2007 A4 HPLC Assay Anal Biochem

8/6/2019 2007 A4 HPLC Assay Anal Biochem

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ANALYTICAL

BIOCHEMISTRY

Analytical Biochemistry 363 (2007) 128134

www.elsevier.com/locate/yabio

0003-2697/$ - see front matter 2007 Elsevier Inc. All rights reserved.

doi:10.1016/j.ab.2006.12.044

Natural substrate assay for chitinases using high-performance liquidchromatography: A comparison with existing assays

Inger-Mari Krokeide, Bjrnar Synstad, Sigrid Gseidnes, Svein J. Horn,Vincent G.H. Eijsink, Morten Srlie

Department of Chemistry, Biotechnology and Food Science, Norwegian University of Life Sciences, P.O. Box 5003, N-1432 Aas, Norway

Received 7 November 2006

Available online 4 January 2007

Abstract

The determination of kinetic parameters of chitinases using natural substrates is diYcult due to low Km values, which require the use

of low substrate concentrations that are hard to measure. Using the natural substrate (GlcNAc)4, we have developed an assay for the

determination ofkcat and Kmvalues of chitinases. Product concentrations as low as 0.5 M were detected using normal-phase high-perfor-

mance liquid chromatography (HPLC) with an amide 80 column (0.20 25 cm) using spectrophotometric detection at 210nm. By means

of this assay, kcat and Kmvalues for chitinases A (ChiA) and B (ChiB) ofSerratia marcescens were found to be 331 s1 and 91 M and

282 s1 and 4 2M, respectively. For ChiB, these values were compared to those found with commonly used substrates where the

leaving group is a (nonnatural) chromophore, revealing considerable diVerences. For example, assays with 4-methylumbelliferyl-(Glc-

NAc)2 yielded a kcat value of 18 2 s1 and a Km value of 306 M. For two ChiB mutants containing a Trp ! Ala mutation in the +1

or +2 subsites, the natural substrate and the 4-methylumbelliferyl-(GlcNAc)2 assays yielded rather similar Km values (5-fold diVerence at

most) but showed dramatic diVerences in kcat values (up to 90-fold). These results illustrate the risk of using artiWcial substrates for char-

acterization of chitinases and, thus, show that the new HPLC-based assay is a valuable tool for future chitinase research. 2007 Elsevier Inc. All rights reserved.

Keywords: Enzymatic assay; Chitinase; Natural substrate; HPLC

Chitin, a -1,4-linked polymer of N-acetylglucosamine

(GlcNAc), is an abundant biopolymer in nature. It is the

most important nonplant structural biopolymer, occurring

in, e.g., the exoskeletons of invertebrates, the cell walls of

fungi, and the digestive tracts of insects. Chitin is easily

derived from waste products such as shrimp shells. So far,

chitin is primarily used as a source for chitosan, a partiallydeacetylated soluble form of chitin, and for glucosamine.

Fragments of chitin or chitosan (chitooligosaccharides)

may inhibit certain chitin-degrading enzymes, giving them

potential as fungicides [1], insecticides [24], and antimala-

rials [5,6]. Chitooligosaccharides are environmentally

friendly because of their fast degradation in nature and,

since chitin does not occur in humans, chitin metabolism is

an interesting target area for development of drugs and

pesticides.

Chitin does not accumulate in nature because the poly-

mer is eVectively degraded by diVerent chitinases belonging

to the glycoside hydrolase enzyme families 18 and 19 [7].

Serratia marcescens has an eYcient chitinolytic machinery

and, when grown on chitin, three family 18 chitinases areexpressed: chitinases A (ChiA), B (ChiB), and C (ChiC) [8].

ChiA and ChiB are processive chitinases that digest the

chitin polymer in opposite directions producing mainly

(GlcNAc)2, while ChiC is a nonprocessive endochitinase

that hydrolyzes the polymer randomly, yielding longer chi-

tooligosaccharides [9,10]. The S. marcescens chitinases have

been characterized in several studies [811], using a variety

of substrates.

Kinetic analysis of chitinases is usually conducted with

artiWcial substrates such as 4-methylumbelliferyl-(GlcNAc)2

* Corresponding author. Fax: +47 64965901.

E-mail address:[email protected] (M. Srlie).
mailto:%[email protected]:%[email protected]:%[email protected]


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Natural substrate assay for chitinases using HPLC / I.-M. Krokeide et al. / Anal. Biochem. 363 (2007) 128134 129

((GlcNAc)2-4MU), (GlcNAc)3-4MU, and p-nitrofenyl-

(GlcNAc)2 ((GlcNAc)2-pNP). These substrates are not

optimal because they present the enzyme with nonnatural

leaving groups and because of substrate inhibition [12].

Because of the length and the nature of the leaving groups,

non-natural substrates are of very limited use when assess-

ing the eVects of mutations meant to aVect natural sub-strate degradation. In addition, the leaving groups 4-MU

and pNP are corresponding bases to weak acids with pKavalues of 7.8 [13] and 7.2 [14], respectively, while acid disso-

ciation constants for sugars areV 12 [15]. At high pH (Glc-

NAc)2-4MU and (GlcNAc)2-pNP yield negatively charged

leaving groups while this will not be the case for a sugar

leaving group. Kinetic characterization with natural sub-

strates is diYcult because (1) low Km values require detec-

tion of low concentrations of substrate and product, (2)

most longer substrates contain more than one hydrolysable

glycosidic bond, (3) degradation products longer than

dimers are also substrates, and (4) some chitinases degrade

longer substrates processively. With respect to the latter

two problems, (GlcNAc)4 is an exception since most chitin-

ases convert this compound exclusively to two dimers,

which are not degraded any further.

We present a chitinase assay based on the use of the natu-

ral substrate (GlcNAc)4 and the use of a sensitive HPLC

setup to monitor substrate and product concentrations. We

have compared the natural substrate assay with existing

assays using wild-type ChiA and ChiB from S. marcescens

and two engineered variants of ChiB. The engineered enzymes

are ChiB-W97A and ChiB-W220A. Trp97 is located in the +1

subsite of the active site of ChiB while Trp220 is located in the

+2 subsite [16,17]. Mutation of these residues will thus aVectthe enzymes interaction with the leaving group.

Materials and methods

Chemicals

Tetra-N-acetylchitotetraose, 4-methylumbelliferyl-di-N-

acetylchitobiose, 4-methylumbelliferyl-tri-N-acetylchitotrii-

ose, para-nitrophenyl-di-N-acetylchitobiose, and acetonitrile

were purchased from SigmaAldrich.

Production and puriWcation of chitinases

ChiA [18], ChiB [19], and mutants of ChiB were puriWed

from periplasmatic extracts of the producer strains by

hydrophobic interaction chromatography, as described

previously [12]. Enzyme purity was veriWed using SDS/

PAGE and was above 95% in all cases. Protein concentra-

tions were determined using the Bradford assay kit

provided by Bio-Rad (Hercules, CA, USA).

Chromatography of chitooligosaccharides

Mixtures of (GlcNAc)4 and (GlcNAc)2 were separated

by normal-phase HPLC using a Tosoh TSK Amide 80

column (0.2025 cm) with an amide 80 guard column. The

sample size was 50L and the chitooligosaccharides were

eluted isocratically at 0.25mL/min with 70% (v/v) acetoni-

trile at room temperature. The chitooligosaccharides were

monitored by measuring absorbance at 210nm and the

(GlcNAc)4 concentrations were quantiWed by measuring

peak areas and by comparing these to those of standardsamples with known concentrations of (GlcNAc)4. Using

these standard samples, it was established that there was a

linear correlation between the peak area and the analyzed

(GlcNAc)4 concentration within the concentration range

0.5300M used in this study.

(GlcNAc)4 assay

Reactions were started by adding 0.5nM ChiA or ChiB

or 1.0 nM ChiB-W220A or 2.0nM ChiB-W97A (Wnal con-

centrations) to 1-mL solutions containing 100, 80, 60, 40,

20, 10, 5, or 2M (for ChiA and ChiB), 500, 400, 300, 200,

100, 50, or 25 M (for ChiBW220A), and 1000, 600, 400,

200, 100, 50, or 25 M (for ChiB-W97A) (GlcNAc)4 in

20 mM NaAc buVer, pH 6.1, and 0.1 mg/mL bovine serum

albumin (Wnal concentrations). After enzyme addition, 50-

L aliquots were transferred to a HPLC vial containing

150L of acetonitrile at appropriate time points. Reaction

conditions and timing were such that the (GlcNAc)4 con-

centration in the sample would not go below 80% of the

starting concentration. An aliquot taken before enzyme

addition was used as the tD 0 sample. The slopes of the

plots of substrate concentrations vs time were taken as the

hydrolysis rate. Then the hydrolysis rates were plotted vs

the substrate concentration in a MichaelisMenten plotand the experimental data were Wtted to the Michaelis

Menten equation using nonlinear Wtting in Origin 7. Alter-

natively, substrate concentrations divided by the hydrolysis

rates were plotted vs substrate concentrations in a Hanes

plot using a standard spreadsheet program to obtain kcatand Km values. The derived values using the two diVerent

approaches were the same within experimental errors.

Assays with nonnatural substrates

The kinetic parameters of ChiB variants for the (Glc-

NAc)2-4MU substrate at pH 6.1 were determined as thor-oughly described elsewhere [12,20] with an enzyme

concentration of 0.2nM and substrate concentrations in the

5 to 50-M range. The kinetic parameters of wild-type

ChiB toward (GlcNAc)2-pNP were determined in the same

way, except that the substrate concentration range was

adapted to the much higher Km for this substrate. ChiB

converts the (GlcNAc)3-4MU substrate exclusively to (Glc-

NAc)2 and (nondetectable) GlcNAc-4MU. The kinetic

parameters of wild-type ChiB toward the (GlcNAc)3-4MU

substrate were determined using a two-step assay based on

detection of substrate depletion, a ChiB concentration of

0.2 nM, and substrate concentrations in the 1 to 10-M

range [21]. In short, reactions were conducted as usual but,


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130 Natural substrate assay for chitinases using HPLC / I.-M. Krokeide et al. / Anal. Biochem. 363 (2007) 128134

before adding the 0.2M Na2CO3 stop solution, the reaction

mixtures were incubated for less than 1 min with a 300-fold

excess of ChiA to convert all remaining substrate to

(detectable) free 4MU. In all cases, the substrate concentra-

tions divided by the hydrolysis rates were plotted vs sub-

strate concentration in Hanes plots using a standard

spreadsheet program to obtain kcat and Km values.

Results

(GlcNAc)4 as substrate

(GlcNAc)4 productively binds in subsites 2 to +2 of

ChiA and ChiB of S. marcescens, yielding two (GlcNAc)2molecules as products that are not subject to further hydro-

lysis [8,12,21]. Longer substrates have several productive

binding modes and yield products that in turn are sub-

strates for the enzymes, complicating kinetic analysis of the

overall reaction. Productive binding of (GlcNAc)3 yields

only products that are not further hydrolyzed, (GlcNAc)2and GlcNAc. However, this substrate is less suitable than

(GlcNAc)4 because it occupies fewer subsites and because

there are two productive binding modes, at least in some

chitinases such as ChiA (binding in 2 to +1 and in 1

to +2) [21]).

HPLC analysis of chitooligosaccharides

Normal-phase HPLC-based quantitative analysis of sac-

charides with an amide 80 column has been used previously

[2124] but, so far, the use of this method for determination

of kinetic parameters (implying measurement of very lowconcentrations of sugar) has not been reported. In the pres-

ent study, the necessary increase in sensitivity was achieved

through a combination of adjustments. First, we used a col-

umn with a considerably reduced diameter (0.20cm vs

0.48 cm used in previous studies). A smaller column diame-

ter yields a smaller degree of sample dilution compared to a

larger column diameter and thus results in sharper peaks.

Second, injection volumes were 50 L. Since water elutes

the chitooligosaccharides, the samples were diluted to 75%

(v/v) acetonitrile before application to allow for retardation

on the column of the chitiooligosaccharides before elution

with 70% (v/v) acetonitrile. Together these adjustments per-mitted detection of (GlcNAc)4 concentrations down to

0.5M. Fig. 1 shows the HPLC chromatograms resulting

from enzymatic hydrolysis of (GlcNAc)4 by ChiB.

Determination of kcat, Km, and eYciency constants (kcat/Km)

for wild-type ChiB with diVerent substrates

Enzymatic hydrolysis of (GlcNAc)4 by ChiB follows

MichaelisMenten kinetics as depicted by Fig. 2A. Kinetic

data for hydrolysis of (GlcNAc)4 and three diVerent artiW-

cial substrates are shown in Table 1. For (GlcNAc)4 hydro-

lysis, the nonlinear Wt of theoretical data to experimental

data yielded kcat and Km values of 282 s1 and 4 2M,

respectively. These values diVer considerably from values

obtained with the often-used substrate (GlcNAc)2-4MU,

which are 18 2 s1 and 316M, respectively [20]. Even

larger diVerences were observed with the (GlcNAc)2-pNP

substrate which yielded kcat and Km values of 1.40.5s-1

and 18135M, respectively. For (GlcNAc)3-4MU (which

is converted to (GlcNAc)2 and GlcNAc-4MU), the kcatvalue is 574 s1 and the Km value is 71M and both are

about two times larger than those for (GlcNAc)4.

The diVerences between the substrates are most apparent

when using eYciency constants (kcat/Km; Table 1). ChiB has

a 700 times greater eYciency toward (GlcNAc)4 compared

to (GlcNAc)2-pNP and a 12 times higher eYciency

compared to (GlcNAc)2-4MU. Results obtained with the

(GlcNAc)3-4MU substrate were similar to those obtained

with (GlcNAc)4.

Determination of kinetic parameters for ChiA with

(GlcNAc)4

The (GlcNAc)2-4MU susbtrate is often used because its

product is easy to detect at low concentrations and because

it tends to display seemingly natural Km values, i.e., in the

lower micromolar range. However, for some enzymes with

extended substrate binding clefts this short substrate may

be suboptimal because of the occurrence of multiple bind-

ing modes and cooperativity between these. This is for

example observed for ChiA [12]. With the (GlcNAc)4 sub-

strate, ChiA displayed normal MichaelisMenten kinetics

(Fig. 2B), yielding kcat and Km values of 331 s1 and

91M, respectively (Table 2).

Fig. 1. HPLC analysis of (GlcNAc)4 hydrolysis by ChiB. The starting con-

centration of (GlcNAc)4 was 40 M, and chromatograms obtained after

hydrolysis of 0% (bottom), approximately 10% (middle), and 20% (top) of

the substrate are shown. Both the tetramer and the dimer peak are split

into two peaks due to the anomer equilibrium of the chitooligosaccha-

rides. The anomers (approximately 60%) elute before the anomers

(approximately 40%; see, e.g., [21]). Due to the small diameter of the col-

umn and the relatively large sample volume, the anomeric forms are only

partly separated. The Wrst 8 min of the chromatograms were omitted for

clarity.

8 10 12 14 16 18 20

0

5

10

15

20

25

Absorbance/mV

Time /min


4/7


Determination of kcat and Km values for engineered chitinases

using (GlcNAc)4 and (GlcNAc)2-4MU as substrates

Activity assays based on natural substrates are particu-larly important for the analysis of the properties of engi-

neered enzymes carrying site-directed mutations near the

catalytic center. To analyze and illustrate this, we have char-

acterized two ChiB variants with mutations in subsite +1

(W97A) and +2 (W220A) (Fig. 3) with both (GlcNAc)4 and

(GlcNAc)2-4MU (Table 2). When (GlcNAc)4 was used, kcatand Km values of 126 2 s

1 and 807 2M were obtained

for ChiB-W97A, while the same values were 81 s1 and

17557M for the (GlcNAc)2-4MU substrate. For ChiB-

W220A, the kcat and Km values were 451 s1 and

71 1M and 0.50.1s1 and 824M when using (Glc-

NAc)4 and (GlcNAc)2-4MU, respectively. Interestingly,while the diVerence between the substrates is modest with

respect to Km values, it is as large as 90-fold with respect to

kcat values.

Discussion

Comparison of the various substrates

Up to now, (GlcNAc)2-4MU, which produces the

Xuorescing 4MU leaving group upon hydrolysis, has been

the most used substrate in chitinase assays [12,20,22,25].

Obvious advantages of the use of (GlcNAc)2-4MU are the

time window and simplicity of the assay and its sensitivity.

Product formation is readily and quickly detected using

Xuorescence spectroscopy. There are also several and seri-

ous disadvantages such as substrate inhibition, a nonnatu-

ral leaving group, and nonlinear kinetics, [12,22,26]. Analternative for (GlcNAc)2-4MU is (GlcNAc)2-pNP, which

yields the yellow chromophore pNP. This substrate has the

same disadvantages as (GlcNAc)2-4MU. The data ofTable 1

show that the kinetic parameters determined with

(GlcNAc)2-pNP are very diVerent from the presumably

more realistic parameters obtained with (GlcNAc)4.

Another alternative is (GlcNAc)3-4MU, which may be

converted either to (GlcNAc)2 and GlcNAc-4MU (as does

ChiB) or to (GlcNAc)3 and 4MU (as does ChiA) [12]. In the

latter case, product formation can readily be determined,

but the method has most of the disadvantages described

above for (GlcNAc)2-4MU. For ChiB, we have been able to

develop a method for measuring the conversion of (Glc-

NAc)3-4MU to (GlcNAc)2 and GlcNAc-4MU under con-

ditions that permit determination of kinetic parameters

[21]. The method is based on measuring product disappear-

ance, which is achieved by converting remaining product

quantitatively to 4MU through a short incubation with a

large excess of ChiA. This assay has the advantage that the

+1 subsite is occupied by a natural leaving group (i.e., a

sugar unit). Not unexpectedly, the results obtained with

(GlcNAc)3-4MU for wild-type ChiB resembled those

obtained with (GlcNAc)4. A serious disadvantage of this

assay is the need to purify two enzymes, especially large

amounts of ChiA, which is time consuming, and it works

Fig. 2. MichaelisMenten plots from HPLC analyses of (GlcNAc)4 hydrolysis with ChiB (A), ChiA (B), and the engineered enzymes ChiB-W220A (C) and

ChiB-W97A (D) at pH 6.1, 37 C. Solid symbols are experimentally determined data for the rate of substrate disappearance vs substrate concentrations

and the solid lines are the best nonlinear Wt using the MichaelisMenten equation.

0 20 40 60 80 100

0,000

0,005

0,010

0,015

0,020

R

ate/(M/s)

(GlcNAc)4/M

0 100 200 300 400 500

0,00

0,01

0,02

0,03

0,04

(GlcNAc)4/M

R

ate/(M/s)

0 20 40 60 80 100

0,000

0,005

0,010

0,015

0,020

Rate/(M/s)

(GlcNAc)4/M

0 200 400 600 800 1000 1200

0.00

0.05

0.10

0.15

Rate/(M/s)

(GlcNAc)4/M

A C

DB


5/7


only for enzymes that exclusively convert (GlcNAc)3-4MU

to (GlcNAc)2 and GlcNAc-4MU.

Although the rate-limiting step in the reaction mecha-

nism of family 18 chitinases is not known [17,20,27], it is

clear that a change in chemical properties of the leaving

group may aVect catalytic eYciency with regard to both the

pKa and the ability to interact with the enzyme. At pH 6.1,

about 2% of 4-methylumbelliferol and about 7% ofpara-

nitrophenol will be negatively charged while virtually all of

the (GlcNAc)2 and GlcNAc-4MU leaving groups will be

protonated and free of charge. It is interesting to note that

the substrate yielding the by far lowest kcat value ((Glc-

NAc)2-pNP) has the most acidic and the smallest leaving

group. ChiB is more eVective toward (GlcNAc)2-4MU

whose leaving group is slightly less acidic and considerably

larger. Several studies have shown that enzymesubstrate

interactions in subsites +1 and +2 are important for

catalytic eYciency [17,22,28,29]. The present data do not

permit discrimination between the roles of the acidity and

Ta le 1

kcat and Km of ChiB from Serratia marcescens for substrates with diVerent leaving groups, at 37 C, pH 6.1

a Ref[21].b Ref[20].

Leaving group (RH) kcat (s1) Km (M) kcat/Km (s

1 M1)

Di-N-acetylchitobiose

28 2 4 2 7

4-Methylumbelliferyl-N-acetylglucosamine

57 4a 7 1a 8

4-Methylumbelliferol

18 2b 31 6b 0.6

ara-Nitrophenol

1.4 0.5 181 35 0.01

O

ROH

OH

NH

O

OOH O

OH

OH

NH

O

O

OHOH

OH

NH

O

OOH O

OH

OH

NH

O

+ RHH2O

O

OH

OH

NH

O

OO

OH

OH

NH

O

OHOH

O

OO

OH

OH

NH

O

OH

O

O OOH

OH

NO

O

Ta le 2

Kinetic parameters for engineered and natural chitinases from Serratia

marcescens at 37C, pH 6.1, using (GlcNAc)4 and 4MU-(GlcNAc)2 as

substrates

a (s1).b (M).c (s1M1).d The combination of sigmoidal behavior and substrate inhibition

precludes determination of kinetic parameters [12].

Chitinase (GlcNAc)4 (GlcNAc)2-4MU

kcata Km

b kcat/Kmc kcat

a Kmb kcat/Km

c

ChiB 28 2 4 2 7 18 2 31 6 0.6

ChiA 33 1 9 1 4 n.d.d n.d.d n.d.d

ChiB-W97A 126 4 807 40 0.2 8 1 175 57 0.03

ChiB-W220A 45 2 71 3 0.6 0.5 0.1 82 4 0.01


6/7


the interaction potential of the leaving group. The data in

Table 1 do suggest though that both the occupancy of the +1

and +2 subsites and a high pKa of the leaving group are

beneWcial for catalytic eYciency because the longer, sugar-

like substrates yielded lower Km and higher kcat values.

The present data show that the use of (GlcNAc)4 to cir-

cumvent the problems connected to other substrates is fully

feasible. Both ChiA and ChiB showed straightforward

MichaelisMenten kinetics without substrate inhibition

and the range of accessible substrate concentrations was

such that kinetic parameters could be determined with rea-sonable accuracy. The results show that ChiA and ChiB

have similar activities toward soluble substrates, in line

with the rather similar architectures of their active sites and

substrate-binding grooves [11,16,17,30]).

Analysis of mutants

The W97A and W220A mutants displayed large

increases in Km with both substrates, as might be expected

upon mutating important interaction partners in subsites

that are known to make a dominant positive contribution

to ligand binding. The increase in Km was larger for the nat-ural substrate (GlcNAc)4, presumably because the wild-

type subsites are optimized for sugar binding rather than

binding of the 4MU group. Not unexpectedly, mutation of

Trp220, which primarily aVects subsite +2, had only a mod-

est eVect on the Km for (GlcNAc)2-4MU.

Mutational eVects on kcat showed a dramatic dependence

on the substrate used (Table 2). With the natural substrate,

both mutations lead to an increase in kcat (4.5 and 1.6 times

higher for ChiB-W97A and W220A, respectively) whereas

the kcat is reduced from 18 to 8s1 for ChiB-W97A and to

0.5s1 for ChiB-W220A with (GlcNAc)2-4MU. For the

ChiB-W220A mutant, the two substrates yield a 90-fold

diVerence in kcat and a 60-fold diVerence in the eYciency

constant kcat/Km. Apart from illustrating the diVerences

between the substrates, these observations also lead to ques-

tions concerning the mechanism and rate-limiting step of

catalysis. For example, the dramatic eVect of the ChiB-

W220A mutation on the kcat obtained with (GlcNAc)2-

4MU must mean that Trp220 is involved in catalysis, even in

the case of short substrates. It is not known how the 4MUgroup binds to the enzyme, but it is clear that the group is

large enough (i.e., larger than a single sugar) to interact with

parts of the +2 subsite, including Trp220. Another puzzling

issue concerns the fact that mutation of the two tryptophans

increased the kcat for the natural substrate. Interestingly,

Watanabe et al. [31] found that mutation of analogous try-

ptophans in chitinase A1 from Bacillus circulans led to

increased speciWc activity toward chitopentaose. Further

kinetic and mutational studies are necessary to Wnd possible

explanations for these observations [32].

In conclusion, the results obtained with wild-type ChiB

and, particularly, with the two mutants clearly show that

the use of easy-to-handle artiWcial substrates for detailed

characterization of family 18 chitinases may lead to wrong

conclusions. Thus, such characterization should be based

on a natural substrate assay, such as the one described here.

Acknowledgments

We are grateful for the help of Dr. Gustav Vaaje-

Kolstad for the making of Fig. 3. Part of this work was

funded by The Norwegian Research Council, Grants

140497 and 140440.

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Fig. 3. Crystal structure of the active site of ChiB with (GlcNAc)5 bound

[17]. Trp97 stacks with the sugar moiety in subsite +1. Similarly, Trp220

stacks with the sugar moiety in subsite +2. Note that the 1 sugar has a

non-chair conformation that resembles a sofa conformation [27], the

occurrence of which is crucial during the catalytic cycle [17,27]. Clearly,

mutations that aVect substrate binding may also aVect this crucial struc-

tural deformation of the sugar. Note. The complex was determined with

an inactive E144Q mutant of ChiB. For illustration purposes, the picture

shows a glutamate.


7/7


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