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High Resolution X-ray Crystallography Shows That Ascorbate Is aCofactor for Myrosinase and Substitutes for the Function of theCatalytic Base*

Received for publication, July 28, 2000, and in revised form, September 5, 2000Published, JBC Papers in Press, September 7, 2000, DOI 10.1074/jbc.M006796200

Wilhelm Pascal Burmeister, Sylvain Cottaz , Patrick Rollin , Andrea Vasella**, andBernard Henrissat

From the European Synchrotron Radiation Facility and Forschungszentrum Julich, BP 220, F-38043 Grenoble cedex, France, the Centre de Recherches sur les Macromolecules Vegetales, CNRS, BP 53, F-38041 Grenoble cedex, France, Institut de Chimie Organique et Analytique, Universite dOrleans, BP 6759, F-45067 Orleans cedex 2, France, the**Laboratorium fur Organische Chemie, ETH Zentrum, Universitatsstrasse 16, CH-8092 Zurich, Switzerland, and Architecture et Fonction des Macromolecules Biologiques, CNRS-IFR1, 31 Chemin Joseph Aiguier, F-13402 Marseille cedex 20, France

Myrosinase, an S -glycosidase, hydrolyzes plant ani-onic 1-thio- -D-glucosides (glucosinolates) consideredpart of the plant defense system. Although O -glycosi-dases are ubiquitous, myrosinase is the only known S -

glycosidase. Its active site is very similar to that of re-taining O -glycosidases, but one of the catalytic residuesin O-glycosidases, a carboxylate residue functioning asthe general base, is replaced by a glutamine residue.Myrosinase is strongly activated by ascorbic acid. Sev-eral binary and ternary complexes of myrosinase withdifferent transition state analogues and ascorbic acidhave been analyzed at high resolution by x-ray crystal-lography along with a 2-deoxy-2-fluoro-glucosyl enzymeintermediate. One of the inhibitors, D-gluconhydroximo-1,5-lactam, binds simultaneously with a sulfate ion toform a mimic of the enzyme-substrate complex. Ascor-bate binds to a site distinct from the glucose binding sitebut overlapping with the aglycon binding site, suggest-ing that activation occurs at the second step of catalysis,

i.e. hydrolysis of the glycosyl enzyme. A water moleculeis placed perfectly for activation by ascorbate and fornucleophilic attack on the covalently trapped 2-fluoro-glucosyl-moiety. Activation of the hydrolysis of the glu-cosyl enzyme intermediate is further evidenced by theobservation that ascorbate enhances the rate of reacti-vation of the 2-fluoro-glycosyl enzyme, leading to theconclusion that ascorbic acid substitutes for the cata-lytic base in myrosinase.

Glucosinolates are anionic -D- S-glucosides found promi-nently in plants of the genusBrassica (cabbage, mustard, rape-seed, and other Cruciferae ). They constitute a large family of S-glucosides that differ by their aglycon (Ref. 1 and Fig. 1a ).The same plants produce myrosinase (thioglucoside glucohy-drolase, EC 3.2.3.1), anS -glucosidase hydrolyzing glucosino-lates. Myrosinase and glucosinolates are stored in differentcompartments of the plant, especially in the seeds. Mixing of enzyme and substrate (for example through mastication) in-duces glucosinolate hydrolysis. The biological function of my-

rosinase and glucosinolates is only partly elucidated; it hasbeen suggested that they represent a defense system of theplant. Glucosinolates may as well serve to store inactive pre-cursors of hormones such as 3-indolylacetic acid. 3-Indolyl ace-tonitrile and related indoles (2) are released from indolyl glu-cosinolates by myrosinase (3). Cleavage of indol-3-ylmethylglucosinolate (glucobrassicin) by myrosinase in the presence of ascorbic acid produces ascorbigen, a condensation product of ascorbic acid with 3-hydroxymethylindole (4). Thus, the my-rosinase system could be involved in the storage and in theinactivation of ascorbic acid. A detailed review on myrosinase isgiven by Bones and Rossiter (3).

Myrosinase hydrolyzes theS -glycosides with retention of theanomeric configuration (5). Retaining glycosidases operate by adouble displacement at the anomeric center promoted by twocarboxylic residues acting as acid/base and as nucleophile, re-spectively. In the first step (glycosylation), the glycosidic oxy-gen is protonated by the carboxyl group acting as general acid,

whereas the second catalytic residue, a carboxylate group, per-forms a nucleophilic attack at the anomeric carbon. A glycosylenzyme intermediate with inverted anomeric configuration isformed via a transition state featuring a (more or less) planar(trigonal) anomeric carbon. In the second step (deglycosyla-tion), the carboxylate group corresponding to the catalytic acidresidue activates a water molecule that attacks at C-1 of theglycosyl enzyme to yield the hemiacetal and the free enzyme,again via a transition state with a trigonal anomeric carbon (6).Many carbohydrate derivatives with a planar anomeric carbonand a half-chair or a distorted half-chair conformation inhibitglycosidases. Three such inhibitors, D-glucono-1,5-lactone(Refs. 7 and 8 and Fig. 1c), nojiritetrazole (Refs. 9 and 10 andFig. 1d ), and D-gluconhydroximo-1,5-lactam (Ref. 11 and Fig.1 e), have been studied. A detailed review is given by Height-man and Vasella (12).

Myrosinase belongs to family 1 of the glycoside hydrolases(1315) but is an unusual member of this family in that it lacksthe acid/base residue in its active site. Although classical gly-cosidases activate the glycosidic oxygen by the catalytic acidresidue in the glycosylation step, no such activation appearsnecessary nor indeed possible for myrosinase. Once the glycosylenzyme intermediate is formed, the glutamine residue thatreplaces the catalytic glutamate residue of the classical -glu-cosidases ensures the correct positioning of a water molecule,without deprotonating it. This positioning is sufficient to allowhydrolysis of the glycosyl enzyme intermediate and the releaseof the products (16).

* The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely toindicate this fact.

To whom correspondence should be addressed. Present address:EMBL, BP 156, F-38042 Grenoble cedex, France. Tel.: 33-4-76-20-72-82; Fax: 33-4-76-20-71-99; E-mail: [email protected].

THE JOURNAL OFBIOLOGICALCHEMISTRY Vol. 275, No. 50, Issue of December 15, pp. 3938539393, 2000 2000 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org 39385


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The covalent glycosyl enzyme intermediate of -glycosidasescan be trapped using substrates that are fluorinated at C-2 (i.e.adjacent to the scissile glycosidic bond) and that carry anaglycon with good leaving group ability (17, 18) (Fig. 1b) Thisapproach has made it possible to observe several relativelystable 2-fluoro-glycosyl enzyme intermediates by x-ray crystal-lography (16, 1921) (Fig. 1 f ).

Even though myrosinase shows considerable activity in ab-sence of L-ascorbic acid, its activity is enhanced by ascorbate, asfirst described by Nagashima and Uchiyama (22). The physio-logical significance of this activation stems from an optimaleffect byL-ascorbate compared with its derivatives (23), sug-gesting that the enzyme has evolved to work with the naturallyoccurring L-ascorbate. A physiological role of the activation byascorbate is further evidenced by the ascorbate concentrationneeded for optimal activation of about 1.5 mM, in the range of the global ascorbate concentration in the concerned plant tis-sues, e.g. 2 mM in horseradish root (24). Ascorbic acid is storedin the vacuoles of plant cells (25) where the local concentrationis much higher (24).

A 25-fold increase of the enzymatic activity of Brassica jun-cea seed myrosinase on the natural substrate sinigrin uponaddition of 1 mM ascorbic acid has been found (26, 27). Ettlinger et al . (23) observed a 400-fold acceleration of the reaction rate

for a crude myrosinase preparation fromSinapis alba grains

upon addition of ascorbate, using sinigrin as the substrate. Variable degrees of activation, ranging from 1.8- to 11-folddepending on the source of myrosinase ( Brassica napus , Bras-sica campestris , and S. alba ), the isoenzyme used, and thesubstrate have been reported by Bjorkman and Lonnerdal (28).These authors also found an increase inK m upon activation byascorbate and concluded that the stability of the enzyme-sub-strate complex was reduced, presumably because of an increasein the rate constant for the formation of the product. Morerecently, Shikita et al . (29) described a 140-fold increase of V maxin the presence of 0.5 mM ascorbate for the cleavage of sinigrinby myrosinase fromRaphanus sativus . This increase in V maxwas paralleled by an increase of K m , an observation that hasbeen interpreted as the result of a noncompetitive activation,because of binding of ascorbate to the enzyme-substrate com-plex. All the authors quoted above noticed the dual behavior of ascorbate, which activates myrosinase at 0.11.0 mM and actsas a competitive inhibitor at higher concentrations, typicallyabove 1.5 mM.

Ettlinger et al . (23) have studied the specificity of myrosinaseactivation by different derivatives of L-ascorbate, concludingthat an acidic group is essential for the activation and thatactivation is independent of the reducing property of ascorbate.These investigators suggested that ascorbate plays the role of

the catalytic acid/base.

FIG. 1. a , structure of glucosinolates.r allyl, sinigrin; r p-hydroxybenzyl,sinalbin, the main glucosinolate inS. albagrains and thus the natural substrate of the myrosinase used in this study.b, 2-F-GTL, the substrate used to produce theglucosyl enzyme ( f ). c e, transition stateanalogues. f , the 2-F-glucosyl group.g,the activator ascorbate. h , nonhydrolyz-able substrate analogue.

Myrosinase Reaction Mechanism39386


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We have initiated a program to analyze the details of themechanism of action of myrosinase and the effect of L-ascorbateusing a myrosinase from white mustard ( S. alba ) grains. Thisenzyme is a dimer with a mass of 130 kDa, of which 30 kDa aredue to glycosylation (16). A detailed view on the enzymaticmechanism of myrosinase is derived from high resolution (1.21.6 ) x-ray crystal structure analysis of binary and ternarycomplexes of myrosinase with inhibitors mimicking the transi-tion state, with ascorbic acid, and with the stable 2-fluoro-glucosyl enzyme intermediate.

MATERIALS AND METHODS Reagents 2-F-GTL1 (Fig. 1b) was synthesized as described previ-

ously (30).D-Glucono-1,5-lactone (Fig. 1c) and ascorbic acid (Fig. 1 g)were purchased from Sigma. Gluco-tetrazole (Fig. 1d ) and gluco-hy-droximolactam (Fig. 1 e) were synthesized as described (9, 11). C-GTL,the C-glycosidic analogue of glucotropaeolin (Fig. 1h ), was synthesizedas a mixture of Z- and E-stereoisomers as described earlier (31).

Data Collection Myrosinase crystals were prepared as described(16) using ammonium sulfate as precipitant. The purified myrosinaseremained suitable for crystallization after storage for 3 years at 4 C in20 mM HEPES buffer, pH 6.5. For cryoprotection, a crystal was trans-ferred to a solution containing 66% (v/v) saturated ammonium sulfate,100 mM HEPES, pH 6.5, and 10% (v/v) glycerol. Before freezing in anitrogen stream at 100 K, the crystal was dipped for a few seconds into

a solution containing 20% glycerol. For some trials glycerol was re-placedby ethylene glycol. The inhibitorswere bound by soakingcrystalsin artificial mother liquor containing the compound. To obtain the2-fluoro-glucosyl enzyme, the native crystals were soaked overnightwith 2.5 mM 2-F-GTL as the hydrolysis of this compound is slow. For theternary complex with 2-F-GTL and ascorbate, the ascorbate was intro-duced subsequently in the cryoprotectant containing 10% glycerol. Datawere collected at the European Synchrotron Radiation Facility(Grenoble, France) on experimental station ID14-3 using a 133-mmMarCCD detector or on ID14-1 using a Mar345 detector (gluconolactonedata set). Data were processed with MOSFLM (32) and the CCP4package (33).

Refinement Statistics of data collection and refinements are givenin Table I. The initial model was entry 1MYR deposited in the ProteinData Bank (16). The native structure has been refined using first X-plor3.1 (34) and then REFMAC (35) to a final resolution of 1.2 . During therefinement a number of changes in the sequence, which was based onan x-ray structure at 1.6 resolution, became apparent and have beencorrected.

The complexes have been analyzed with SIGMAA weightedF obs F calc maps calculated with model phases. The model is based on thenative high resolution structure from which the water and glycerolmolecules in the active site have been removed. A direct interpretationof a F deriv F nat Fourier synthesis is hampered by the presence of aglycerol molecule and several well ordered water molecules in the activesite. The structure of the complexes have been refined as described forthe native structure. Coordinates and structure factors have been de-posited in the Protein Data Bank (see Table I for accession numbers).

Enzyme Kinetics Time-dependent reactivation of the 2-fluoro-glu-cosyl enzyme covalent intermediate in absence or in presence of ascor-bic acid was determined essentially as described previously (5). Myrosi-nase was inactivated with 2.5 mM 2-F-GTL (30) for 18 h at 40 C,followed by four successive ultrafiltrations on Nanosep 30 kDa (PallFiltron Corp.) to remove the excess of inactivator. The inactivatedenzyme was incubated at 25 C without or with ascorbic acid (at con-centrations of 0.1 and 1 mM), and reactivation was monitored usingaliquots (20 l) from the solutions of inactivated enzyme at appropriatetime intervals. The samples were assayed by usingp-nitrophenyl- -D-glucopyranoside (36) as the substrate, following the variation of absorb-ance at 430 nm ( 7002 M 1 cm 1 for 4-nitrophenol). First-order rateconstants (k react ) were determined by fitting the recovered activity as afunction of time.

The inhibition constants for gluco-tetrazole and gluco-hydroximolac-tam have been determined with sinigrin as substrate. The hexokinase/ glucose-6-phosphate dehydrogenase coupled enzyme system was usedto assay myrosinase activity by measuring glucose release, followingthe variation of absorbance at 340 nm (37). Myrosinase was preincu-

bated at 34 C (10 l at appropriate dilution) in the absence or presenceof inhibitor (38 l at final concentrations of 0.4, 0.8, and 2 mM for thegluco-tetrazole and 0.125, 0.5, and 1 mM for the gluco-hydroximolac-tam). Reactions were initiated by addition of substrate (332 l at finalconcentration of 0.17, 0.24, 0.35, and 0.7 mM in 50 mM Mes buffer, pH6.5, containing 3 mM MgCl2 , 0.55 mM ATP, 0.72 mM NADP, 0.56 unit/mlhexokinase, and 0.35 unit/ml glucose-6-phosphate dehydrogenase). Ki-netic data were fitted to a competitive inhibition model (38). All re-agents were purchased from Sigma.

RESULTSThree established or putative transition state analogues,

gluco-tetrazole,D-glucono-1,5-lactone, and gluco-hydroximolac-tam were soaked into the crystals. The resulting electron den-sities (Fig. 2,ac ) show that the three inhibitors bind similarly,with all hydroxyl groups involved in identical hydrogen bonds.This recognition is similar to that observed for the 2-fluoro-glucosyl enzyme intermediate (Fig. 2d ), except that the threeinhibitors display a somewhat distorted half-chair conforma-tion, whereas the glucose ring in the 2-fluoro-glycosyl enzymehas a clear 4 C 1 chair conformation (Fig. 3).

D-Glucono-1,5-lactone binds in a distorted half-chair confor-mation (Fig. 3) with O-1 of the lactone in van der Waals contactwith O 1 of Gln 187 (Fig. 2a ). The binding of D-glucono-1,5-

lactone with full occupancy at a concentration of 20 mM in theactive site at the exact position of the glucose moiety of thesubstrate is in contradiction with the experiments by Bottietal . (36), who concluded that theD-glucono-1,5-lactone is a non-competitive inhibitor with aK i of 5 mM.

The inhibition constants for gluco-tetrazole ( K i 0.7 mM)and gluco-hydroximolactam ( K i 0.6 mM) were determinedusing sinigrin as substrate (data not shown). The gluco-tetra-zole binds in a pure half-chair conformation (Figs. 2b and 3).There is a hydrogen bond from Gln187 O 1 to N1 of the tetrazolefor which there is no equivalent for the gluconolactone inhibi-tor. This hydrogen bond is in the plane of the tetrazole moietyand may explain why the gluco-tetrazole inhibitor is bound ata slightly higher position in the active site than the otherinhibitors. It is noteworthy that the residue equivalent to my-rosinase Gln187 in related O-glucosidases, such as the cyano-genic -glucosidase from white clover (39), is the catalytic acid/ base glutamate that would be in an appropriate position for anin plane protonation of the substrate as predicted by Height-man and Vasella (12).

The structure of the complex with gluco-hydroximolactamshows a slightly distorted half-chair conformation for the in-hibitor (Fig. 3). In addition to the inhibitor, a sulfate ion wasfound bound in the active site. This sulfate ion forms saltbridges and hydrogen bonds to Arg259 N 2, Gln187 N 2, and thehydroxyl group of the hydroximo group (Fig. 2c). The N1 nitro-gen atom of this group is in van der Waals contact with theoxygen O1 of the carbonyl group of Gln187 .

Several, albeit unsuccessful, experiments have been under-taken to observe substrate binding directly. The diastereomericmixture of C-GTL (Fig. 1h ) did not inhibit at concentrations upto20mM (enzyme assays; data not shown), nor did it bind in thecrystal at concentrations of up to 100 mM. We also attempted totake advantage of the slow hydrolysis of 2-F-GTL by myrosi-nase and to trap the enzyme-substrate complex using flashfreezing of the crystals. Cottazet al . (5) determined inactiva-tion parameters K i of 0.9 mM and k i of 0.083 min 1 for 2-F-GTL.These data suggest that it should be possible to observe theenzyme-substrate complex. A number of experiments usingdifferent soaking times (520 min) and different pH values of the buffer (pH 4.26.5) did not lead to the observation of anyelectron density corresponding to the substrate analogue. Thenonglucosylation of Glu409 confirms that myrosinase is only

very slowly inactivated by 2-F-GTL.

1 The abbreviations used are: 2-F-GTL, 2-F-glucotropaeolin; Mes,

4-morpholineethanesulfonic acid.

Myrosinase Reaction Mechanism 39387


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To study the intriguing activation of myrosinase by ascor-bate, myrosinase crystals were soaked with ascorbate alone.Omit maps showed clearly the presence of ascorbate togetherwith a glycerol molecule in the active site (Fig. 4a ). Ascorbate isrecognized by a salt bridge between Arg259 N and the O-1oxygen and by a hydrogen bond between the hydroxyl group atposition 2 and Arg259 N 2. O-3 forms a hydrogen bond with N2of Gln187 (Fig. 4a ). A portion of the ascorbic acid molecule islocated in the hydrophobic pocket, which binds the hydrophobicpart of the glucosinolates. This pocket is formed by the residuesIle257 , Phe331 , Tyr330 , Phe371 , and Phe473 , which are invariantin the nine known myrosinase sequences with exception of Phe331 . Another conserved residue, Arg194 (Lys in one se-quence), is not directly in contact but may play an electrostaticrole. The binding of ascorbate is remarkably similar to thebinding of the sulfate ion in the gluco-hydroximolactam inhib-itor structure. A soak with a mixture of gluco-hydroximolactamand ascorbic acid showed that there is indeed competitionbetween the binding of sulfate and ascorbate. The structureshows a partial occupancy for ascorbate (q 0.6) and forsulfate (q 0.4) (Fig. 4b). Two of the oxygen atoms of thesulfate ion are in positions similar to O-2 and O-3 of ascorbate,interacting with N 2 of Gln187 and Arg259 N 2.

From their common position, it is obvious that ascorbate andthe intact substrate cannot bind together in the active site.However, the presence of both ascorbate and gluco-hydroximo-lactam (Fig. 4b) or of ascorbate in the 2-fluoro-glucosyl enzyme(Fig. 4c) shows that ascorbate can bind once the aglycon of thesubstrate has diffused away. In the presence of the 2-fluoro-glucosyl group or of the gluco-hydroximolactam, a hydrogenbonds is formed between the O-6 hydroxyl group of glucose andthe O-6 hydroxyl group of ascorbic acid (Fig. 4,b and c). Ascor-bate is placed ideally to act as a catalytic base and to activatea water molecule, substituting for the general acid/base in thecanonical mechanism of retaining glycoside hydrolases. Such awater molecule is visible in Fig. 4c. It is bound at a distance of 2.56 to O-3 ofascorbate, whereas the distance to the C-1 atomof the 2-fluoro-glucosyl enzyme is 3.22 . The water molecule isplaced 3.70 and 3.99 away from Gln187 N 2 and O 1, respec-tively, making a contribution of this residue unlikely. In the2-fluoro-glucosyl enzyme and in the absence of ascorbate, thewater molecule is closer to Gln187 (3.27 and 2.79 away fromN 2 and O 1, respectively) and 3.40 from C-1 of the glucosering.

The activation of the hydrolysis of the glucosyl enzyme byascorbate has been verified using the 2-F-GTL-inactivated en-zyme. In the absence of ascorbate, half-reactivation required53 h. Addition of 1 mM ascorbate resulted in a 14-fold activa-tion, shortening the half-reactivation time to 3.6 h (Fig. 5) anddemonstrating that activation concerns hydrolysis of the glu-cosyl enzyme.

DISCUSSIONThe good affinity displayed by the transition state analogous

inhibitors (gluco-tetrazole and gluco-hydroximolactam) is inagreement with the hypothesis that the reaction mechanismcatalyzed by myrosinase is similar to the one for the related

-O-glucosidases (12). The position of the sulfate ion in thegluco-hydroximolactam complex is close to the one suggestedfor the sulfate group of glucosinolates, as modeled in a putativeenzyme-substrate complex (16). It is possible to model a sini-grin molecule using the position of the gluco-hydroximolactaminhibitor and of the sulfate ion and to visualize the enzyme-substrate complex (Fig. 6). The position of the sulfate groupand of the glucose moiety determines the position of the hydro-phobic moiety of the aglycon that is expected to bind in the

hydrophobic pocket formed by the three phenylalanine resi-

T A B L E I

S t a t i s t i c s o n t h e d a t a s e t s a n d t h e r e f i n e d s t r u c t u r e s

V a l u e s

f o r t h e

h i g h e s t r e s o

l u t i o n s h e l

l a r e g i v e n

i n p a r e n t h e s e s .

A l l s t r u c t u r e s w

i t h

t h e e x c e p t i o n o f t h e s o a k w

i t h 2 - F

- G T L h a v e

b e e n r e

f i n e d

i n c l u

d i n g c o n s t r a

i n e d

i n d i v i d u a l a n

i s o t r o p i c

B - f a c t o r

r e f

i n e m e n t . T h e c o n t r i b u t i o n o f t h e

h y d r o g e n a t o m s o f t h e p r o t e i n

h a s

b e e n

i n c l u

d e d . R

f r e e

i s b a s e

d o n 5 % o f t h e r e

f l e c t i o n s

( 4 4 ) .

D a t a s e t

N a t

i v e

G l u c o t e t r a z o l e

( 1 0 m M

)

G l u c o

h y -

d r o x

i m o l a c t a m

( 1 0 m M

)

G l u c o n o l a c t o n e

( 2 0 m M

)

2 - F

- G T L

( 2 . 5

m M

)

A s c o r

b i c a c

i d

( 5 m M

)

G l u c o

h y -

d r o x

i m o l a c t a m

( 5 m M

) ,

a s c o r b a t e

( 1 0 m M )

2 - F

- G T L ( 2

. 5 m M

) ,

a s c o r b

i c a c

i d ( 1 0 m M

)

P r o t e i n D a t a

B a n

k

1 c 4 m

1 c 6 q

1 c 6 s

1 c 6 x

1 c 7 0

1 c 7 1

1 c 7 2

1 c 7 3

R e s o l u t i o n

( )

1 . 2 0 ( 1

. 2 0 1 . 2 6 )

1 . 3 5 ( 1

. 3 5 1 . 4 2 )

1 . 3 5 ( 1

. 3 5 1 . 4 2 )

1 . 6 0 ( 1

. 6 0 1 . 6 6 )

1 . 6 5 ( 1

. 6 5 1 . 7 4 )

1 . 5 0 ( 1

. 5 0 1 . 5 8 )

1 . 6 0 ( 1

. 6 0 1 . 6 6 )

1 . 5 0 ( 1

. 5 0 1 . 5 8 )

U n

i q u e

2 2 3 8 0 2

1 5 1 0 9 2

1 4 1 3 0 8

9 9 7 8 3

9 0 0 1 7

1 1 0 2 3 9

8 4 6 6 2

9 8 7 7 6

R e d u n

d a n c y

4 . 5 ( 4

. 0 )

3 . 1 ( 3

. 0 )

2 . 7 ( 2

. 5 )

3 . 3 ( 3

. 0 )

3 . 3 ( 2

. 4 )

3 . 7 ( 3

. 0 )

4 . 2 ( 3

. 3 )

4 . 2 ( 3

. 3 )

C o m p l e t e

( % )

9 9 . 6

( 9 9 . 6 )

9 5 . 8

( 9 4 . 9 )

8 3 . 7

( 7 3 . 6 )

9 7 . 1

( 9 1 . 3 )

8 8 . 5

( 7 6 . 2 )

9 5 . 6

( 9 1 . 5 )

9 9 . 7

( 9 9 . 7 )

8 2 . 7

( 7 1 . 4 )

R m e r g e

( % )

7 . 6 ( 3 8 . 6 )

7 . 2 ( 2 7 . 8 )

7 . 5 ( 4 0 . 0 )

8 . 9 ( 3 3 . 7 )

8 . 5 ( 3 5 . 0 )

6 . 5 ( 2 9 . 4 )

7 . 6 ( 2 5 . 6 )

6 . 3 ( 3 5 . 2 )

R c r y s t

( % )

1 2 . 4

( 1 9 . 5 )

1 1 . 9

( 1 5 . 4 )

1 2 . 0

( 1 8 . 6 )

1 3 . 8

( 2 0 . 1 )

1 6 . 9

( 2 9 . 3 )

1 2 . 3

( 1 4 . 8 )

1 2 . 9

( 1 6 . 3 )

1 3 . 4

( 1 8 . 1 )

R f r e e

( % )

1 4 . 2

( 2 0 . 7 )

1 4 . 6

( 1 9 . 6 )

1 5 . 2

( 2 4 . 3 )

1 7 . 8

( 2 8 . 6 )

1 9 . 5

( 3 4 . 0 )

1 5 . 1

( 1 9 . 1 )

1 5 . 7

( 1 9 . 8 )

1 6 . 7

( 2 4 . 0 )

R o o t m e a n s q u a r e

b o n

d

l e n g t

h s

( )

0 . 0 1 5

0 . 0 1 7

0 . 0 1 9

0 . 0 1 6

0 . 0 1 4

0 . 0 1 4

0 . 0 1 2

0 . 0 1 4



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dues Phe331 , Phe371 , and Phe473 , with residue Tyr330 at thebottom. It should be noted that the substrate has to adopt adistorted boat conformation similar to that described by Davies et al . (19) to avoid a steric clash with amino acid side chains. According to modelization, the sulfate group of the glucosino-late is bound to Arg259 N 2 and Gln187 N 2. In myrosinase,departure of the aglycon without assistance from an enzymatic

acid/base residue can be attributed either to substrate assist-

ance via the sulfate group of the aglycon or to the good leavinggroup ability of the aglycon. According to our model based onthe position of sulfate in the complex with gluco-hydroximolac-tam, a contribution of the sulfate group to catalysis is unlikely,because the oxygen atom next to the sulfur atom of the thio-glycosidic linkage is that involved in the covalent bond to thenitrogen atom of glucosinolates. However, rotation of the sul-

fate group toward the thioglucosidic linkage during catalysis

FIG. 2. View of the active site with different inhibitors. F o F c electron density maps contoured at 3 based are based on a model of thenative structure from which solvent molecules in the active site have been removed. Water molecules are shown asred spheres . The refinedstructures with the bound inhibitor are superposed, including water molecules, sulfate ions, inhibitor, and active site residues. The figure wasgenerated with the program O (45).a , D-glucono-1,5-lactone.b , gluco-tetrazole.c, gluco-hydroximolactam. In presence of the inhibitor an additionalsulfate ion is bound to the active site, and its hydrogen bonds are shown asdotted lines . d , 2-F-glucosyl enzyme covalently bound to Glu409 . Thefluorine atom is shown inyellow . The pink arrow marks the water molecule that is believed to hydrolyze the glucosyl enzyme.

FIG. 3. Conformation of different inhibitors bound to the active site of myrosinase. The nucleophile is shown as well as Gln187 , whichis at the position of the acid/base. The structure of the gluco-tetrazole complex is shown inred , the gluco-hydroximolactam inhibitor is inblue ,together with the bound sulfate molecule. The gluconolactone is shown ingreen , and the covalently linked 2-F-glucose is inmagenta . The drawingwas made with MOLSCRIPT (40).



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could allow a better positioning of the sulfate group in the vicinity of the glycosidic sulfur atom. Because of its low p K a ,the sulfate group is very unlikely to be protonated at neutralpH, which is optimal for the reaction (28, 41, 42). Nevertheless,intramolecular participation of the sulfate group cannot befully excluded, because its p K a could be shifted significantly inthe enzyme active site. The observation that desulfo-glucocap-parin, a substrate analogue that lacks the sulfate group, isneither an inhibitor nor a substrate (23) does not help to re-solve the issue of the possible intramolecular participation of the sulfate group of glucosinolates; conceivably, this sulfategroup is used by active site residues to induce the distortedconformation of the substrate required for catalysis. Myrosi-nase is inactivated by 2-F-GTL with a rate constant of 0.083min 1 and a K i of 0.9 mM (5). The aglycon of 2-F-GTL isidentical to that of glucotropaeolin, the natural substrate, evi-dencing that the aglycon does not need any additional pull. Again, this observation cannot be taken as evidence for oragainst a participation of the sulfate group, because optimalbinding interactions of the enzyme with the aglycon can con-siderably increase the leaving group ability of the aglycon (43).

The enzyme-substrate complex must be very transient, be-cause there is a difference of several orders of magnitude be-

tween the K m of the myrosinase-catalyzed reaction and theK iof the nonhydrolyzable substrate analogue C-GTL. On the onehand, it has not been possible to observe any electron density inthe crystals of myrosinase for the slowly hydrolyzed substrateanalogue 2-F-GTL whereK m for the inactivation is 0.9 mM (5),even at a soaking concentration of 100 mM. This suggests aK iabove 100 mM, considering this compound a competitive inhib-itor. Because the glycosylation reaction can be carried out inthe crystal this shows that the packing does not prevent catal-ysis. Furthermore, effects of the crystal packing on the activesite are unlikely, because of the absence of crystal contacts inits vicinity and the rigidity of the active site reflected by lowatomic temperature factors. On the other hand, hydrolysis of 2-F-GTL (k i 0.083 min 1 ) is sufficiently slow that the en-zyme-substrate complex can be observed in crystals that havebeen soaked with the inhibitor. The lowK m value for theinactivation in the presence of 2-F-GTL shows that substraterecognition is not abolished by the replacement of the 2-hy-droxyl group by fluorine. Nevertheless, there is an increase in K m of 1 order of magnitude compared with the natural sub-strate ( K m 0.075 mM) (41). We have to conclude that in thecase of myrosinase, the short lifetime of the Michaelis complexbetween myrosinase and glucosinolate is compensated by ahigh commitment toward hydrolysis.

The precise positioning of a water molecule above C-1 of theglucosyl enzyme intermediate by hydrogen bonding to O1 (d2.79 ) and N2 (d 3.27 ) of Gln187 promotes the last step of the reaction to a sufficient but not to an optimal extent (Fig.7a ). Binding of ascorbate slightly affects the position of thiswater molecule that now interacts with O-3 of ascorbate (d2.56) and no longer with Gln187 (d 3.99 and 3.70 , Fig. 7b);it may thus be activated by (partial) deprotonation by ascorbateacting as a base. The p K a of 4.1 of ascorbate at 25 C suggestsan optimum activation around that pH value, but we observedthe same flat pH dependence of activity as described by Bjork-man and Lonnerdal (28) or Palmieriet al. (41) in the presenceor absence of 0.5 mM ascorbate using sinigrin as substrate (data

FIG. 4. The binding of ascorbate. The structures have been ob-tained on crystals soaked with ascorbic acid and different inhibitors.Electron density maps as described for Fig. 2. Water molecules areshown as red spheres . The refined structures are shown, includingascorbate, water molecules, sulfate ions, glycerol, inhibitors, and activesite residues. Hydrogen bonds involved in ascorbate recognition are

shown as dotted lines . a , soak with ascorbate, the glycerol molecule

comes from the cryoprotectant.b, ascorbate and gluco-hydroximolac-tam. The ascorbate competes with the sulfate ion that has both partialoccupancies of 0.6 for the ascorbate and 0.4 for the sulfate.c, ascorbatebound to the 2-F-glucosyl enzyme. The water molecule that is activatedby the ascorbate for an attack on the C-1 carbon of the glucose is

indicated by apink arrow .



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not shown). As described earlier for the sinigrin- S. alba my-rosinase system (41), the activation is about 2-fold. We assumethat the p K a of the bound ascorbate is shifted toward neutralpH, so that the activation effect is not distinguishable from thegeneral pH dependence of the activity.

So far there is no satisfactory explanation of the large re-ported differences in the degree of activation, ranging from 1.8-(28) to 400-fold (23) and depending on the source and isoform of myrosinase and on the substrate. The possible influence of other factors, such as the purification protocol or buffer systemis as yet unclear. There might be minor differences in the activesite of isoforms that could affect the relative affinities for sub-strate and activator and dramatically influence the fine bal-ance between activation and inhibition, explaining the differ-ent degrees of activation. These differences do not show up inthe few known sequences of myrosinases. The key residues of the active site involved in the interaction with ascorbic acid(Arg259 and the hydrophobic residues Phe371 , Phe473 , Tyr330 ,and Ile257 ) are strictly conserved.

We assume that the reaction catalyzed by myrosinase pro-ceeds according to Fig. 8 (a and b) in the absence or presence of ascorbate. After formation of the enzyme-substrate (ES) com-plex governed byk 1 and k1 , the glucosyl enzyme (GE) isformed with rate constant k 2 . The glucosyl enzyme is hydro-

lyzed into free glucose and free enzyme with rate constantk3 .

The back reactions for the hydrolysis of the glucosyl enzymeand the cleavage of the substrate have been neglected. Withthese assumptions, Equations 1 and 2 describe the kineticparameters of the reaction (38).

K mk 1 k2

k1

k3k2 k3

(Eq. 1)

V maxk2k3

k2 k3(Eq. 2)

An action of ascorbate at the stage of substrate cleavagegoverned byk2 can be excluded, since aglycon and ascorbatecannot bind at the same time in the active site. If k2 wasrate-limiting (k2 k3 ), then V max and K m would remain con-stant if k3 increases. If, however, deglycosylation is rate-limit-ing (k3 k2 ), then an increase in ks 3 in the presence of ascorbate should lead to an increase in V max and K m , bothproportional to k3 , as indeed observed: Michaelis-Menten con-stants for sinigrin of 23 and 250 M have been measured forR.sativus myrosinase in absence and presence of 0.5 mM ascor-bate, respectively (29). In the same time,V max increased from2.06 to 280 mol/min/mg. A similar increase of K m in parallel toV max has been observed by Bjorkman and Lonnerdal (28).These results confirm a model where the deglycosylation step

of the reaction catalyzed by myrosinase is rate-limiting and

FIG. 5. Kinetics of reactivation inabsence ( ) or in presence of ascorbic

acid ( , 0.1 m M ; , 1 m M). The plot showsrecovered activityversus time.

FIG. 6. A model of substrate bind-ing. The model of the active site residuesincluding the ball-and-stick model of bound sinigrin is shown ( yellow ). The po-sitions of the gluco-hydroximolactam in-hibitor and of the bound sulfate ion in therefined model on which the modeling isbased are shown in cyan . The sinigrinmolecule hasbeenplaced by hand and hasbeen submitted to a crystallographic en-ergy minimization using the gluco-hy-droximolactam data set. The refinementconstrained the sulfate and glucose partof the model to the experimental electrondensity while the geometry wasregularized.



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where this step is activated by ascorbate.When a high concentration of ascorbate is used, there is

binding competition between ascorbate and the substrate, re-sulting in a competitive inhibition, as observed by Shikitaet al.

(29). The additional hydrogen bond of the hydroxyl group at C-6

of the glucosyl group with the hydroxyl group at C-6 of ascor-bate (Fig. 4, b and c) may increase the affinity of the bindingsite for ascorbate once the glucosyl enzyme has been formed.This is an advantage for the enzymatic function, considering

the competition between binding of substrate and ascorbate.

FIG. 7. Hydrolysis of the glucosyl enzyme. Its structure in the absence (a ) and the presence (b) of ascorbate. The refined myrosinase structureis shown. Apink arrow shows the direction of attack of the water molecule, which is positioned ina by Gln187 held by its hydrogen bond network( green dotted lines ). In b this molecule is positioned and activated by the ascorbate molecule. The hydrogen bond directly involved in the activationin shown in bright green .

FIG. 8. Schematic reaction mecha-nism of myrosinase in the absence ( a )and presence ( b and c ) of ascorbicacid. E , enzyme; S , substrate; GE , glu-cosyl enzyme;A, ascorbate; k 1 , k 2 , etc.,dissociation constants not involvingascorbate; k 3 , rate constant in presence of ascorbate;k A1 , k A 1 , etc., dissociation con-stants of ascorbate. The less importantback reactions are not shown.



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This increase in affinity is schematized in Fig. 8b, where therate constants k A1 and k A 1 for ascorbate binding to the glu-cosyl enzyme (GE) are not the same as the rate constantsk A2and k A 2 for ascorbate binding to the enzyme alone.

A scheme of the ascorbate-assisted cleavage of the thioglyco-sidic linkage by myrosinase is given in Fig. 8c. The glucosino-late binds to the active site, but the enzyme-substrate complexis very unstable and short-lived (I). The bound glucosinolatecan be cleaved by the action of the nucleophile Glu409 , withoutenzymatic activation of the thioglycosidic linkage (II). Uponformation of the glucosyl enzyme, a molecule of ascorbate bindsto the active site and acts as a base, abstracting a proton froma water molecule (III) and thus enhancing its nucleophilicattack at the anomeric center (IV). Finally, glucose and ascor-bate are released from the active site.

In typical retaining glycosidases, the nucleophile and gen-eral acid/base residues are separated by 4.8 to 5.3 (6). Inmyrosinase, Gln187 is located precisely at the same position (5.0 from the nucleophilic oxygen) as the acid/base residue of related O-glycosidases. In contrast, ascorbate is placed with itsO-3 oxygen 7.0 away from Glu409 . This larger distance iseasily explained by the alleviated distance constraints forascorbate, as compared with the acid/base residue of O-glyco-sidases, which must be at a position that also allows activationof the leaving group by proton transfer to the glycosidic oxygen.

To the best of our knowledge, myrosinase is the first exampleof an enzyme catalyzing a hydrolysis where an external cofac-tor is recruited in the middle of a reaction path to ensure basecatalysis. Furthermore, activation of myrosinase appears toalso be the first case where ascorbic acid is not used as reducingagent. The effect of ascorbate in contrast to 2-dehydroascorbate(26) could be the basis of a regulation mechanism for myrosi-nase activity as a function of the redox potential in the cell.

Acknowledgments We thank R. Iori and S. Palmieri who suppliedthe protein, S. Arzt and H. Belrhali for the collection of diffraction data,and E. P. Mitchell for the critical reading of the manuscript.

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