Structural and functional evidence for two separate oligosaccharide binding sites of Pasteurella mul

Vol.1, No.4, 97-111 (2013) Advances in Enzyme Research

http://dx.doi.org/10.4236/aer.2013.14011

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

which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

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 s−1 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 mM−1·s−1,

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 mM−1·s−1

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

1. INTRODUCTION

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

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,

18,21].

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. EXPERIMENTAL

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

Electrophoresis

Reaction mixtures were analyzed on 20% TBE poly-

acrylamide gels (Invitrogen) by gel electrophoresis and

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

marker.

2.3. PmHAS Activity in Single-Step

Elongations

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

Menten

vHA

vKHA





 (1)

max

Hill

vHA

vKHA





 (2)

max

substrate

inhibition

vHA

vKHAHAK





 (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

F. K. Kooy et al. / Advances in Enzyme Research 1 (2013) 97-111

100

fold structure. All structural images were generated with

PyMOL version 0.99 (Delano Scientific LLC, San Carlos,

California, USA).

2.7. Polymerization Reactions with

HA4-Fluor

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. RESULTS AND DISCUSSION

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-

tions.

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

F. K. Kooy et al. / Advances in Enzyme Research 1 (2013) 97-111

101

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

OPEN ACCESS

F. K. Kooy et al. / Advances in Enzyme Research 1 (2013) 97-111

102

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

PmHAS.

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

HA6

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

HA8

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

HA7

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

HA9

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 (s−1) KM (mM) kcat/KM (mM−1·s−1)

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 (-)

3456789

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-

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

mM−1·s−1 for HA5 and HA7; after that, a slight decrease

is observed to 34.1 mM−1·s−1for 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 mM−1·s−1. 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

6·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-

F. K. Kooy et al. / Advances in Enzyme Research 1 (2013) 97-111

104

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

OPEN ACCESS

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

F. K. Kooy et al. / Advances in Enzyme Research 1 (2013) 97-111

106

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

pink).

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-

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-

centrations.

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-

(A)

(B)

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.

F. K. Kooy et al. / Advances in Enzyme Research 1 (2013) 97-111

108

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.

4. CONCLUSION

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-

strates.

5. ACKNOWLEDGEMENTS

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).

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ABBREVIATIONS

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

α1,3-N-galactosaminyltransferase;

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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