A

HfoTNocrp©

E

K

1

Ett[ttxo

Op

s

0d

Enzyme and Microbial Technology 41 (2007) 440–446

Probing the active site environment of alkaliphilic family 11 xylanasefrom Penicillium citrinum: Evidence of essential

histidine residue at the active site

Tanmay Dutta, Rupam Sahoo, Sougata Sinha Ray,Arindam Bhattacharjee, Rajib Sengupta, Sanjay Ghosh ∗

Department of Biochemistry, University College of Science, Calcutta University, 35,Ballygunge Circular Road, Kolkata 700019, West Bengal, India

Received 26 December 2006; received in revised form 23 March 2007; accepted 26 March 2007

bstract

Alkaliphilic xylanases are not only important for their biotechnological applications, but also for their implications in protein structure–function.ere, we present for the first time the presence of single active site histidine residue in the microenvironment of GH-family 11 alkaliphilic xylanase

rom extremophilic fungus Penicillium citrinum MTCC 6489 using chemical modifications. The kinetic studies showed a time dependent inactivationf xylanase by OPTA or DEPC resulting in a pseudo-first-order kinetics with a second-order rate constant of 49.8 and 5.08 min−1 M−1, respectively.he difference spectrum of DEPC modified versus native protein exhibit an absorbance maximum at 244 nm characteristic of the formation of-carbethoxyhistidine, which is completely reversed by neutralized hydroxylamine implying the presence of histidine residue. Moreover, the ratef inactivation shows pH dependence with an inflection point at 6.2. CD studies reveal no significant change in the DEPC modified xylanase

onformation. Substrate dependent protection (0.5% xylan) from DEPC inactivation phenomenon conclusively proves the presence of histidineesidue in the active site. To explore the presence of tryptophan in the active site xylanase is modified with NBS, which reveals its position in closeroximity to active site, but not involved in catalysis.

2007 Elsevier Inc. All rights reserved.

Extre

bmaaorbf

.C. number: 3.2.1.8

eywords: GH-family 11 xylanase; Penicillium citrinum; Active site histidine;

. Introduction

Cellulase-free xylanases (1,4-�-d-xylan xylanohydrolase,.C. 3.2.1.8) from extremophiles are gaining importance due

o their biotechnological applications in paper and pulp indus-ries and as useful model systems for structure–function studies1,2]. Xylanases act on �-1,4-linked xylopyranosyl residues ofhe xylan backbone and in conjunction with cellulases convert

he cellulosic biomass to sugars [3]. A number of family 10ylanases with higher pH optima have been isolated from vari-us bacteria and fungi [4–6]. Family 10 and 11 xylanases have

Abbreviations: GH, glycosyl hydrolase; DEPC, diethylpyrocarbonate;PTA, o-phthalaldehyde; TNBS, 2,4,6-trinitrobenzenesulfonic acid; PHMB,-hydroxymercuribenzoic acid; NBS, N-bromosuccinamide∗ Corresponding author. Tel.: +91 33 2461 5445; fax: +91 33 2476 4419.

E-mail addresses: ghoshs71@hotmail.com,gbioc@caluniv.ac.in (S. Ghosh).

iatang[

e

141-0229/$ – see front matter © 2007 Elsevier Inc. All rights reserved.oi:10.1016/j.enzmictec.2007.03.012

mophilic fungus; Substrate protection; Isoindole fluorescence

een reported from a number of Bacillus sp., with a pH opti-um of pH 8–10 [1,5,7–9]. The pH activity profiles of enzymes

re highly dependent on the pKa of the catalytic residues whichre themselves dependent on the local environment and hencen the nature of the amino acids in the vicinity of the catalyticesidues [1]. Many of the conserved amino acids of xylanases areelieved to be structurally important for confirming the correctolding and packing [10].

In family 11 xylanases, two glutamate residues have beenmplicated in the catalytic mechanism. These two carboxyliccid residues suitably located in the active site are involved inhe formation of the intermediate; one acts as a general acid cat-lyst by protonating the substrate, while the second performs aucleophilic attack, which results in the departure of the leaving

roup and the formation of the �-glycosyl enzyme intermediate5].

Chemical modification is one of the versatile tools to delin-ate requirement for catalytic activity of an enzyme. The identity

robial

omkcDeci

wtcotasHfr

shD

2

2

ohcCa

2

sspb

2

Tbats

2

dwrxoao

2

iphAa5ft

2N

iFptdesa03Dfif3t

(pauo

2

fmxpo

2

moei1

2

temperature using a cell of 1 mm path length. Replicate scans were obtained at

T. Dutta et al. / Enzyme and Mic

f the functional amino acid(s) in the active site environ-ent in alkaliphilic xylanase has therefore been explored by

inetic and chemical modification studies using diethylpyro-arbonate, o-phthalaldehyde and N-bromosuccinamide [11–14].iethylpyrocarbonate is specific for histidine at neutral pH. Pres-

nce of tryptophan residue in the microenvironment of xylanasesould be identified by using tryptophan specific chemical mod-fier N-bromosuccinamide [15,16].

Extremophilic Penicillium citrinum secrets a low moleculareight novel xylanase (22 kDa) with a pI of 3.6 and belongs to

he family G/11 according to the numerical classification of gly-osyl hydrolase [17]. The enzyme exhibited stability and activityver a wide pH range of 4–10 with a pH optimum of 8.5. Despitehe importance of extremophilic xylanases in biotechnologicalpplication no reports are documented deciphering the activeites and the microenvironment of fungal alkaliphilic xylanases.ence, the characterization of active site residues of family 11

ungal alkaliphilic xylanase from P. citrinum would be the firsteport in that list.

In this paper, we present evidence by chemical modificationtudies that a xylanase loses its catalytic activity when a singleistidine residue is modified with the histidine selective reagentEPC.

. Materials and methods

.1. Materials

Birchwood xylan, dinitrosalicylic acid, diethylpyrocarbonate (DEPC),-phthalaldehyde (OPTA), 2,4,6-trinitrobenzenesulfonic acid (TNBS), p-ydroxymercuribenzoic acid (PHMB), N-ethylmaleimide, N-bromosu-cinamide (NBS) and hydroxylamine were purchased from Sigma Chemicalo. (St Louis, MO, USA). All other chemicals used in this work were ofnalytical grade.

.2. Microorganisms and growth conditions

P. citrinum (MTCC-6489) is an alkali tolerant fungus. It was isolated fromoil from the Dhapa situated near Kolkata, India. P. citrinum was cultivated inolid-state fermentation. The enzyme was extracted from solid matrix and wasurified as described previously [17]. The purity of the enzyme was confirmedy SDS-PAGE and gel filtration chromatography [17].

.3. Purification of xylanase

The P. citrinum was grown at 30 ◦C for 96 h for the production of xylanase.he enzyme was purified to homogeneity from the extracellular culture filtratey 0–80% ammonium sulfate precipitation (w/v), followed by phenyl sepharoseffinity chromatography. Native molecular weight was determined on gel fil-ration chromatography using Amersham Pharmacia Biotech Superdex-200 HRize-exclusion column [17].

.4. Xylanase assay

The xylanase assay was carried out by incubating 0.3 mL of appropriatelyiluted enzyme in 0.05 M phosphate buffer, pH 7.0, with 0.3 mL of 1% birchood xylan (w/v) in a final volume of 0.6 mL, at 50 ◦C for 30 min. The released

educing sugar was determined by the dinitrosalicylic acid method using d-ylose as standard [17]. One unit of xylanase activity was defined as the amountf enzyme that produced 1 �mol of xylose equivalent per min from xylan underssay conditions. Protein concentration was determined according to the methodf Bradford [18] using bovine serum albumin as standard.

0w1mb

Technology 41 (2007) 440–446 441

.5. Kinetic inactivation and isoindole formation by OPTA

Fresh OPTA solution was made in methanol for each experiment. The mod-fication was carried out by incubating 2.5 �M of xylanase in 0.05 M potassiumhosphate, pH 7.0, with varying concentrations of OPTA, at 25 ± 2 ◦C. Methanolad no effect on the activity of the enzyme and was always less than 2 % (v/v).t different time intervals, an aliquot was withdrawn from the reaction mixture

nd the residual activity was measured after stopping the reaction by adding�L of 0.01 M cysteine. The formation of xylanase–isoindole derivative was

ollowed spectrofluorimetrically by monitoring the increase in fluorescence withhe excitation wavelength fixed at 338 nm [13].

.6. Reaction of xylanase with DEPC, PHMB and-ethylmaleimide

Xylanase (2.5 �M) in potassium phosphate buffer, 0.05 M, at pH 7.0 wasncubated with varying concentrations of DEPC (0.002–0.01 M) at 25 ± 2 ◦C.reshly prepared DEPC in absolute ethanol was used; samples were removederiodically at different time intervals and the reaction was arrested by the addi-ion of 50 mL of 0.01 M imidazole buffer, pH 7.5. The residual activity of theiluted enzyme derivative was determined under standard assay conditions andxpressed as a percentage of the control. The exact concentration of the stockolution was calculated from the increase in the absorbance at 230 nm when anliquot of the DEPC solution was added to a solution of 0.01 M imidazole in.05 M potassium phosphate buffer, pH 7.5, using an extinction coefficient of200 M−1 cm−1 [11,12]. For pKa, determinations, xylanase was incubated withEPC at various pH values (pH 5.5–8). The modification reaction is specific

or an unprotonated histidine residue between pH 5.5 and 7.5 [11]. The sto-chiometry of the formation of N-carbethoxyhistidine residues was calculatedrom the increase in absorbance at 244 nm using the extinction coefficient of200 M−1 cm−1. The reaction was initiated by the addition of DEPC and waserminated when the maximum absorbance at 244 nm had been attained.

Xylanase (2.5 �M) was incubated with different concentrations of PHMB0.01–0.05 M) in 0.05 M potassium phosphate buffer, pH 7 at 25 ± 2 ◦C. Sam-les were removed at different time intervals and assayed for residual xylanasectivity. Control tubes having only enzyme or only inhibitor were incubatednder identical conditions. Similar experiments were performed in the presencef N-ethylmaleimide.

.7. Substrate protection against inactivation by DEPC

The xylanase (5 �g) was incubated with different amounts of xylan (1–5 mg)or 10 min at 4 ◦C. A 10 �L aliquot of DEPC (0.01 M) was added and the reactionixture was incubated at 25 ± 2 ◦C for 15 min in a total volume of 250 �L. The

ylanase activity was estimated by adding xylan to a final concentration of 5 mger reaction mixture. Parallel controls for enzyme activity for various amountsf xylan in the absence of DEPC were run.

.8. Fluorescence measurements

Fluorescence measurements were performed with a Hitachi F3010 auto-atic recording spectrofluorimeter with an excitation and emission bandwidth

f 4 mm in a quartz cuvette. An excitation wavelength of 295 nm was used. Theffect of NBS on the activity and fluorescence of xylanase was determined afterncubation of enzyme (2.5 �M) with different aliquots of NBS at 25 ± 2 ◦C for5 min.

.9. Circular dichroism spectroscopy

CD spectra were recorded in a Jasco-J715 spectropolarimeter at ambient

.1 nm resolution, 0.1 nm bandwidth and a scan speed of 50 nm min−1. Spectraere averages of six scans with the baseline subtracted spanning from 250 to95 nm in 0.1 nm increments. The CD spectra of the native and DEPC (0.01 M)odified xylanase (25 �g mL−1) were recorded in 0.05 M potassium phosphate

uffer (pH 7.0).

4 robial Technology 41 (2007) 440–446

3

3O

fOAr[ets4ucw[bIerwc

3

ateelc

FoistSc

Fig. 2. Kinetics of the inactivation of xylanase with DEPC. (A) Calculationof the pseudo-first-order rate constant of inactivation of xylanase. Xylanase(2.5 �M) was incubated with different concentrations of DEPC at 25 ± 2 ◦C

42 T. Dutta et al. / Enzyme and Mic

. Results

.1. Kinetics of inactivation of alkaliphilic xylanase byPTA

The incubation of purified xylanase from the alkali tolerantungi P. citrinum MTCC 6489 with increasing concentration ofPTA resulted in a time dependent decrease in enzyme activity.plot (Fig. 1) of ln(A/A0) against time was used to estimate the

ate of inactivation according to the equation ln(A/A0) = Kobs × t19]. When the pseudo-first-order rate constants obtained atach concentration were replotted against OPTA concentra-ion, a linear relationship was observed (inset of Fig. 1). Thelope of the plot yields a second-order rate constant equal to9.8 min−1 M−1. The linearity of this curve indicates a bimolec-lar reaction between the enzyme and OPTA without reversibleomplex formation [20]. Analysis of the order of inactivationith respect to OPTA concentration by the method of Levy et al.

21] yielded a slope of ∼1 indicating that one molecule of OPTAinds to one molecule of the enzyme at the active site (Fig. 1).t was observed that xylanase was not inactivated in the pres-nce of high concentration of PHMB and N-ethylmaleimide andetained complete activity (data not shown). So cysteine groupas absent in the active site of the alkaliphilic xylanase of P.

itrinum.

.2. Inactivation kinetics of alkaliphilic xylanase by DEPC

When incubated with DEPC, xylanase looses its catalyticctivity. Both time and concentration dependent inactivation ofhe enzyme was observed with excess DEPC dissolved in 8%

thanol at neutral pH value. The enzyme was not inactivated bythanol alone. The ln(A/A0) was plotted against time to obtaininear first-order plot (Fig. 2A). When pseudo-first-order rateonstants obtained at each concentration were replotted against

ig. 1. Kinetics of inactivation of xylanase by o-phthalaldehyde. Pseudo-first-rder plots for the inactivation of xylanase by OPTA. Xylanase (2.5 �M) wasncubated with different concentrations of OPTA and control in 50 mM potas-ium phosphate buffer pH 7.0 at 25 ± 2 ◦C. Aliquots were removed at indicatedime intervals and the reaction was terminated by 10 mM cysteine. (Inset)econd-order plot of pseudo-first-order rate constants as a function of OPTAoncentration.

in 50 mM potassium phosphate buffer, pH 7.0, and the data were plotted asdescribed in the text. The curves were best fit by least square analysis. Theconcentrations of DEPC used were indicated in parentheses. (Inset) Determi-nation of the second-order rate constant of inactivation of xylanase by DEPC.The slopes of the straight lines obtained in panel (A) were plotted against con-centrations of DEPC. The slope of this curve indicates the second-order rateconstant of inactivation, which is 5.08 min−1 M−1. (B) CD spectra of nativeaar

Dos

3

poic

3

t

nd DEPC-modified xylanase. Far-UV CD spectra were recorded for nativend DEPC-modified xylanase from 250 to 195 nm at 25 ± 2 ◦C. Each spectrumepresents the average of six scans.

EPC concentration, a linear relationship was observed (insetf Fig. 2A). From the slope of the curve in inset of Fig. 2A, theecond-order rate constant was calculated to be 5.08 min−1 M−1.

.3. CD spectra of native and DEPC modified xylanase

Circular dichroism spectra of the native xylanase was com-ared with that of the DEPC modified xylanase. The CD spectraf native and DEPC modified xylanase were identical, suggest-ng that modification by DEPC does not result in a significanthange in the enzyme structure (Fig. 2B).

.4. Isoindole derivative formation by OPTA

The inactivation of xylanase by OPTA resulted in a concomi-ant increase in fluorescence at 415 nm (excitation wavelength

robial Technology 41 (2007) 440–446 443

3dibattidOibnTix(h

3v

stssns[

3h

ficvhtdcDehai(wa(

3m

f

Fig. 3. Effect of hydroxylamine on optical absorbance at 244 nm. Xylanase(2.5 �M) was incubated with 10 mM DEPC in 50 mM phosphate buffer, pH 7.0,and the change in absorbance against control enzyme was monitored at the timeindicated. At the end of 40 min, hydroxylamine was added and the decreaseinm

ubtDepooa

3x

As the unprotonated amino acid residues of a protein aremodified by DEPC, valuable information on the nature of themodified group may be obtained from the studies of pH depen-

T. Dutta et al. / Enzyme and Mic

38 nm), which is a characteristic for the formation of an isoin-ole derivative. Upon complete inactivation, an average of onesoindole derivative per molecule of the enzyme was found,ased on the increased absorbance at 338 nm using a molarbsorption coefficient of 7.66 mM−1 cm−1 [13]. The increase inhe amount of isoindole derivative formed correlated well withhe decrease in enzyme activity, suggesting that OPTA causesnactivation of xylanase by the formation of a single isoindoleerivative (data not shown). The inactivation of xylanase byPTA suggested the involvement of a lysine or histidine residue

n the active site. However, involvement of lysine residue cane ruled out by the fact that lysine specific modifier TNBS didot show any inhibition of xylanase activity (data not shown).he involvement of a histidine residue in the formation of an

soindole ring was confirmed by the inability of DEPC modifiedylanase to form a xylanase isoindole derivative with OPTAdata not shown). These results indicated the involvement ofistidine residue in the formation of an isoindole derivative.

.5. UV difference spectrum of DEPC modified xylanaseersus native xylanase

The optical difference spectrum of the DEPC modified ver-us native xylanase showed a peak at 244 nm characteristic ofhe formation of N-carbethoxy histidine (data not shown). Noignificant change in absorbance at 280 nm was evident in thepectrum. Thus, the inactivation of xylanase by DEPC couldot be due to formation of an o-carbethoxy derivative of tyro-ine residues, which shows a decrease in absorbance at 280 nm22].

.6. Reaction of DEPC modified xylanase withydroxylamine

Hydroxylamine removes the ethoxyformyl group from modi-ed histidine and tyrosine residue but not from the ethoxyformylysteine and ethoxyformyl lysine residues [22]. DEPC inacti-ated xylanase was completely reactivated on treatment withydroxylamine (data not shown), suggesting that the inactiva-ion was due to modification of the histidine residue and notue to modification of cysteine or lysine. This result also indi-ates that the ethoxyformyl histidine did not react further withEPC, resulting in the cleavage of the imidazole ring [22]. Oth-

rwise, the loss of activity would have been irreversible withydroxylamine. Moreover, the absorbance at 244 nm attainedfter 40 min falls immediately after addition of hydroxylamine,ndicating the reversal of the modified histidine residue onlyFig. 3). However, modification of tyrosine residues with DEPCas ruled out by the absence of any significant change in

bsorbance at 280 nm characteristics to tyrosine modificationinset of Fig. 3).

.7. Correlation between the number of histidine residues

odified and the loss of catalytic activity

The number of histidine residue modified can be estimatedrom the absorbance change at 240 nm (ε240 = 3200 M−1 cm−1)

Ftbao

n absorbance was noted as a function of time. The inset of the figure showso change of absorbance at 280 nm with time as an indication of no tyrosineodification.

pon addition of DEPC. Fig. 4 represents the correlationetween the extent of enzyme inactivation and the extent of his-idine modification over an incubation period of 40 min withEPC. The increase in absorbance at 244 nm represents the

xtent of modification, whereas inactivation is expressed as theercentage of residual activity. The figure shows that after 40 minf incubation of xylanase with DEPC, there was complete lossf activity when total histidine is modified. This suggests thatlkaliphilic xylanase contains a single histidine residue.

.8. pH dependence of the inactivation of alkaliphilicylanase by DEPC

ig. 4. Correlation between the extent of inactivation and the extent of modifica-ion. Xylanase (2.5 �M) was incubated with 10 mM DEPC in 50 mM phosphateuffer, pH 7.0. The percent of the residual activity and the increase in thebsorbance at 244 nm as a measure of modification were plotted as a functionf time as indicated.

444 T. Dutta et al. / Enzyme and Microbial Technology 41 (2007) 440–446

Fig. 5. The pH dependence of the inactivation rate of xylanase with DEPC. (A) Xylanase (2.5 �M) was incubated with 6 mM DEPC in 50 mM phosphate buffer, pH 5–8.T e obtr he slo

dis

K

w

K

w(n

t(fwtam

3s

ociprr

3D

o2emc

otxmc

4

etsnfsdcbfigidTaocto

tosrtti

he pseudo-first-order rate constants obtained were plotted against pH. The curvate constants were plotted according to Eq. (1). The pKa value obtained from t

ence of inactivation of xylanase by DEPC. The extent of thenactivation of the enzyme by DEPC is dependent on the pH, ashown in Fig. 5A. The data may be expressed by

obs = (Kobs)max

1 + ([H+]/Ka)(1)

hich in linear form may be represented as

obs[H+] = Ka(Kobs)max − KaKobs (2)

here Ka is the dissociation constant of the reacting group andKobs)max is the pseudo-first-order rate constant of the unproto-ated reacting group [23].

The pseudo-first-order rate constant was determined and plot-ed against pH (Fig. 5A). By plotting Kobs[H+] against Kobsvalue taken from Fig. 5A) a straight line is obtained (Fig. 5B)rom which the value of (Kobs)max = 0.037 min−1, pKa = 6.20ere calculated from the ordinate intercept and the slope of

he line, respectively. The apparent pKa value of 6.20 offersdditional evidence that the inactivation of xylanase is due toodification of histidine residue.

.9. Evidence for the presence of the histidine in the activeite

A progressive protection of inactivation by DEPC wasbserved with increasing amount of xylan (data not shown), indi-ating that xylan and DEPC compete for the same binding site,.e. the histidine residue in the active site. Xylan offered 100%rotection against inactivation of the enzyme by DEPC. Theseesults confirm that histidine is present at the substrate-bindingegion of the enzyme.

.10. Tryptophanyl fluorescence analysis of native andEPC modified xylanase

The native and NBS modified xylanase were found to flu-resce with an emission maximum of 339 nm on excitation at

95 nm (data not shown). The fluorescence spectrum of nativenzyme showed a rapid decrease on addition of NBS up to aolar ratio of 1:10 (xylanase:NBS). The tryptophanyl fluores-

ence analysis showed that the substrate (0.5%) quenched 30%

fioea

ained was a theoretical one. (B) The experimentally obtained pseudo-first-orderpe was 6.20 and the (Kobs)max value was 0.037 min−1.

f the native protein tryptophan fluorescence. However, on addi-ion of graded concentration of NBS there was no change in theylanase activity indicating that tryptophan residue or residuesay be present in the microenvironment but not involved in the

atalysis.

. Discussion

In an attempt to understand the catalytic mechanism of annzyme, it is essential to study the structural elements and thehree-dimensional conformation of the active site. It is true thatome chemical modification reagents may have some degree ofone-selectivity. But in most of the cases, the results obtainedrom DEPC modified histidine residue correlated well with theite directed mutagenesis data as well as the crystallographicata. Some times a single point mutation may cause a globalonformational change of the protein that is why CD spectra ofoth wild type and mutated protein could give definitive proofor non-alteration of the secondary structure. Similarly, chem-cal modification study would be an important tool when theene sequences are not available. The utility of chemical mod-fication is greatly enhanced by its use in conjunction with siteirected mutagenesis, which mutually supplement each other.hough extensive studies have been carried out on the industrialpplications of xylanases, there are comparatively fewer reportsn the molecular enzymology of this fungal alkaline family 11lass of xylanases. Present findings thus remain strongly indica-ive towards the presence of histidine residue in the active sitef alkaline xylanase.

The results presented in this paper provide the first evidencehat one essential histidine residue is located at the active sitef family 11 alkaline xylanase from P. citrinum. Our result alsohowed the presence of a tryptophan residue in the microenvi-onment of xylanase active site. Although, upon modification ofryptophan residue in presence of chemical modifier NBS andhe substrate xylan showed quenching, but it did not showed anynhibition in catalysis. The inactivation of xylanase due to modi-

cation of lysyl and cysteine residues with OPTA has been ruledut. The inactivation of xylanase by DEPC requires a large molarxcess of reagent in order to counteract its rapid hydrolysis inqueous medium. The increase in absorption at 244 nm follow-

robial Technology 41 (2007) 440–446 445

ioTtbDkitecwie

tpttbbsBshcbotfaargtiin[a(Icrlttoohc(ttfiHa

Sb

ffpkgtccbmitCmiatpiSiabd

T. Dutta et al. / Enzyme and Mic

ng incubation with DEPC and its reversal by hydroxylamineffers strong evidence for the formation of carbethoxyhistidine.he formation of dicarboxyethylated imidazole derivative due

o ring cleavage is excluded, as this derivatization would note reversed by hydroxylamine. The inactivation of xylanase byEPC is a time dependent bimolecular reactions as evidenced byinetic studies. The rate of inactivation shows pH dependence,ndicating that the inactivation is due to the modification of aitratable residue with a pKa value of 6.20. This is also strongvidence for histidine derivatization by DEPC, and the value isonsistent with the pKa value for the histidine residues modifiedith DEPC in other enzymes [12,14]. The inability of DEPC to

nactivate the enzyme in the presence of xylan provides strongvidence that the modified histidine is in the active site.

Family G/11 xylanases are generally characterized by theirypical �-jelly roll fold structure [24]. The structure consistsrincipally of �-pleated sheets formed into a two-layered troughhat surrounds the catalytic site [5]. There are increasing datahat indicate a role for H-bonds and salt bridges in protein sta-ilization [9,25–27]. In family 11 xylanases, the number of saltridges varies between 2 and 12. There is one completely con-erved salt bridge between C-terminal Glu (or Asp) of �-strand7 and A6 [9]. Alkaliphilic xylanases also have large number of

alt bridges. It has been proposed that alkaliphilic proteins mayave a tighter internal packing with smaller and less numerousavities, the increased of packing efficiency increases hydropho-icity. Tighter packing can be achieved through the formationf hydrophobic clusters and enhanced van der waals interac-ions [9]. Tertiary structure analysis of family 11 xylanasesrom Trichoderma reesei and Aspergillus niger indicated thatdaptation to low pH is brought about by an increase in neg-tive charge and a substitution and reorientation of aromaticesidues in the active sites [28–31]. In contrast, a random muta-enesis study of Neocallimastix patriciarum xylanase indicatedhat an increased negative charge and increased hydrophobicityncreased the pH optimum of this enzyme [32]. Xylanases stablen alkaline condition are typically characterized by a decreasedumber of acidic residues and an increased number of arginine33]. A number of reports on sequence homology of xylanasesre documented [1]. In general, sequences from the same family10 or 11) were closely related and exhibited more homology.n family 11 xylanase, serine and histidine residues are highlyonserved. NMR studies revealed His149 to be an importantesidue in establishing the conformation of the Bacillus circu-ans xylanase. Mutagenesis of H149 to Phe and Gln did not alterhe active site, which showed that histidine was not present inhe active site of B. circulans xylanase. However, the stabilityf the folded protein was found to decrease [1]. The active sitef xylanase A of Streptomyces lividans contains three importantistidine residues, two of which (H81 and H207) are completelyonserved in family 10 xylanase. Not only that these two residuesH81 and H207) are also involved in hydrogen bonding network inhe vicinity of the two catalytic residues [34,35]. The X-ray crys-

al structure of family 10 glycoside hydrolase of Cellulomonasmi identified one histidine residue around the key carboxylates.is205 is being hydrogen bonded to both the nucleophile, Glu233,

nd to Asp235. These trio residues are highly conserved within

co

o

cheme 1. Mechanism of histidine modification by DEPC and removal of car-ethoxy group from modified histidine by hydroxylamine.

amily 10 enzymes [36,37]. Without cloning the xylanase generom P. citrinum MTCC 6489, it is not possible to get the fullrotein sequence of this xylanase enzyme. At present it is notnown about the resemblance of this enzyme with other fun-al xylanases having acidic pH optima, still it is conceivablehat alkaliphilic xylanase of P. citrinum MTCC 6489 may alsoontain conserved glutamate residues and these acidic residuesould be joined to the active site histidine residue through H-onding. In presence of DEPC, this hydrogen bonding networkay be disrupted and thereby inhibit the catalysis by chang-

ng the microenvironment of the active site without affectinghe secondary structure of xylanase which is evidenced by theD measurement in presence of DEPC. Two conserved gluta-ate residues are involved in double displacement mechanism

n which a covalent glycosyl–enzyme intermediate is formednd subsequently hydrolysed via oxocarbenium-ion-like transi-ion state [37]. Carbethoxyhistidine formation may inhibit theroduct formation by altering the anomeric configuration, whichs very important in this catalysis. The mechanism proposed incheme 1 for the formation of carbethoxyhistidine formation

s based on well-established chemical reactions. Nucleophilicttack from histidine nitrogen is made at the carbonyl car-on atom of DEPC resulting in the formation of carbethoxyerivative of protein histidine residue. Hydroxylamine removesarbethoxy group from modified histidine through a nucle-

philic attack to carbonyl carbon atom of carbethoxy group.

Future work on cloning, expression and characterizationf this alkaline xylanase from P. citrinum will reveal the

4 robial

sap

A

IapR

R

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

[

46 T. Dutta et al. / Enzyme and Mic

tructure–function relationship of family 11 alkaline xylanasend site directed mutagenesis data of this residue would com-lement the chemical modification technique.

cknowledgements

We thank Dr. Soumen Basak and Mr. Kalyan Giri of Sahanstitute of Nuclear Physics, Kolkata, for expert assistance andccess to circular dichroism spectroscopy. This work was sup-orted by the grant from Council for Scientific and Industrialesearch (CSIR), Govt. of India.

eferences

[1] Kulkarni N, Shendye A, Rao M. Molecular and biotechnological aspectsof xylanases. FEMS Microbiol Rev 1999;23:411–56.

[2] Viikari L, Kantelinen A, Sundquist J, Linko M. Xylanases in bleaching:from an idea to the industry. FEMS Microbiol Rev 1994;13:335–50.

[3] Subramaniyan S, Prema P. Biotechnology of microbial xylanases:enzymology, molecular biology and application. Crit Rev Biotechnol2002;22:33–46.

[4] Horikoshi K. Alkaliphiles: some applications of their products for biotech-nology. Microbiol Mol Biol Rev 1999;63:735–50.

[5] Collins T, Gerday C, Feller G. Xylanases, xylanase families andextremophilic xylanases. FEMS Microbiol Rev 2005;29:3–23.

[6] Wong KKY, Tan LUL, Saddler JN. Multiplicity of �-1,4-xylanase inmicroorganisms: functions and applications. Microbiol Rev 1988;52:305–7.

[7] Nakamura S, Wakabayashi K, Nakai R, Aono R, Horikoshi K. Purificationand some properties of an alkaline xylanase from alkaliphilic Bacillus sp.41M-1. Appl Env Microbiol 1993;59:2311–6.

[8] Gessesse A. Purification and properties of two thermostable alka-line xylanases from an alkaliphilic Bacillus sp. Appl Env Microbiol1998;64:3533–5.

[9] Hakulinen N, Turunen O, Janis J, Leisola M, Rouvinen J. Three dimensionalstructures of thermophilic �-1,4-xylanases from Chaetomimum ther-mophilum and Nonomuraea flexuosa. Eur J Biochem 2003;270:1399–412.

10] Yang VW, Zhuang Z, Elegir G, Jeffries TW. Alkaline-active xylanaseproduced by an alkaliphilic Bacillus sp. isolated from kraft pulp. J IndMicrobiol 1995;15:434–44.

11] Lundblad RL, Noyes CM. Chemical reagents for protein modification, vol.1. Boca Raton, FL: CRC Press Inc.; 1984.

12] Bhattacharyya DK, Bandyopadhyay U, Banerjee RK. Chemical and kineticevidence for an essential histidine in horseradish peroxidase for iodideoxidation. J Biol Chem 1992;267:9800–4.

13] George SP, Rao MB. Conformation and polarity of the active siteof xylanase I from Thermomonospora sp. as deduced by fluorescentchemoaffinity labeling. Eur J Biochem 2001;268:2881–8.

14] Olano J, Soler J, Busto F, Arriaga D. Chemical modification ofNADP-isocitrate dehydrogenase from Cephalosporium acremonium. EurJ Biochem 1999;261:640–9.

15] Bandivadekar KR, Deshpande VV. Structure–function relationship ofxylanase: fluorimetric analysis of the tryptophan environment. BiochemJ 1996;315:583–7.

16] Ladokhin AS. Fluoroscence spectroscopy in peptide and protein analysis.In: Encyclopedia of analytical chemistry. John & Wiley Sons Ltd., 2000,

p. 1–15.

17] Dutta T, Sengupta R, Sahoo R, Sinha Ray S, Bhattacharjee A, GhoshS. A novel cellulase free alkaliphilic xylanase from alkali tolerant Peni-cillium citrinum: production, purification and characterization. Lett ApplMicrobiol 2007;44:206–11.

[

Technology 41 (2007) 440–446

18] Bradford MM. A rapid and sensitive method for detecting microgramamounts of protein utilizing the principle of protein–dye binding. AnalBiochem 1976;72:248–54.

19] Blanke SR, Hager LP. Chemical modification of chloroperoxidase withdiethylpyrocarbonate. Evidence for the presence of an essential histidineresidue. J Biol Chem 1990;265:12454–61.

20] Church FC, Lundblad RL, Noyes CM. Modification of histidines inhuman prothrombin. Effect on the interaction of fibrinogen with throm-bin from diethyl pyrocarbonate-modified prothrombin. J Biol Chem1985;260:4936–40.

21] Levy HM, Leber PD, Ryan EM. Inactivation of myosin by 2,4-dinitrophenol and protection by adenosine triphosphate and otherphosphate compounds. J Biol Chem 1963;238:3654–9.

22] Miles EW. Modification of histidyl residues in proteins by diethylpyrocar-bonate. Methods Enzymol 1977;47:431–42.

23] Takeuchi M, Asano N, Kameda Y, Matsui K. Chemical modifi-cation by diethylpyrocarbonate of an essential histidine residues in3-ketovalidoxylamine A C–N lyase. J Biochem (Tokyo) 1986;99:1571–7.

24] Gotz F, Borriss R, Heinemann U. Structure and function of Bacillus 486hybrid enzyme GluXln-1: Native like jellyroll fold preserved after insertionof autonomous globular domain. Proc Natl Acad Sci USA 1998;95:6613–8.

25] Joshi MD, Sidhu G, Pot I, Brayer GD, Withers SG, McIntosh LP. Hydro-gen bonding and catalysis: a novel explanation for how a single aminoacid substitution can change the pH optimum of a glycosidase. J Mol Biol2000;299:255–79.

26] Wakarchuk WW, Campbell RL, Sung WL, Davoodi J, Yaguchi M. Muta-tional and crystallographic analyses of the active site residues of Bacilluscirculans xylanase. Protein Sci 1994;3:467–75.

27] Joshi MD, Sidhu G, Pot I, Brayer GD, Withers SG, McIntosh LP. Dissect-ing the electrostatic interactions and ph dependent activity of a family 11glycosidase. Biochemistry 2001;40:10115–39.

28] Torronen A, Rouvinen J. Structural comparison of two major endo-�-1,4-xylanase from Trichoderma reesei. Biochemistry 1995;34:847–56.

29] Torronen A, Harkki A, Rouvinen J. Three dimensional structure endo-�-1,4-xylanase II from Trichoderma reesei: two conformational states in theactive site. EMBO J 1994;13:2493–501.

30] Krungel U, Dijkstra BW. Three dimensional structure of endo-�-1,4-xylanase I from Aspergillus niger, molecular basis of its low pH optimum.J Mol Biol 1996;263:70–8.

31] Fushinobu S, Ito K, Konno M, Wakagi T, Matsuzawa H. Crystallogra-phy and mutational analysis of an extremely acidophilic and acid-stablexylanase; biased distribution of acidic residues and importance of Asp 37for catalysis at low pH. Protein Eng 1998;11:1121–8.

32] Chen YL, Tang TY, Cheng KJ. Direct evolution to produce an alkaliphilicvariant from a Neocallimastix patriciarum xylanase. Can J Microbiol2001;47:1088–94.

33] Turunen O, Vourio M, Fenel F, Leisola M. Engineering of multiple argininesin to the Ser/Thr surface of Trichoderma reesei endo-�-1,4-xylanase IIincreases the thermo tolerance and shifts the pH optimum towards alkalinepH. Protein Eng 2002;15:141–5.

34] Roberge M, Shareck F, Morosoli R, Kluepfel D, Dupont C. Characteri-zation of two important histidine residues in the active site of xylanasea from Streptomyces lividans, a family 10 glycanase. Biochemistry1997;36:7769–75.

35] Roberge M, Shareck F, Morosoli R, Kluepfel D, Dupont C. Site-directedmutagenesis study of a conserved residue in family 10 glycanases:histidine 86 of xylanase A from Streptomyces lividans. Protein Eng1998;11:399–404.

36] Schmidt A, Schlacher A, Steiner W, Schwab H, Kratky C. Structure of

xylanase from Penicillium simplicissimum. Protein Sci 1998;7:2081–8.

37] Notenboom V, Birsan C, Nitz M, Rose DR, Warren RAJ, Wither SG.Insights into transition state stabilization of the �-1,4-glycosidase Cexby covalent intermediate accumulation in active site mutant. Nature1998;5:812–8.