β1,4-galac-
tosyltransferase-I with a pentasaccharide and trisaccha-
rides of the N-glycan moiety. Journal of Molecular Bio-
logy, 353, 53-67.
[54] Pedersen, L.C., Dong, J., Taniguchi, F., Kitagawa, H.,
Krahn, J.M., Pedersen, L.G., Sugahara, K. and Negishi, M.
(2003) Crystal structure of an 1,4-N-acetylhexosami-
nyltransferase (EXTL2), a member of the exostosin gene
family involved in heparan sulfate biosynthesis. Journal
of Biological Chemistry, 278, 14420-14428.
[55] Zhang, Y., Swaminathan, G.J., Deshpande, A., Boix, E.,
Natesh, R., Xie, Z.H., Acharya, K.R. and Brew, K. (2003)
Roles of individual enzyme—Substrate interactions by
α-1,3-galactosyltransferase in catalysis and specificity.
Biochemistry, 42, 13512-13521.
[56] Patenaude, S.I., Seto, N.O., Borisova, S.N., Szpacenko,
A., Marcus, S.L., Palcic, M.M. and Evans, S.V. (2002)
The structural basis for specificity in human ABO(H)
blood group biosynthesis. Nature Structural Biology, 9,
685-690. http://dx.doi.org/10.1038/nsb832
[57] Persson, K., Ly, H.D., Dieckelmann, M., Wakarchuk,
W.W., Withers, S.G. and Strynadka, N.C.J. (2001) Crystal
structure of the retaining galactosyltransferase LgtC from
Neisseria meningitidis in complex with donor and accep-
tor sugar analogs. Nature Structural Biology, 8, 166-175.
[58] Breton, C., Bettler, E., Joziasse, D.H., Geremia, R.A. and
Imberty, A. (1998) Sequence-Function relationships of
prokaryotic and eukaryotic galactosyltransferases. Jour-
nal of Biochemistry, 123, 1000-1009.
[59] Tarbouriech, N., Charnock, S.J. and Davies, G.J. (2001)
Three-Dimensional structures of the Mn and Mg dTDP
complexes of the family GT-2 glycosyltransferase SpsA:
A comparison with related NDP-sugar glycosyltransfe-
rases. Journal of Molecular Biology, 314, 655-661.
[60] Jing, W. and DeAngelis, P.L. (2003) Analysis of the two
active sites of the hyaluronan synthase and the chondroi-
tin synthase of Pasteurella multocida. Glycobiology, 13,
661-671. http://dx.doi.org/10.1093/glycob/cwg085
[61] Pedersen, L.C., Darden, T.A. and Negishi. M. (2002)
Crystal structure of b1,3-glucuronyltransferase I in com-
plex with active donor substrate UDP-GlcUA. Journal of
Biological Chemistry, 277, 21869-21873.
[62] Gastinel, L.N., Bignon, C., Misra, A.K., Hindsgaul, O.,
Shaper, J.H. and Joziass, D.H. (2001), Bovine a1,3-gala-
cto-syltransferase catalytic domain structure and its rela-
tionship with ABO histo-blood group and glycosphin-
golipid glycosyltransferases. EMBO Journal, 20, 638-649.
[63] Ramakrishnan, B., Boeggeman, E. and Qasba, P.K. (2004)
Effect of the Met344His mutation on the conformational
dynamics of bovine b-1,4-galactosyltransferase: Crystal
structure of the Met344His mutant in complex with chito-
biose. Biochemistry, 43, 12513-12522.
[64] Negishi, M., Donga, J., Dardenb, T.A., Pedersenb, L.G.
and Pedersen, L.C. (2003) Glucosaminylglycan biosyn-
thesis: What we can learn from the X-ray crystal struc-
tures of glycosyltransferases GlcAT1 and EXTL2. Bio-
chemical and Biophysical Research Communications, 303,
[65] Fondeur-Gelinotte, M., et al. (2007) Molecular basis for
acceptor substrate specificity of the human β1,3-glucu-
ronosyltransferases GlcAT-I and GlcAT-P involved in gly-
cosaminoglycan and HNK-1 carbohydrate epitope bio-
synthesis, respectively. Glycobiology, 17, 857-867.
[66] Zhang, Y., Deshpande, A., Xie, Z.H., Natesh, R., Acharya,
K.R. and Brew, K. (2004) Roles of active site tryptophans
in substrate binding and catalysis by α-1,3 galactosyl-
transferase. Glycobiology, 14, 1295-1302.
[67] Mulders, K.J.M. and Beeftink, H.H. (2013) Chain length
distribution and kinetic characteristics of an enzymatic-
cally produced polymer. e-Polymers, 24, 1-12.
F. K. Kooy et al. / Advances in Enzyme Research 1 (2013) 97-111 111
α3GT: α1,3-galactosyltransferase;
β4GAlT1: β1,4-galactosyltransferase;
EXTL2: α1,4-N-acetylhexosaminlytransferase;
GlcAT1: β1,3-glucuronosyltransferase;
GlcAT-P: β1,3-glucuronosyltransferase;
GlcNAc: N-acetylglucosamine;
GlcUA: glucuronic acid;
HA: hyaluronan;
HA4: hyaluronantetrasaccharide;
hGTA: blood group A
hT2: UDP-GalNAc:polypeptide
α-N-acetylgalactosaminyltranserase T2;
K4CP: K4CP chondroitin polymerase;
LDH: lactate dehydrogenase;
LgtC: α1,4-galactosyltransferase;
MALDI-TOF MS: Matrix-Assisted Laser Desorption/
Ionization Time of Flight Mass Spectrometry;
NAc-transferase domain: domain in PmHAS that
elongates UDP-GlcNAc to the oligosaccharide;
PmHAS: Pasteurella multocida hyaluronan synthase;
PK: pyruvate kinase;
SeHAS: Streptococcus equisimilishyaluronan synthase;
UA-transferase domain: domain in PmHAS that elon-
gates UDP-GlcUA to the oligosaccharide;
XlHAS: Xenopuslaevis hyaluronan synthase.
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