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Protein Engineering vol.9 no.12 pp. 1191-1195, 19% Identification of two glutamic acid residues essential for catalysis in the {3-glycosidase from the thermoacidophilic archaeon Sulfolobus solfataricus Marco Moracci1, Luisa Capalbo2, Maria Ciaramella and Rossi3 Institute of Protein Biochemistry and Enzymology—CNR, Via Marconi 10, 80125, Naples, Italy 2 Present address: Biology Department, University of Utah, Salt Lake City, UT 84112, USA 3 Dipartimento di Chimica Organica e Biologica, Universita di Napoli, Via Mezzocannone 16, 80134 Naples, Italy 'To whom correspondence should be addressed The Sulfolobus solfataricus, strain MT4, p-glycosidase (SsPgly) is a thermophilic member of glycohydrolase family 1. To identify active-site residues, glutamic acids 206 and 387 have been changed to isosteric glutamine by site-directed mutagenesis. Mutant proteins have been purified to homogeneity using the Schistosoma japonicum glutathione Stransferase (GST) fusion system. The proteolytic cleavage of the chimeric protein with thrombin was only obtainable after the introduction of a molecular spacer between the GST and the SsP-gly domains. The Glu387 -> Gin mutant showed no detectable activity, as expected for the residue acting as the nucleophile of the reaction. The Glu206 —» Gin mutant showed 10- and 60-fold reduced activities on aryl-galacto and aryl-glucosides, respectively, when compared with the wild type. Moreover, a significant A^m decrease with p/o-nitrophenyl-P-n-glucoside was observed. The residual activity of the Glu206 -> Gin mutant lost the typical pH dependence shown by the wild type. These data suggest that Glu206 acts as the general acid/base catalyst in the hydrolysis reaction. Keywords: chimeric enzymes/p-glucosidase/glycosyl hydrolase active site/site-directed mutagenesis/'Sulfolobus solfataricus Introduction The Sulfolobus solfataricus P-glycosidase (SsP-gly; EC 3.2.1.x) shows broad specificity against (3-(l-4)-, P-(l-3)- and p-(l-6)-(9-glucosides, and remarkable exo-glucosidase activity against oligosaccharides, which are hydrolyzed from the nonreducing end (Nucci et al., 1993). This enzyme hydrolyzes P-glycosides, maintaining the anomeric configuration of the substrate; for this reason it can be classified as a retaining glycosyl hydrolase (Moracci et al., 1994). Like other enzymes from hyperthermophilic Archaea, Ssp-gly is extremely stable to heat with a half-life of 48 h at 85CC, and displays optimal activity at temperatures >85°C (Moracci et al., 1995a). The amino acid sequence places the enzyme in glycosyl hydrolase family 1 (Henrissat, 1991), along with archaeal, bacterial and eukaryal enzymes. Recently the protein has been crystallized in its native form (Pearl et al., 1993), and resolution of its 3-D structure is currently under way (C. Aguilar et al., manuscript in preparation). Retaining glycosidases operate by means of a two-step reaction involving a glycosyl-enzyme intermediate, supported © Oxford University Press by two residues in the active site (often carboxylic acids), where one acts as a general acid/base catalyst and the other as a nucleophile (Sinnot, 1990). In the Agrobacterium P-glucosidase (Abg), belonging to family 1, the attacking nucleophile and the general acid/base have been identified as conserved glutamic acid residues by covalent modification with a suicide substrate and by site-directed mutagenesis, respectively (Withers et al., 1990; Wang et al., 1995). 3-D structure comparisons and hydrophobic cluster analyses have shown that this system of two catalytic glutamic acid residues in a conserved a/p-barrel fold is common to several glycosyl hydrolase families (Henrissat etal, 1995; Jenkins et al, 1995). More recently, the resolution of the 3-D structure of two glycohydrolases of family 1 confirmed these observations, showing that the two catalytic residues are separated by the distance expected for retaining glycosidases. The 3-D structure also provided an insight into the active site of the enzymes of this family (Barret et al., 1995; Wiesmann et al., 1995). In particular, the analysis of the active centre confirms the observation of Davies and Henrissat (1995), that small differences in the structure rather than the global fold may be responsible for the large variety of substrate specificities observed among glycosidases. Here we report the construction of SsP-gly mutants which are severely impeded in their activity. A kinetic analysis provides evidence that the mutated residues act as the activesite nucleophile and the general acid/base catalyst. Materials and methods Protein expression and purification Wild-type and mutant enzymes were expressed as fusions with glutathione S-transferase (GST). Fusion proteins were purified from Escherichia coli JM 105 transformed with vectors pGEXGly and pGEX-K-Gly, which were constructed by inserting the S.solfataricus lacS gene from pDAFl (Moracci et al., 1995a) into pGEX-2T and pGEX-2TK (Pharmacia, Uppsala, Sweden), respectively. Bacterial strains expressing GST-Ssp-gly chimeric proteins were grown in Luria-Bertani medium with 50 ug/ml ampicillin at 37°C to 0.6 OD at 600 nm, induced by adding 0.1 mM isopropyl-p-D-thiogalactopyranoside and grown overnight. Cells were harvested by centrifugation at 4000 g at 4°C, resuspended in buffer A [50 mM sodium phosphate buffer, pH 7.3; 150 mM NaCl; 1% (v/v) Triton X-100], and broken by three passes through a French press (American Instrument Company, Travenol Laboratories Inc., Silver Spring, MD). The extract was clarified by centrifugation at 17 000 g, passed through a 0.45 (im filter and loaded onto a glutathione sepharose 4B column equilibrated with buffer A. Binding of the chimeric proteins was monitored by measuring the GST activity in the fractions at room temperature, according to the manufacturer's instructions (Pharmacia). The column was eluted widi buffer B (0.5 M Tris-HCl, pH 8.0; 10 mM reduced glutathione), and fractions displaying GST activity were pooled and incubated with 1191 M.Moracci et aL thrombin protease (10 U/mg protein) overnight at 25°C to separate GST and Ssfi-gly. Thrombin cleavage was monitored by SDS-PAGE. Digested samples were applied to a Superdex 200 HG 26/60 FPLC column (Pharmacia) and eluted in buffer A without Triton but with 1 mM dithiothreitol. The wild-type and mutant enzymes displayed the same retention time on this column. Pooled fractions from gel filtration (which SDSPAGE showed to be >95% pure) were concentrated and dialyzed against 50 mM sodium phosphate buffer, pH 6.5, with 50% (v/v) glycerol, and used in every subsequent characterization. When stored at -20°C, the enzyme preparation was stable for several months. The yield from this procedure was ~ l - 2 mg/1 of bacterial culture. Protein concentrations were determined by the method of Bradford (1976), using bovine serum albumin as standard. Spectrophotometrically, a 10 mg/ml SsP-gly solution in 0.1 M sodium phosphate buffer, pH 6.5, showed an absorbance of 28.78 OD at 280 nm at room temperature. Enzyme characterization Standard assays of Ssp-gly activity against nitrophenyl-glycoside and disaccharide substrates were performed as reported previously (Nucci et aL, 1993). Kinetic parameters for Ss(3gly were measured at the indicated pHs and temperatures using aryl-glycoside substrate concentrations ranging from 0.05 to 20 mM. The protein concentrations in the reaction mixture were 0.2-2.0 (Xg/ml for the wild type and 2-10 (J.g/ml for the mutants. The effect of pH on activity was studied using 50 mM sodium citrate (pH 3.0-5.5), 50 mM sodium phosphate (pH 6.08.0) and 50 mM potassium chloride/50 mM boric acid (pH 8.210.3) buffers (pH values were measured at the temperature of the assay). The stability of the enzyme at each pH was tested before the assay, and activity values were determined only at pH values at which the enzyme was stable at least for 5 min. Thermal activity experiments were performed as reported previously (Moracci et aL, 1995a). All kinetic data were plotted and refined using the program GraFit (Leatherbarrow, 1992). Mutagenesis The vector pBluMSl used as a template for the site-directed mutagenesis was prepared by ligating a BamH\-Pst\ fragment of pDAFl containing the lacS gene into pBluescript n KS(+) (Stratagene, La Jolla, CA). Site-directed mutagenesis was performed by the method of Mikaelian and Sergeant (1992) based on the PCR. The oligonucleotides used were T7 and 'reverse' sequencing primers (Promega and Stratagene) and the following synthetic oligonucleotide (Primm, Milan, Italy): 5'-TATTATAGCGAGGTCGACGGTATCG-3', where the bold 5' end represents mismatched nucleotides. Mutagenic oligonucleotides, designed according to Kuipers et aL (1991), were purchased from Pharmacia and were as follows (mismatches are in bold): E206Q, 5'-ACTCAACAATGAATCAACCTAACGTTGTT-3'; E387Q, 5'-CTATATGTACGTTACTC A AA ATGGTATTGCGG A-3'. Amplified DNA fragments were purified by agarose gel electrophoresis, digested with Xbal and cloned in vector pGEM3 (Promega). Mutant clones were identified by direct sequencing, and restriction fragments containing the mutation were completely re-sequenced and substituted for the corresponding fragments in the wild-type pGEX-K-Gly. Results and discussion Family 1 of glycosyl hydrolases includes enzymes with different substrate specificities: p-glycosidases with broad specificity, 1192 6-phospho-p-gluco and (3-galactosidases, myrosinases and lactases. These retaining enzymes utilize a double displacement mechanism. During the first step, a carboxylic acid residue acting as the nucleophile displaces the glycosidic oxygen and releases the aglycon, in a process that requires the assistance of a general acid catalyst, resulting in a covalent glycosylenzyme intermediate. During the second step, the general base catalyst promotes water attack to the anomeric centre of the glycosyl-enzyme intermediate, releasing the sugar and the free enzyme. The double role of a general acid/base catalyst is played by the same carboxyl group. The two carboxylic residues essential for catalysis in Sspgly were searched for among the glutamic and aspartic acids conserved in mesophilic and thermophilic members of family 1. The glutamic acid 206 and 387 residues of SsP-gly, found in the two fully conserved motifs Asn-Glu-Pro and Glu-AsnGly (Figure 1), respectively, correspond to the general acid/ base catalyst and the nucleophile identified previously in the active site of Abg (Withers et aL, 1990; Wang et aL, 1995). Glu206 and Glu387 were changed to glutamine by sitedirected mutagenesis. The substitution of glutamate by the isosteric glutamine was chosen to delete the charge without introducing major changes in the local structure of the sites. To facilitate the purification of proteins expected to have impaired enzyme activity, wild-type and mutant enzymes were expressed by fusing their N-termini to the Schistosoma japonicum GST. Unfortunately, the GST-SsP-gly chimeric proteins, expressed by the pGEXGly vector, were found to be resistant to thrombin hydrolysis, thus indicating that the SsPgly N-terminus is not accessible to thrombin. A similar result was reported for a barley P-glucanase (Chen et aL, 1995). This problem was overcome by constructing the pGEX-K-Gly vector in which the recognition sequence of the cAMPdependent protein kinase acts as a spacer between the thrombin cleavage site and the N-terminus of the SsP-gly (Moracci et aL, 1995b). Chimeric proteins expressed from this vector were cleaved efficiently, producing recombinant or 'long' forms with a seven-residue extension at their N-terminus. The recombinant enzyme containing the wild-type Ssp-gly coding sequence did not differ significantly from the native form with regards to the pH optimum, thermostability, thermophilicity and kinetic parameters (data not shown). Hence, the SsP-gly 'long' form was used in the subsequent characterization and will be referred to as 'wild type' throughout this paper. The use of the recognition sequence of the protein kinase as a spacer between domains in GST fusions may be applied to other enzymes with buried N-termini. Table I compares the specific activity of the wild type and of the two mutants on several substrates at 65°C. The Glu387 —> Gin mutant is totally inactive against all the substrates tested. The complete inactivity showed the absence of contamination with the wild-type enzyme, and did not allow reliable estimates of kinetic parameters to be obtained. The mutation did not affect the overall structure of the molecule, as tested by farUV CD spectra at 20 and 75°C (data not shown). The Glu387 is fully conserved in family 1; the mutation of the corresponding glutamic acid residue to glutamine in the Pyrococcus furiosus p-glucosidase led to a reduction in the specific activity of >103-fold (Voorhost et aL, 1995). These data are consistent with the function of Glu387 as the attacking nucleophile. The Glu206 —» Gin mutation strongly affects, but does not completely abolish, glycosidase activity. Although the mutant was found to be completely inactive on disaccharides (Table S.solfataricus [J-glycosidase active site Sgly 1 Sgol 1 Pfu 1 Tm Cth Abg BpA BpB CM Bet Cbg 1 1 1 1 1 1 1 1 HYSFP K F R F O S * GFQSEHGTPG SEDPNTD1YK WVKDPBMU GLVSGOLPEN GPGYWGNYKT FHDNAQKKa KIARLNVBTC HLSFP KGFKFGHSQS GRJSEMGTPG SEDPMSMHV WVHDRBffVS QWSGDLPEN GPGYKNVKR FHDEAEHGL NAVRINVB»S HKFP KHFHFCYSWS GFQFEMGLPG SE.VESDWV HVHWENIAS GLVSGDLPEH GPAYBfl-YO} DHDIA3XGM DCIRGGIEKA WWKKFP HSKITFP HT DPKTLAARFP KTIIHJFP KSEHTFIFP 1O4SFP HSIHHFP FKPLP1SFOD FSDURSCFA EGFLWGVATA KDFIWGSATA GDF1.FGVATA QDFMGTATA ATFUKTSTS KGFLHGAATA SDfWGVATA PGFVFGTASS SYQIEGSPU AYQIEGAYNE SFQIECSTKA AYQIEGAYQE SYQIEGGTDE SYQIEGASNE AYQIEGAYNE AFQYKAAFE DGAGHSIIWT DGKGESTJDR DGRKPSIM* DGRGLSIMJT GWH*>HIUI DGKGESTJOt DGRGMSIWT DGKGPSUDT FSHT PG WKNGDTGDV F S H T . . . . P G fflADGHTGDV FO« PG HVFGRHHGDI FAHT....PG KVFHGDNOW F C Q I . . . . P G KVIGGDCGDV FTHQ... .KR WLYGHHGDV FAHT PG KVKHGDNGMV F n * . . . Y P E lUKDRTHGDV ACDHYNRWE ACDHYHRYEE ACOHYNRWEE ACDSYHRYn ACOHFHHFKE ACDHYHRfEE ACDSYHRVEE AIDEYtRYKE DIEHEW.GV DIKIMKHGI DLDLIKEHGV OIRLHKELQ DVqLMKqLGf DVSUOCELGL DVQLUUXGV DIGIWDMM. KAYRFSISKP KSYRFSISWP EAYRFSLAWP RTYRFSVSKP LHYRFSVAKP KAYRFSIAWT ICVYRFSISUP DAYRFSISWP Sgly 86 Sgal 86 Pfu 8 4 RIFFHPLPRP Q. ..HFDESK CPVTEVHHE NEURLDEYA KKDAUffffiE IFKDLXSRGL YFILNKYWP LPLWLHDPIR VRR.GDFT6P SGWLSTRTVY RIFPRPLPKP EHQTGTDKEN SPYISVDUrt SKLREHONYA ItfEALSHYRH ILEOLRKRGF HIVLNHYWrT LPIWLHDPIR VRR.GDfTGP TGWLNSRTVY RIFPKPTFW KVDVEKDEE. QCIISVDVPE STIKaEKIA WEALEHYRX IYSOKERGK TFILNLYWP LPUflHDPIA VRKLGPORAP AGWLDaCTW Tm Cth Abg BpA BpB CM Bd Cbg RILPEG..TG RIFPEG. .TG RIIPDG..FG RIFPNG. .DG RIWAA...G RIFPOG. .FG RVLPQG. -TG RVLPKQCLSG 84 84 89 84 86 83 84 98 RV KL PI EV II TV EV GV NQKGLDFYHR HQKGLDFYKR NEKOLDFYDR NQEGLDYYHR NEEO.LFYEH KJCGLEFYDR NRAGLDYYHR NREGIKITYNN IIDTLLEKGI LTKLLLEHGI LVDGCKARGI WDLUCNGI LLDHEUGL LINKLVEMGI LVDELLANGI LIKEVLANGM S g l y 1 8 2 EFARFSAYIA HKFDOLVDEY STHMEEHWG GLGYVGVKSG FPPGYLSFEL S S g a l 1 8 5 EFARFSAYVA IKLDOLASEY ATWE0WVW GAGYAFPRAG FPPMITLSFRL S P f u 1 8 3 EFVKFAAFVA YHLDDLVDW SDfJEENWY NOGYINLRSG FPPGYLSFEA A Tun Cth Abg BpA BpB CM Bet Cbg 1 4 2 WFAEYSRVLF 1 4 2 YFTEYSEVIF 1 4 7 AFQRYAKTVM 1 4 2 AFVOFAETW 1 4 3 HFKTYASVI1I 1 4 1 YYFDYANLVI 1 4 2 AFAEYAEU4F 1 5 9 DFRDYA£LCF S g l y 2 6 6 SF S g a l 2 6 9 SY Pfu 2 6 7 WH Tin Cth Abg BpA BpB CM Bet Cbg ENfGDRVXNi KNLGDIVPIW ARLGDRLDAV REFHOaQH* DRFGERIMMI mYKOCVWOI KELGGKIKQW KEFGDRVKHi nLME0»WA FTHiEeGWS ATFME0KAV LTFHE0KIA NTINiEYCAS nFHEEYCIA nFffiPJICm ITI MFPWCTrt •• T HAPGHRDIYV HAPaKDLRT HAPGERtMA HAPGLTN1.QT HAPGHEHWE HAPGIKDFKV HAPGKKDLQl FAPGRCSOTL 2 2 5 YF EP ASEKEEDIRA 2 2 5 YH YP ASEKAEDIEA 2 3 8 SA I P ASDGEAOLKA 2 2 5 WA VP YSTSEEDKAA 2 2 6 HV DA ASERPEDVAA 2 2 4 PVYLQTERLG YKVSEIEREH 2 2 S WA VP YRRTKEDffiA 2 5 7 WF EP ASKEKADVDA VRFMHQFNNY AELSFSLAG. AERAFQFHHG CARTISLHS. AIRRDGRN. VSLSSQLDN. ORVNGSSG. AKRGLDFH.L 3 9 9 KVS FVE R 3 1 9 GFSPA.NSIL. E 3 1 3 EFPATWAPA V 3 9 8 GFLQS.EEIN M 3 1 9 SLLQV.EflVH H 3 1 8 WIFPI.RWEH P 3 1 9 GHLSS.EAIS M 3 4 9 ARPAIQTDSL I PLFLNPIYRG RWYLDPVLKG AFF.OPVFKG DWFLQPTYQG RWFAEPLFNG QLFLDPVUG DWLDPIYFG GWFWPLTKG DYPELVLEFA RYPENAULY EYPAEWEAL SYPOFLVDWF KYPSXVBIY SYPOKLLDYL EYPKFWJ3WY RYPESWYLV AFR AVrtfl. SU VSttiL ALA UQVI AID VOtHL AFT AAttfl A» WHSL AID VStffl. ItLNaGCDSG REPYLAAHYQ RE «KGI GOR AEQGA GT YLN VOXDLLDSQK EHLGY RKR LPfALQL LPIHLQD LPLTLHG LP(>LQD LPTJIED LPqCLOD LPQ4LQD VPQALED K. GGWANRE1AD K. GGWKNROTTD D. GOUSRSTAH A. GGWGNRRTT.Q E. GGWTORETIQ I . GGWAHPEIVN Q. GGWGSRITID EY RGFLGRNIVD RRHHYHI KJWARAYD5 IKSVSWCP.. . . V Q I Y A N S EIAMNI IQAHARAYDA IKSVSKKS.. ..VGIIYANT EKAKFNL IQAHIGAYDA IKEYSEKS.. . .VGVIYAFA QPLTDKOeA VEHA.EfCNR WKFFOAIIRG ETJRGteaV ROD YPLRPQDNEA VEIA.ERLW WSFFDSIIKG ETrSEGQN.V RED DPUEEYKDE VE...EIRHC DYEFVTILHS S g l y 3 3 6 SLGGYGHaE RNSVSUGLP TSDFGWE S g a l 3 3 8 TLPGYGDRCE RNSLSLANLP TSDFGWE P f u 3 2 1 PLPGYGFMSE RGGFAKSGRP ASDFGWE Tna Cth Abg BpA BpB CM Bet Cbg IVGHLY. .GV LLGHR..GI WLSHIY. .GV FLSIM...GV ILGYGT..GE FLGYFH..GI FLSNYL. .GV HNAYAY..GT TPFVTIYMD WAIUYWO (CTYATLYrtH) EPFCTLYHID IPMLTLYHWD EPWTLYWD EPFCTLYHWD (FYVTLFhM) •T LRAHARAVXV LLSHGKAVH. NUHGFGVEA LVAHaSVRR LHCHGIASNL MLSHFKWKA LVAHGRAVTL UAHAAAARL F R . . .ETVXD FR.. .EHNID S R . . .HVAPK F R . . .ELGTS HK.. .EKGLT VK...EIHID FR.. .ELGIS YICTXYVSON GHGIVFNNG AQIGIALNLS VPVaVLHAH GQIGIAPNVS GKIGITLWE VEVGITLNLT GHGIAPHTS GIIGITLVSH T UGRLD WIGVNYYTRT WtCRTEKGYV LRNRLD WIGVNYYTRT WTKAESGYL KGKLD WIGVNYYSRL VY6AKTXH.V YLPEHYKD &SFPE. . . D MPWEAE TVPK3D...G GLDFVQ. .PG ALSMQQEVKE KPPIVD...G LPKFSTE DMSEIQBaD DUOJSOPID DLGIISQKLD CHDIIGEPID WEUQQPGD NFIF....PD DXEUHQPID ESKELTGSFD FF PEGLYDVLTK YWRYH..LY HYVTfliGJA FF PFGIYDVUK YIWRYG..IP 1YVHFMGIA Hi PKLEMJJCY UfWYE. .LP W n f l f i i W DLP laAHGWE IV PEGIYfflUK VKEEYNP.PE VYITQifi.AA KFE KTDHGH IY TCGLYDLLHL LDROYGK.PN IVISfiifi.AA SOV KTDIGWE VY APALHTLVET LYERYD.LPE CYTTafi.AC GLP VTDIGWP VE SRGLYEVLHY .LQKYGN.ID IYITfHS..AC EEP VTDHGWE IH PESFYKLLTR IEKDFSKGLP ILTTHK-AA AGE YTEHGWE VF PQGLFDLLIW IKESYPQ.IP IYnfiJfi.AA GAP KTDIGW IY AEGLYOLLRY TADKYGN.PT LYTTHK.AC KAT FE)«GKPLGP HAASSWLCIY PQGIRXLaY VMWYNN.PV IYnfJfiiRNE »•• FVaKYYSGH FIAFNKYSSE WWGLNYYTPH WGIHYYSHS FLQHYYTRS FLGINYYTRA FIMNYYTSS FLGLNtTSSY LVKFDPDAPA FIKYDPSSES RVADOATPGV VNRFNPE..A IIR..STNDA VRLYD.ENSS HNRYNPGEAG YAAKAPRIPN DDADYQRPYY LVSHVYQVHR DDADYQRPYY LVSHIYQVHR DAADRYRPHY LVSHLKAVYN FDO.WSEOG FKD.HGSHG YIW.GV.ENG IKD.EV.VMG WD.a.VHG YND.IVTEDG YND.GLSLDG FNDPTLSLQE RVHJQNRIDY LKAHIGQAKK KIEDTKRIQY LKDYLTQAHR QVNDQPRLDY YAEHLGIVAD KVQODRRISY MQQHLVQVHR QIEDTGRHGY IEEHLKACHR KVWSKRIEY LKQHFEAARK RimQRRIDY LAMHLIO/kSR SLLDTPRIDY YYRHLYYVLT S g l y 4 1 2 AINSGADVRG YUKSLADNY EWASGFSHRF GLUCVDY.NT O0.YWRPSAL VYReATNGA TTDEIEHmS VPPVKPtRH S g a l 4 1 4 ALNEGVDVRG YLHTSLADMY BISSGFSHRF GU-KVDY.LT KRLYWRPSAL VYREITRSKG IPEELEHLKR VPP1KPLRH P f u 3 9 7 AWCEGADVRG YLH«SLTDMY EWAQGFRHRF GLVYVDF. ET WCRYLRPSAL VFREIATQKE IPEELAHLAD LKFVTRK. . TBO Cth Abg BpA BpB Cto Bet Cbg 3 8 5 AIQEGVPUG 3 8 9 AIQDGVNLKA 3 9 2 LIRDGYPWG 3 8 5 TIHXLHVKG 3 8 9 FIEEGGQLKG 3 9 7 AIENGVDLRG 3 8 9 AIEDGINUCG 4 3 3 AIGDGVNVKG YFVWSLLDHF YYIWSLLDNF YFAWSLHDHF YHAHSLLDHF YFVWSFLDNF YFVWSLMDNF YMEWSLHDNF YFAWSLFDW BMEGYSKRF EHAYGYHCRF BUEGYRHRF BlAEGYtMRF EXAWCYSKRF BIAHGYTKRF BMEGYGWIF EIDSGYTVRF GWYVDY.ST GIVHVHF.DT GLVHVDY.QT G«HVDF.RT GIVHINY. ET GIIYVDY. ET GLVHVDY.DT GLVFVDFKtM QKRIVXDSGY LERHICDSGY QVRTVKHSa QVRTP1CESYY QERTPKQSAL QKRIKKDSFY LVRTPKDSfY LMWKLSAH WYSHWKNNG WYKEVIIOtW WYSALASGFP WYRNWSMH WFKQXMKNG FYQQYIKEHS WYKGV1SRGW WFKSFLKK LED F ICGNHGVAKG LETRR F LOL Fig. 1. Amino acid sequence alignment of enzymes of the glycohydrolase family 1. Alignment was performed using the program PILEUP. Numbers on the left represent the residue numbers of the first amino acid of each line. The glutamic acid residues changed to glutamine by site-directed mutagenesis in SsfJ-gly are indicated by an asterisk. The conserved motives Asn-Glu-Pro and Glu-Asn-Gly are underlined. Residues that make up the active centre in Cbg of T.repens and are invariant in all sequences are labeled by a circle. Trpl38, Glyl86 and Val254 in the Cbg of T.repens are marked by arrows. Abbreviations used and Swiss-Prot Data Bank accession numbers are as follows: Abg, Agrobaclerium sp. fi-glucosidase (P12614); Bci, B.circulans p-glucosidase (Q03506); BpA, B.polymixa fi-glucosidase A (P22073); BpB, B.polymixa p^glucosidase B (P22505); Cbg, T.repens cyanogenic p^-glucosidase; Csa, Caldocellum sacchawlyiicum fi-glucosidase A (P10482); Cth, Clostridium thermoceUum p^-glucosidase A (P26208); Pfu, Rfuriosus P-glucosidase (U37557); Tma, Thermotoga maritima P-glucosidase A (Q08638); Sgal, S.solfaiaricus (strain DSM1616) (5-galactosidase (P14288); Sgly, S.solfataricus (strain MT4) fi-glycosidase (P22498). The amino acid sequence of T.repens Cbg was obtained from the Brookhaven Protein Data Bank because the sequence in the Swiss-Prot Data Bank did not correspond to that reported by Barret et al. (1995). 1193 M.Moracci et al. I), and lacked any transglycosylating activity (M.Moracci and A.Trincone, unpublished results), it showed a residual activity on p/o-nitrophenyl-P-D-glycosides (Table I). Kinetic constants were determined for the Glu206 —> Gin mutant at 65°C and compared with values obtained for the wild type (Table II). A 10- to 30-fold decrease in the Km value and a 60-fold decrease in the k^ value on p/oNpGlu occurred upon mutation, suggesting that the glycosyl-enzyme intermediate accumulates during the reaction, k,.^ values on p/oNp-fJ-D-galacto and fucoside (/j/oNpGal and Fuc) were found to have lower reduction levels. Furthermore, the tccJKm ratio decreased to different extents using the different substrates; whereas values obtained with aryl-gluco- and aryl-fucosides were barely affected (1.6- and 6.0-fold reductions, respectively), a 14- and 33fold reduction occurred respectively with aryl-galactosides. These results suggest that p/oNp-gluco- and -galactoside substrates make different interactions with the Glu206 residue in the Ssp-gly active site. This may reflect the differences in enantioselectivity towards secondary hydroxyl groups of 1,2diols shown by Ssp-gly in the transglycosylation reaction using phenyl-p-D-gluco- and -galactosides as glycoside donors (Moracci et al, 1994). It has been reported that adding small nucleophiles such as azide partially restores the activity of the Abg mutant in which the general acid/base residue has been changed to glycine (Wang et al., 1995). In contrast, the addition of azide and other nucleophiles to the reaction mixture did not significantly affect the activity of the Glu206 -> Gin mutant (data not shown). This difference may be ascribed to the different nature of the substituting residues, because the glycine may permit the access of small nucleophiles to the active site whereas the larger glutamine may not. Proof that the general acid/base catalyst of a glycohydrolase has been removed is indicated by the change in activity versus pH upon mutation. Therefore we determined the pH dependence of the hydrolysis of pNpGal substrate for the wild type and the Glu206 -> Gin mutant at 65°C (Figure 2). The wild-type SsP-gly showed a typical bell-shaped curve, with maximum activity at pH 6.5, suggesting that two amino acid side chains with pKa values of 6.0 and 7.6 are involved in the catalysis. This result is in agreement with the abnormally high pKa value (6.8) found for the glutamic acid acting as the general acid/base in the active site of the Bacillus circulans xylanase (Davoodi et al., 1995). The Glu206 -» Gin mutation strongly affected the activity dependence on pH, confirming that the residual activity measured is due to the mutant itself and not to contamination with the wild type. The low activity of Glu206 —» Gin on pNpGal showed the same thermal activation of the wild type. In addition, no anomalous Arrhenius behavior was detected over the whole temperature range for both enzymes (Figure 3). These results, and far-UV CD spectra at 20 and 75°C (data not shown), suggest that the Glu206 -» Gin mutation specifically affects catalysis with minor changes to the general 3-D structure. The results presented strongly suggest that glutamic acid 206 in Ssp-gly works as the general acid/base catalyst in the hydrolysis reaction. The studies reported so far on glycohydrolases mutated in the general acid/base catalyst demonstrate that the kinetic parameters can be affected to different extents. In Abg, the mutation Glu -» Gly resulted in a lO^fold reduction in the value of kml of the hydrolysis reaction on pNpGlu (Wang et al., 1995); in the Staphylococcus aureus 6-phosphop-galactosidase, the corresponding mutation to glutamine Table I. Specific activity of wild-type and mutant SsfJ-gly* pNpGal oNpGal pNpGlu oNpGlu pNpFuc pNp-tio-Gal pNp-tio-Glu Cellobiose Laminartibiose Gentiobiose Wild type Glu206 -> Gin Glu387 -> Gin 55 6 60.1 54.8 48.0 58.2 NA 3.1 27.1 37.9 36.3 3.9 6.9 0.97 0.94 8.6 NA 0.4 NA NA NA NAb NA MA NA MA HA MA UA NA NA 10 •Values are shown as U/mg at 65°C. b NA (no measurable activity) means that, using concentrations of enzyme of 10 Hg/ml in the assay, the rates of change in absorbance did not vary by altering the substrate concentrations, and were approximately the same as in the control without substrate (typically <0.001 pH Fig. 2. Comparison of the pH dependence on pNpGal substrate of wild-type (O) and Glu206 -> Gin mutant ( • ) SsP-gly at 65°C. Table II. Kinetic constants of hydrolysis at 65°C by wild-type and mutant Glu206 -» Gin Substrate oNpGal pNpGal oNpGlu pNpGlu pNpFuc 1194 Afm(mM) Wild type Mutant Wild type Mutant Wild type Mutant 0.95 1.17 1.01 0.30 0.45 1.53 4.79 0.03 0.03 0.09 295 275 252 240 263 34.0 ± 0.8 31.0 ± 0.4 4.46 ± 0.13 4.40 ± 0.06 35.0 ± 0.4 310 234 251 830 584 22 7 160 132 382 ± 0.08 ± 0.06 ± 0.24 ± 0.04 ±0.11 ±0.10 ±0.17 ± 0.006 ± 0.003 ± 0.005 ± ± ± ± ± 6 4 12 7 12 S.solfataricus fj-glycosidase active site 400 1 1 1 1 1 i 1 r • [• i •- - 100 1° 300 '_ t Jr , £> ' '"-Si -V t t - 60 I 200 < Zt J I 11 - 40 <s 10 I ° - 20 40 60 Temneralure (°C) Fig. 3. Companson of the temperature dependence of wild-type (O, D) and Glu206 —> Gin mutant ( • , • ) Ssp"-gly. Activities are shown as U/mg ( • , 0 ) and as percentage relative to maximum activity at 85°C ( • , • ) . Inset: Arrhenius plot of wild-type (O) and Glu206 -» Gin mutant ( • ) resulted in a 103-fold reduction of the km value on o-nitrophenyl-p-r>galactoside-6-phosphate (Witt et al, 1993). However, in the exoglucanase/xylanase from Cellulomonas fimi, the mutation of the general acid/base catalyst to glycine resulted in only a 200- and 20-fold reduction for the kcat and KJKm values, respectively, on p-nitrophenyl-p-D-cellobioside (MacLeod et al, 1994). The reasons for these differences are not clear; it is possible that enzymes with different substrate specificities require the assistance of the general acid/base catalyst to varying extents (Wang et al, 1995). Non-covalent interactions play a crucial role in the catalysis of glycohydrolases (Namchuk and Withers, 1995); therefore, residues surrounding the fully conserved active-site amino acids may affect the catalytic mechanism. The analysis of the 3-D structure reported recently for a phospho-fi-galactosidase from Lactococcus lactis and a cyanogenic (3-glucosidase from Trifolium repens (Cbg) provided more information on the amino acids involved in the catalysis of family 1 enzymes (Barret et al, 1995; Wiesmann et al, 1995). In Cbg, three residues (Trp, Gly and Val, indicated by an arrow in Figure 1) are thought to contribute to the increase in pKa of the general acid/base catalyst Glul83 (Barret et al, 1995). We noticed that the glycine and valine are not perfectly conserved among family 1 enzymes, and in SsP-gly they are substituted by valine and alanine, respectively. Interestingly, Val209 is invariant in archaeal and bacterial thermophilic (3-glycosidases, and AIa263 is fully conserved among archaeal thermophilic enzymes. 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(1993) Protein Engng, 6, 913-920. Received April 24, 1996; revised July 24, 1996; accepted July 25, 1996 Acknowledgements We thank Mr Giovanni Imperato for his technical assistance, and the helpful critical reading of Dr Carlo A.Raia is gratefully acknowledged. This work was supported by CNR Target Project Biotechnology and Bioinstrumentation, 1195