THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc

Vol. 269, No. 20, Issue of May 20, pp. 14530-14535,1994 Printed in U S A

Identification of Active Site Carboxylic Residues in BaciZZus Zicheniformis 1,3-1,4-P-~-Glucan 4-Glucanohydrolase by Site-directed Mutagenesis*

(Received for publication, November 12, 1993, and in revised form, February 23, 1994)

Miquel Juncosa’CS, Jaume PonsSP, Teresa Dotlll, Enrique QuerolS, and Antoni PlanasSn** From the Unstitut de Biologia Fonamental V Villar Palasi and Department de Bioquimica i Biologia Molecular, Universitat Autonoma de Barcelona, 08193 Bellaterra, Barcelona, Spain and the W E T S Znstitut Quimic de Sarria, Universitat Ramdn Llull., 08017 Barcelona, Spain

Active site residues of 1,3-1,4-P-n-glucan 4-glucanohy- drolase (EC 3.2.1.73) from Bacillus licheniformis have been identified by site-directed mutagenesis. Previous work revealed that Glu-134 was essential for enzymatic activity, and it was proposed as the catalytic nucleophile by affinity labeling of the highly homologous Bacillus amyloliquefaciens enzyme. To search for the general acid catalyst, the Asp and Glu residues conserved among the Bacillus isozymes have been mutated to Asn and Gln, re- spectively. Out of the 14 positions studied, only the E138Q mutation yielded an inactive enzyme, whereas the E134Q and D136N mutants retained less than 0.6% of the wild type activity. Based on the three-dimensional structure of a hybrid B. amyloliquefaciens-Bacillus macerans 1,3- 1,4+-~-glucan 4-glucanohydrolase, Glu-134, Asp-136, and Glu-138 are the only carboxylic acid residues that are properly located into the active site cleft to participate in catalysis. Glu-138 appears as the most likely candidate to function as the general acid catalyst, while Asp136 may affect the pK, of the catalytic residues.

1,3-1,4-p-~-glucan 4-glucanohydrolase (p-1,3-1,4-gl~canase)~ (EC 3.2.1.73) is a glycosidase that hydrolyzes p-glucans con- taining mixed P-1,3 and P-1,4 linkages as lichenan and barley P-glucan. The enzyme-catalyzed depolymerization of the latter yields 3-~-O-ce~lobiosyl-~-glucopyranoside and 3-P-0-cellotrio- syl-D-glucopyranoside in a 2.2 to 1 ratio, defining the cleavage specificity at P-1,4-glycosyl linkages on 3-0-substituted gluco- pyranose units (Parrish et al., 1960; Anderson and Stone, 1975; Woodward et al., 1983; Buliga et al., 1986). Bacterial P-1,3-1,4- glucanases are the best characterized, and a number of genes from different Bacillus species have been cloned (Cantwell and McConnell, 1983; Hofemeister et al., 1986; Borriss et al., 1988; Bueno et al., 1990; Gosalbes et al., 1991; Lloberas et al., 1991; Schimming et al., 1992; Louw et al., 1993). They show a high degree of sequence similarity (Fig. 11, but no apparent homol- ogy was detected when compared with bacterial endo-P-1,4- glucanases or with barley P-1,3-1,4-glucanases (Fincher et al., 1986; Litts et al., 1990).

Initial attempts to identify active site residues were based on sequence comparison to eukaryotic and viral lysozymes. Bor- r i ss et al. (1990) first found a limited sequence similarity be- tween Bacillus P-1,3-1,4-glucanases and the sequence sur- rounding the catalytic Glu-11 and Asp-20 of T4 lysozyme. Our studies on the P-1,3-1,4-glucanase from Bacillus licheniformis were directed to test this hypothesis by site-directed mutagen- esis of the putative essential residues (Planas et al., 1992a). Substitution of Glu-134 with Gln yielded an enzyme with nearly abolished activity suggesting its essential role in cataly- sis. Based on the T4 lysozyme model, this residue was tenta- tively proposed as the general acid catalyst. However, H0j et al. (1992) have reported for the highly homologous Bacillus amyloliquefaciens enzyme that Glu-105 (the equivalent residue to Glu-134 in B. licheniforrnis) is covalently modified by mecha- nism-based inhibitors (epoxyalkyl P-oligoglucosides), proposing that residue as the likely catalytic nucleophile. Additional evi- dence to discard the initial T4 lysozyme-like model is shown here when the conserved residue Asp-143, which matches the position of the catalytic nucleophile Asp-20 of T4 lysozyme, proves not to be an essential residue. Taken together with the fact that the B. licheniformis enzyme is a retaining glycosidase (Malet et al., 1993), Glu-134 might be the oxocarbonium-stabi- lizing residue or the true nucleophile in the hydrolytic mecha- nism, depending on whether the reaction intermediate exists as an oxocarbonium ion-paired to an enzyme carboxylate or as a covalent glycosyl-enzyme adduct.

The identity of the general acid remains unknown, and it may not be inferred from sequence similarity analyses. Here, we approach the search of other essential residues by a screen- ing mutagenesis strategy. It is generally accepted that the mechanism of retaining glycosidases is initiated by a general acid (Sinnott, 1990) that is a carboxyl group (Asp or Glu) in most of the cases (Svensson and Segaard, 1993). Thus, all the individual Asp and Glu residues in the B. licheniformis enzyme that are conserved among the Bacillus P-1,3-1,4-glucanases are mutated to the isosteric and uncharged Asn and Gln residues, respectively, with the aim of finding a single mutant with com- pletely abolished glycosidase activity. The results are discussed bv examining the location of the mutated residues on the re- - cently solved x-ray structure of the hybrid B. amyloliquefa-

* This work was supported by Grant BI091-0477 from Ministerio de ciens-~acillus enzyme (Keitel et al., 1993). Educaci6n y Ciencia, Spain. The costs of publication of this article were defrayed in part by the payment of page charges. This article must MATERIALS AND METHODS therefore be hereby marked “aduertisernent” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

cia, Spain.

Bacterial Strains and Culture Media-Escherichia coli TG1 (supE

for plasmid propagation, transformation with the mutagenic PCR reac- I1 Supported by the CETS Institut Quimic de Sarria. tion, and protein expression. E. coli HBlOl (supE44 hsdS2O ** To whom correspondence should be addressed. “el.: 34-3-2038900, (rB-m,-)recA13 ara-14 prom lacy1 galK2 rpsL20 xyl-5 rntl-1) was the

* The abbreviations used are: P-1,3-1,4-glucanase, 1,3-1,4-P-~-glucan grown in 2YT medium. Ampicillin at 100 pg/ml was added when appro-

p Recipient of a fellowship from the Ministerio de Educaci6n cien- hsdA5 thi A(lac-proAB) F‘ [traD36 pros+ lad9 l a c z m I 5 1 ) Was used

F a : 34-3-2056266. host strain for protein expression. For plasmid isolation, bacteria were

4-glucanohydrolase; PCR, polymerase chain reaction; wt, wild type. priate.

14530

Active Site Carboxylic Residues of 1,3-1,4-P-Glucanase 14531

Chemicals and Enzymes-Restriction endonucleases, T4 DNAligase, and Taq polymerase were from Boehringer Mannheim, and Deep Vent" polymerase was from New England Biolabs Inc. 35S-a-ATP was pur- chased from Amersham Corp. DNA sequencing was performed by the T7 sequencing kit from Pharmacia LKB Biotechnology Inc. Oligonucleo- tides were synthesized by CLONTECH or by the DNA facility at the University of California, Berkeley. Barley p-glucan was from Sigma.

Site-directed Mutagenesis by PCR-The gene coding for B. licheni- formis p-1,3-1,4-glucanase cloned into pUC119 as a 1.21-kilobase SucV SphI fragment (Planas e t al., 1992a) was used as template for the mutagenic PCR following the method of Landt et al. (1990) in two separate steps. The first comprises the use of the mutagenic primers and the forward universal primer flanking the 3' end of the p-1,3-1,4- glucanase gene. The primers were the following: D51N, 5'- GGCAAAAAGCWTGGGTACTCG A-3'; D89N, 5"TATAATAAGTT- TaCTGCGGAGAA-3'; E92Q, 5"TTGACTGCGGAcAAAAC CGCT- CCG-3'; E105Q, 5'-TATGGGCTATATcAAGTCAACATG-3'; D136N, 5" TGGGATGAAATCUCATCGAATTT-3'; E138Q, 5"GAAATCGACAC- - CAATTTCTAGGA-3'; D143N, 5'-TAAACTGAACCT'ITGTCGTAlY3Tl"

CAAACT-3'; D190N, 5'-AAATGGTATGTG@CGGTCAATTA-3'; and D219N, 5'-GGTGCAGGTGTC&TGAATGGCTCG-3'. The second step uses the product of the first PCR as a primer and the reverse universal primer to yield the whole p-1,3-1,4-glucanase gene with the desired mutation. For these purposes, Deep Vent@ polymerase, which has a 3'+5' proofreading activity, gave the best results. Preliminary com- parative experiments showed an increased single point mutation efi- ciency and amplification fidelity over Tuq polymerase, which lacks proofreading activity. The final amplified DNA was cut with EcoRI1 Hind111 and ligated again to a pUC119 vector. Mutated plasmids were transformed into E. coli TGl cells. Transformants were screened by DNA sequencing using appropriate primers located about 100 bases from the mutation point. Positive clones were confirmed by complete sequencing of the entire gene.

Protein Expression and Purification of wt and Mutant Enzymes- These procedures were basically as described before (Planas et al., 1992b). Proteins were purified from the extracellular medium of E. coli TG1 cultures harboring the mutagenized plasmids. The enzymes were analyzed by SDS-polyacrylamide gel electrophoresis according to La- emmli (1970) and by fast protein liquid chromatography on an ionic exchange TSK CM-3SW column at pH 5.6.

Enzyme Assay and Kinetics-/3-1,3-1,4-Glucanase activity on plates was detected by the Congo red staining assay after growing E. coli HBlOl cells containing the mutagenized plasmids on LB plates supple- mented with 0.04% (w/v) barley p-glucan (Teather and Wood, 1982). Activity in the extracellular medium of liquid cultures and enzyme activity of purified proteins were determined as previously reported (Planas et al., 1992a) by measuring the net release of reducing sugars from barley 6-glucan as substrate by the dinitrosalicylic acid reagent (Hinchliffe, 1984). Kinetic parameters were derived by fitting the data to a rectangular hyperbola by nonlinear regression.

Protein Fluorescence-Fluorescence spectra of wt and E138Q pro- teins were recorded on a Perkin-Elmer LS 50 spectrofluorimeter at 20 "C in 1 x 1-cm cells. Excitation was at 282 nm (3-nm slit), and the emission spectra were recorded from 290 to 440 nm (8-nm slit). Samples were dissolved in 0.1 M HEPES pH 7.0 at a final concentration of 28 pg/ml for wt and 44 pgml for E138Q mutant. Solutions in 8 M urea, 0.1 M HEPES pH 7.0 at the same final enzyme concentration were prepared and the spectra recorded at different times (0, 1, 3, and 3% h).

Antiserum-Polyclonal antibodies were obtained by immunizing a 4-month-old New Zealand White female rabbit by three consecutive subcutaneous injections. The first contained 200 pg of pure enzyme in 1 ml when Freund's complete adjuvant was added. The other two had incomplete Freund's adjuvant and 100 pg of p-1,3-1,4-glucanase in the same final volume. The rabbit was injected 10 and 40 days after the first injection and bled 10 days later. The isolated serum was titrated by standard enzyme-linked immunosorbent assay methods (Harlow and Lane, 1988) using goat anti-rabbit Ig conjugated to horseradish peroxi- dase (Nordic Immunology).

Zmmunoblotting-Western blots of extracellular extracts of strains expressing mutated genes were performed according to standard pro- cedures (Harlow and Lane, 1988). After SDS-polyacrylamide gel elec- trophoresis, proteins were transferred to nitrocellulose (Schleicher & Schuell). The blots were rinsed and blocked with phosphate-buffered saline-Tween, 1% bovine serum albumin. After incubation with anti-p- 1,3-1,4-glucanase serum, followed by incubation with horseradish per- oxidase coupled to anti-rabbit Ig, the blots were stained with the 4-chloronaphthoVH202 reagent.

TCCTAGAAATTCGATG-3'; D168N, 5"AACCTTGGTTTT&4TGCAG-

RESULTS

Choice of Mutagenized Residues-10 out of the 18 carboxylic residues (8 Glu and 10 Asp) present in the B. licheniformis @-1,3-1,4-glucanase are strictly conserved among the Bacillus isozymes (Fig. 1) and also in the closely related enzyme from Clostridium thermocellum. Six other residues, Glu-37, Asp-51, Asp-89, Asp-179, Asp-190, and Glu-220, are located in highly conserved positions, whereas Asp-128 is replaced by a histidine in the B. macerans and Bacillus polymyxa enzymes and Glu-75 is only maintained in the most homologous Bacillus subtilis and B. amyloliquefaciens proteins. The region between Trp-132 and Asp-168 shows an extensive sequence identity where most of the conserved Asp and Glu are located. Individual replace- ment of these acidic amino acids into the isosteric Asn or Gln will allow the identification of the residue functioning as the proton donor only if one mutant enzyme is inactive while the remaining replacements yield functional proteins.

Mutations at positions Asp-133, Glu-134, Glu-160, and Asp- 179 have already been reported (Planas et al., 1992a1, but they are included in the tables for comparison. The other mutated residues are Glu-92, Glu-105, Asp-136, Glu-138, Asp-168, and Asp-219, covering all the strictly conserved carboxylates, and Asp-51, Asp-89, and Asp-190 as highly conserved.

Screening Mutagenesis-The named mutants were prepared by site-directed mutagenesis using the PCR methodology by Landt et al. (1990) and a polymerase with 3'-.5' proofreading activity (Deep Venta). 50 clones for each mutagenic reaction were screened on plates by the Congo red activity assay with barley p-glucan as substrate. Fig. 2 shows the activity halos of one selected clone for each mutagenized plasmid. In addition to E134Q already identified as an essential residue, only the bac- terial colony containing the E138Q mutation lacks the activity halo. Specific activities could not be quantified because of the non-homogeneous growth of all the colonies on the plate. A preliminary characterization was carried out by determining the activity of extracellular extracts of the mutant clones grown to stationary phase. Expression of recombinant p-1,3-1,4-glu- canase in E. coli is constitutive for wt and other mutants, being 60-70% of the total enzyme secreted to the extracellular me- dium (Planas et al., 1992a). Western blots using an antiB. licheniformis ~-1,3-1,4-glucanase serum showed that the ex- pression level of the inactive E138Q mutant was similar to other mutants (Fig. 3). Relative activities to wt, determined with barley p-glucan as substrate, are compiled in Table I.

Three types of mutants were obtained. Seven clones retained at least 60% of the wild type activity; D51N, E92Q, D133N, and E160Q had reduced activity (3-30%); and E134Q, D136N, and E138Q were nearly or completely inactive under the assay conditions.

Enzyme Purification and Kinetic Characterization-D136N, E138Q, and D143N mutant proteins were purified up to 95% purity as judged by SDS-polyacrylamide gel electrophoresis, following the procedure described for the wt enzyme (Planas et al., 199213). Catalytic parameters were determined a t 55 "C and pH 7.0 (Table 11); this corresponds to the optimal conditions for the wt enzyme and is identical to those used in the previous characterization of D133N, E134Q, E160Q, and D179N pro- teins (Planas et al., 1992a). The Asn for Asp replacement at position 143 led to an enzyme with 65% of wt V,, and a slightly lower K,. E138Q is completely inactive, whereas D136N re- tains 0.5% activity (in terms of v,,) and has a K, similar to wt. D136N and E138Q were also assayed at lower temperatures (Fig. 4). The residual activity of D136N parallels the profile of the wt enzyme, and E138Q shows essentially no activity over the entire temperature range (25-65 "C), indicating that the phenotype of the inactive mutant was not due to thermal inac-

14532 Active Site Carboxylic Residues of 1,3-1,4-P-Glucanase

1 B. lich MSYRVKRULM LLVTGLFLSL B .mac .. .MKKKSCF TLVTTFAFSL B . amy .... MKRVLL ILVTGLFMSL B . sub . MPYLXRVLL LLVTGLFMSL B.pOly .."KKKSWF TLMITGVISL B .brev .. .MVKSKYL VFISVFSLLF C.therm ... MKNRVIS LLMASLLLVL

92 105 B.lich CGBNRSVQTY GYGLYEVNMK B.mac CABYRSTNIY GYGLYgVSMK B.amy CGENRSVQTY GYGLYBWK B.Sub CGENRSVQTY GYGLYEVRMK B.pOly CGEYRSTNNY GYGLYBVSMK B.brev AGELRTNDFY HYGLFEVSMK C.therm SGBYRTKSFF GYGYYgVRMK

190 B.lich YVDGQLKHTA TTQIPQTPGK B.mac YVDGVLKHTA TANIPSTPGK B.amy YVDGQLKHTA TTQIPAAPGK B.sub YVDGQLKHTA TNQIPTl'LGK B.pOly YVDGVLKHTA TTNIPSTPGK B.brev YVNGEAVHTA TENIPQTPQK C.therm W K K W R G TRNIPVTPGK

30 51 STFAAS.... .ASAQT.... GGSFYEPFNNYNTGLWQKAD GYSNGNMFNC I...FS.... .VSALA.... GSVFWEPLSYFNRSTWEKAD GYSNGGVFNC CGITSS.... .VSAQT.... GGSFFEPFNSYNSGLWQKAD GYSNGDMFNC FAVTAT.... .ASAQT.... GGSFFDPFNGYNSGFWQKAD GYSNGNMFNC F...FS.... .VSAFA.... GNVFWEPLSYFNSSWQKAD GYSNGQMFNC GVFWGFSHQ GVKAEEERPM GTAFYESFDAFDDERWSKAG WJTNGQMFNA SVIVAPFYKA EAATW.... NTPFVAVFSNFDSSQWEKAD .WANGSVFNC

134 138 PAKNVGIVSS FFTYTG..PT DGTPWDEIDIEFLGKDTTKV QFNYYTNGVG PAIWTGIVSS FFTYTG..PA HGTQWDEIDIEFLGKDTTKV QFNYYTNGVG PAKNTGIVSS FFTYTG..PT EGTPWDEIDIEFLGKDTTKV QFNYYTNGAG PAKNTGIVSS FFTYTG..PT DGTPWDEIDIEFLGKDTTKV QFNYYTNGAG PAKNTGIVSS FFTYTG..PS HGTQWDEIDIEFLGKDTTKV QFNYYTNGVG PAKVEGTVSS FFTYTGEWDW DGDPWDEIDIBFLGKDTTRI QFNYFTNGVG AAKNVGIVSS FFTYTG..PS DNNPWDEIDIEFLGKDTTKV QFNWYKNGVG

219 IMMNLWNGAG VDEWLGSYNG VT.PLYAHYNWVRSTKR... .......... IMMNLWNGTG VDDWLGSYNG AN.PLYAEYDWKYTSN... .......... IMMNLWNGTG VDDWLGSYNG VN.PIYAHYDWMRYRKK... .......... IMMNLWNGTG VDEWLGSYNG VN.PLYAFYDWVRYTKK... .......... IMMNLWNGTG VDSWLGSYNG AN.PLYAEYDWKYTSN... .......... IMMNLWPGVG VDGWlCVFDG DNTPVYSYYDWVRYTPLQNY QIHQ ...... IMMNLWPGIG VDEWLGRYDC: RT.PLQAEYEWKYYPNGVP QDNPTPTPTI

89 TWRANNVSMT SLGEMRLSLT S..PSYNKFD TWRANNYNFT NEGKLKLGLT S . . SAYNKFD TWRANNVSMT SLGEMRLALT S . . PSYNKFD TWRANNVSMT SLGEMRLALT S . . PAYNWD TWRANNVNFT NDGKLKLSLT S. . PANNKFD TWYPEQV..T AELMRLTIA KKTTSARNYK VWKPSQVTFS N-GKMILTLD REYGGSYPYK

160 168 179 NHBKIVNLGF DAANSYHTYA FDWQPNSIKW GHEKVISLGF DASKGFHTYA FDWQPGYIKW NHBKFADLGF DAANAYHTYA FDWQPNSIKW NHEKIVDLGF DAANAYHTYA FDWQPNSIKW GHEKIINLGF DASTSFHTYA FDWQPGYIKW GNEFYYDLGF DASESFNTYA FEWREDSITW GNBYLHNLGF DASQDFHTYG FXWRPDYIDF

..............................

..............................

..............................

..............................

..............................

.............................. APSTPTNPNL PLKGDVNGDG * * * * * * * *

FIG. 1. Amino acid sequence alignment of highly conserved bacterial ~-1,3-1,4glucanases. B. lich, B. licheniformis (Lloberas et al.,

McConnell, 1983); B. poly, B. polymyxa (Gosalbes et al., 1991); B. breu, Bacillus brevis ( h u w et al., 1993); C. therm, C. thermocellum (Schimming 1991); B. mac, B. macerans (Borriss et al., 1988); B. amy, B. amyloliquefaciens (Hofemeister et al., 1986); B. sub, B. subtilis (Cantwell and

et al., 1992). Amino acids corresponding to the signal peptides are in italics. The mutated positions are indicated in boldface characters. The C. thermocellum sequence is not complete on the C-terminal end.

wt I , D51N

El 05Q

Dl 33N

El 60Q

Dl 68N

Dl 36N FIG. 2. Activity on plate of the mutant clones. Congo red activity

halos (clearing zones) on a plate of E. coli HBlOl cells expressing the mutagenized genes coding for B. licheniformis P-l,3-1,4-glucanase are shown. The plate contained 0.04% barley P-glucan, and the colonies were grown for 8 h a t 37 "C before staining for activity.

tivation. Nevertheless, the inactive E138Q mutant presents a folded structure as was evidenced by its fluorescence spectra (Fig. 5). Both w t and E138Q had similar emission spectra, and their fluorescence intensities were partially quenched upon ex- posure to 8 M urea.

DISCUSSION Glycosidases operate by a single displacement reaction (in-

verting enzymes) or by a double displacement mechanism (re- taining glycosidases) where the departure of the aglycon is facilitated by general acid catalysis (Koshland, 1953; Sinnott, 1990). Asp, Glu, and Tyr residues, but not His, have been iden-

1 M 2 3 4 5 6

ing the D136N and E1386 mutants. Three different clones express- FIG. 3. Western blot of extracellular extracts of clones express-

ing D136N are in lanes 1,2, and 3, and clones expressing E1386 are in lanes 4,5, and 6. M, molecular weight markers (prestained, Boehringer Mannheim).

tified as proton donors in those enzymes for which three-di- mensional structure determinations, chemical labeling, and site-directed mutagenesis studies have provided cumulative evidence of the involvement of single amino acid residues in catalysis (Svensson and S~gaard, 1993). Replacement of the general acid Asp or Glu in human lysozyme (Muraki et al., 19871, T4L (Anand et al., 1988), hen egg white lysozyme (Mal- colm et al., 1989), cellobiohydrolase I1 from Dichoderma reesei (Rouvinen et al., 1990), glucoamylase (Sierks et al., 1990), endo- p-glucanase CelD from Clostridium thermocellum (Chauvaw et al., 19921, and xylanase (Luthi et al., 1992) leads to essen- tially complete inactivation. Only for p-galactosidase, a residue other than an Asp or Glu was proven to be the proton donor (Tyr residue) (Ring and Huber, 1990).

Following the protonation of the glycosidic oxygen by the general acid, the mechanism of retaining glycosidases involves the development of a positively charged oxocarbonium ion that

Active Site Carboxylic Residues of 1,3-1,4-/.3-Glucanase 14533

TABLE I Enzyme activity in extracellular extracts of mutant clones

E. coli HBlOl containing the mutagenized plasmids were grown to stationary phase, and the activity of the cell-free extracellular media was assayed by the dinitrosalicylic acid method with 5 mg/ml barley

tive to wild type. Values are the average of three determinations with p-glucan a t 55 "C. Percentages of residual activity are expressed rela-

independent clones.

Mutant Activity Mutant Activity

wt D51N D89N E92Q E1056

E134Q D133N

D136N

I 100 30 85

3 50 15 0.2 0.5

I

E138Q 0 D143N El60Q

65 15

D168N 60 D179N 80 D190N 70 D219N 100

TABLE I1 Kinetic parameters of purified wt and mutant B. licheniformis

p-1,3-1,4-glucanases Conditions were as follows: phosphate citrate buffer, pH 7.0, 55 "C,

0.2-9 mg/ml barley p-glucan, and 15-80 ng/ml enzyme (E134Q, D136N, and E138Q were assayed at 1-5 pg/ml).

Enzyme vm, Km

wt D133N" E134Q" D136N E138Q D143N E160Q"

% 100 20 <0.3

0.5 0

65 30

mg I ml 1.30 k 0.15 1.75 f 0.25

1.40 r 0.22

0.85 f 0.20 7.0 t 1.20

Planas et al. (1992a).

I 1

20 - 20 30 40 50 60 70

D136N E138Q T PC1 rn 14 0 - m Y

4 I

20 30 40 50 60 70

Temperature (*C) FIG. 4. Activity versus temperature profile of wt (O), D136N (O),

and E138Q ( 0 ) mutant P-1,3-1,4-glucanases. Activities are given in

cation of the D136N (0) and E138Q ( 0 ) activity-temperature profile. percentage relative to wt maximum activity (at 55 "C). Inset, magnifi-

Conditions: citrate-phosphate buffer, pH 7.0, 6 mg/ml barley p-glucan; enzyme concentration, 10 ng/ml for wt, 0.46 pg/ml for D136N, and 4.6 pg/ml for E138Q.

is stabilized by a nucleophilic amino acid at the catalytic site (Asp or Glu) either by electrostatic stabilization or by formation of a covalent glycosyl-enzyme adduct (Svensson and Sogaard, 1993).

The initial prediction of putative active site residues of Ba- cillus @-1,3-1,4-glucanases was based on sequence similarity to the sequence surrounding the catalytic Glu-11 and Asp-20 of T4 lysozyme (Borriss et al., 1990). For the B. licheniformis enzyme, Glu-134 matches the position of the general acid Glu-11 of T4L, and Asp-143 corresponds to the catalytic nucleophile Asp-20 in

286 334 382 430 286 334 382 430

Wavelength (nm) Wavelength (nm)

FIG. 5. Fluorescence spectra of wt and E138Q mutant P-1,3-1,4- glucanases. Curves in both plots are: 1, native enzymes in 0.1 M HEPES pH 7.0; 2, in 8 M urea, 0.1 M HEPES pH 7.0 after 1 h; 3, same as curve 2 after 2 h; and 4, same as curve 2 after 31/2 h. Enzyme concentration was 28 pg/ml for wt and 44 pg/ml for E138Q.

TABLE I11 Spatial neighboring residues to mutated Asp and Glu in

B. licheniformis ~-1,3-1,4-glucanase based on three-dimensional structure of hybrid H(A16M)

Residues and numbering are for the B. licheniformis enzyme.

Residue Distance to Neighboring residuesb active site" (d < 4.5 A)

Asp-51 Asp-89

Glu-92

Glu-105

Asp-133

Glu-134

Asp-136

Glu-138

Asp-143

Glu-160

ASP-168

Asp-179 Asp-I90 Asp-219

A 22.5 21.5

11.0

22.5

14.5

4.5

4.5

3.5

18.5

8.0

21.5

21.5 23.0 16.5

Ala-50, Gly-52, Trp-63, Arg-64, Ala-65,' H,O' Thr-62, Arg-64," Asn-67, Thr-81, Pr0-83~(S'),

Ala-50, Tyr-53, Trp-63, Gly-91d(A),Asn-93d(Y), Phe-88, Cys-90, H,O'

Arg-94," Met-209, Met-210, A~n-211 ,~ H,O,' H,Oe

Leu-103, m-104 , Val-106, Thr-175, 'Qr-176, Ala-177, Trp-237, Arg-23gd(K), H,O,C H,O'

Thr-124. Pro-131d(~). lh-132. Glu-134, Tyr-152, Thr-I53,-&n-i54', Pro-202, Gln-203d(Sc), Thr-204"

Phe-121, Thr-122, Tyr-123, Trp-132,'ASp-133, Ile-135, Asp-136,' Tyr-152, Pro-202, H20c

Phe-120, Phe-121, Glu-134,' Ile-135, Ile-137, Glu-138, Asn-150, 'Qr-151

Val-117, Ser-119, Asp-136, Ile-137, Phe-139, Leu-140, Gln-148, Trp-221, H,O,' H,O'

Lys-112, Leu-140, Lys-142, Thr-144, Thr-145,'

Gln-148, Phe-149, Asn-150, Asn-I58'j(G),

Lys-112, Thr-144,' Thr-145, Ala-17Od(SC),

Leu-103, Gln-181,' Se1--184~(Y), lj~-188, H,OC Phe-167, His-174, Thr-175, Val-189 Ser-224, H,O'

LYS-146

His-159, Lys-161, H,O,' H,O'

Asn-171d(K)

Average distance between both carboxyl oxygens of the correspond- ing Asp or Glu residue and the glycosidic oxygen of the scissile linkage of a hypothetic substrate modeled into the active site.

Residues at a distance shorter than 4.5 A to the side chain atoms of the corresponding Asp or Glu residue. The residue found in the crystal structure of H(A16M) at a d < 4.5 A is denoted in parentheses.

Hydrogen bonding residue to the carboxylate. Residue that is not conserved in the B. Zicheniformis and H(A16M)

enzymes.

T4L. Cumulative evidence has demonstrated that this is not the case. (i) Glu-134 (B. licheniformis) (Planas et al., 1992a) or the equivalent Glu-105 (B. macerans) (Keitel et al., 1993) proved to be essential by site-directed mutagenesis, but it is likely to be the catalytic nucleophile as demonstrated by mechanism-based affinity labeling of the B. amyloliquefaciens enzyme (Hgj et al . , 1992). (ii) The recently determined three- dimensional structure of a covalent protein-inhibitor complex of a hybrid @-1,3-1,4-glucanase (B. amyloliquefaciens-B. mac- erans) with 3,4-epoxybutyl-~-~-cellobioside (Keitel et al., 1993)

14534 Active Site Carboxylic Residues of 1,3-1,4-P-Glucanase

I 135 CDl ' J

\

Q 148 CA

I 135 CD1

FIG. 6. Structure of the B-strand framework with the DroDosed active site catalytic residues. The protein coordinates are for the H(A16M) crystallographic stkcture (Keitel et al., 1993) (see tegt). - showed that the inhibitor binds to the corresponding Glu-105. (iii) As shown here, Asp-143 (B. licheniformis), tentatively aligned with the nucleophile Asp-20 in T4 lysozyme, is not essential since the mutation D143N leads to retention of 65% of the wt activity.

Considering that carboxylic residues act as general acids in most of the glycosidases, the search for the residue that might function as the proton donor was limited to Asp and Glu. The B. licheniformis enzyme has a quite broad pH optimum with pK, values on V,, of 4.8 and 8.7 for the enzyme-substrate complex (barley p-glucan as substrate, data not shown). Being the low pK, due to the catalytic nucleophile Glu-134, the high pK, of the proton donor may not be represented by an Asp residue but rather by another glutamic acid residue.

Among the mutations studied, only those at positions 134 and 138 inactivated B. licheniformis p-1,3-1,4-glucanase; therefore, Glu-138 is likely to be the general acid. The low activity of the D136N mutant points to an important role of Asp-136, which is in close proximity to both catalytic residues. The recent determination of the three-dimensional structure at 2.0-A resolution of a hybrid p-1,3-1,4-glucanase consisting of amino acids 1-16 of B. umyloliquefuciens and the rest derived from B. maceruns (H(A16M))' (Keitel et al., 1993) provides the structural basis to analyze the effect of each mutation. The H(A16M) and B. licheniformis enzymes share 77% of sequence identity. Molecular modeling based on the H(A16M) crystallo- graphic structure (data not shown) suggests that only minor local adjustments are required to model the latter. Because of the close similarity, the crystal structure is used in the follow- ing discussion, A hypothetic substrate was built into the cleft of the free enzyme to position approximately the scissile glyco- sidic bond by using the crystallographic structure of the cova- lent enzyme-inhibitor complex between H(A16M) and 3,4- epoxybutyl-p-D-cellobioside. Distances from the modeled glyco- sidic oxygen to the carboxylate oxygens of the Asp and Glu

The H(A16M) crystallographic structure (codes lAYH and 1BYH) has been deposited in the Protein Data Bank, Brookhaven National Laboratory, Upton, NY.

residues are summarized in Table 111, which also includes the spatial neighboring residues to the conserved Asp and Glu that have been mutated in the B. licheniformis enzyme. No signifi- cant changes in the atomic coordinates are observed between the structure of the free H(A16M) protein and the enzyme- inhibitor complex, so that the above distances are significant, even allowing for moderate de5ations. Only Glu-134, Asp-136, and Glu-138 are closer than 5 A to the catalytic site centered at the scissile glycosidic bond. Except for Glu-160, the rest are more than 10 apart, and therefore they do not participate in catalysis.

As reported previously (Keitel et ul., 1993), the polypeptide stretch between Trp-132 and Leu-140 (B. licheniformis num- bering) is in a p-strand and has three acidic side chains (Glu- 134, Asp-136, and Glu-138) pointing toward the cleft. Shown in Fig. 6 is a stereo view of this stretch that is presented as the catalytic machinery. In the free enzyme, the side chain of Asp- 136 hydrogen bonds to the carboxylate of Glu-134, whereas Glu-138 forms at least two hydrogen bonds with water mol- ecules located within the active site cleft. When compared with the structure of the enzyme-inhibitor complex where the cor- responding Glu-134 is covalently bound to the aglycon of the inhibitor, Asp-136 exchanges the former hydrogen bond for a new interaction with the carboxylate of Glu-138, concomitant with the loss of water molecules from the cleft. Although yet unknown, this hydrogen bond pattern may be responsible for the different pK, values of the nucleophile and general acid together with the different hydrophobic environment of both residues. The mutational analysis reported here fits into this picture of the catalytic machinery. Replacement of the carbox- ylic side chain of Glu-138 completely abolishes activity, which is consistent with its proposed role of general acid. Asp-136 is important but not essential since the mutation D136N retains some activity (0.5%). It may assist in catalysis as a neighbor group affecting the microscopic pK, values of the catalytic resi- dues. Asp-133, previously shown to have an effect on the en- zyme activity (Planas et ul., 1992a1, does not directly partici- pate in catalysis. Its side chain is oriented away from the active

Active Site Carboxylic Residues of 1,3-2,4-P-Glucanase 14535 site cleft because it is part of the same @-strand 132-140. The mutation E92Q significantly reduces the activity (to 3% of wt, Table I), but it does not interact with catalytic residues. How- ever, as observed in the H(A16M)-inhibitor complex structure, the cellobiose unit of the inhibitor forms a hydrogen bond be- tween the 6-hydroxyl group and the side chain of Glu-92. It may therefore be important for substrate binding to position the substrate or to lower the Kd for productive binding. Likewise, the side chain of Glu-160 is located on a subsite of the cleft that might accommodate a glucose moiety on the reducing end site of a polymeric substrate. That subsite is empty in the H(A16M)- inhibitor complex since the inhibitor only partially fills the cleft. The reduced activity and higher K, of the E160Q mutant assayed with barley e-glucan may reflect the participation of Glu-160 in substrate binding when the cleft is entirely occu- pied. The rest of the mutated positions (Table I) have little effect on activity and are located away from the active site cleft.

The fidelity of this analysis for the B. Zicheniformis enzyme based on the H(A16M) structure is somehow supported by in- spection of the neighboring residues to the conserved acidic amino acids in the three-dimensional structure when aligning the primary sequences. As shown in Table 111, most of the residues a t a distance shorter than 4.5 A to the carboxylate groups in H(A16M) are conserved in the B. licheniformis en- zyme. Only Pro-83, Gln-203, and Ala-170 in the latter substi- tute three serines in H(A16M) that seem to form hydrogen bonds to Asp-89, Asp-133, and Asp-168, respectively.

H0j and co-workers (Chen et al., 1993) have recently reported the identification of catalytic amino acids of barley P-1,3-1,4- glucanase isozyme 11. This enzyme is not related to the bacte- rial p-1,3-1,4-glucanases, but it possesses a high degree of structural similarity to barley 1,3-/3-glucan endohydrolase. Carbodiimide-mediated modification identified Glu-288 as the putative proton-donating residue, whereas mechanism-based affinity labeling with epoxyalkyl P-oligoglucosides revealed Glu-231 as the catalytic nucleophile. Once more, the general acid catalyst is represented by a Glu residue as here also pro- posed for the Bacillus endo-1,3-1,4-/3-~-glucan 4-glucanohyd1-0- lases.

Acknowledgments-We are indebted to Dr. Udo Heinemann (Freie Universitat Berlin) for providing the coordinates of the hybrid p-1,3- 1,4-glucanase before they were released to the Brookhaven Protein Data Bank, for having T. D. in the laboratory for a preliminary model- ing of the B. licheniformis enzyme, as well as for helpful discussions.

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