111] ~ - G L Y C O S I D A S E F R O M S. solfataricus 201

Like other organisms, hyperthermophiles appear to have a larger reper- toire of glycosyltransferases than of glycoside hydrolases. Aeropyrum pernix and Archaeoglobus fulgidus, whose genomes are totally devoid of known glycosidases, contain 5 and 15 potential glycosyltransferases (Table II), respectively. Even if some of the putative glycosyltransferases discovered in genomes turn out to be inactive or not even enzymes, the trend toward glycosyltransferases will probably continue.

There is accumulating evidence for an important gene transfer between archaeal and bacterial hyperthermophiles. 26'27 The most spectacular exam- ple is the recent bacterial genome of Thermotoga maritima bearing 24% of genes most similar to archaeal onesY Even if there is evidence suggesting a lower degree of horizontal transfer from bacteria to archaea, 26 one would expect the majority of glycosidases to be already "sampled" in both archaea and bacteria. In fact, no glycosidase (and transglycosidase) families are purely archaeal, but the reverse is not true. Therefore, the division of Tables I and II into archaeal and bacterial enzymes does not necessarily reflect the "real" origin of a given enzyme.

26 L. Aravind, R. L. Tatusov, Y. I. Wolf, D. R. Walker, and E. V. Koonin, Trends Genet. 14, 442 (1998).

27 K. E. Nelson, R. A. Clayton, S. R. Gill, M. L. Gwinn, R. J. Dodson, D. H. Haft, E. K. Hickey, J. D. Peterson, W. C. Nelson, K. A. Ketchum, L. McDonald, T. R. Utterback, J. A. Malek, K. D. Linher, M. M. Garren, A. M. Stewart, M. D. Cotton, M. S. Pratt, C. A. Phillips, D. Richardson, J. Heidelberg, G. G. Sutton, R. D. Fleischmann, J. A. Eisen, O. White, S. L. Salzenberg, H. O. Smith, J. C. Venter, and C. M. Fraser, Nature 399, 323 (1999).

[I 11/3-Glycosidase f r o m Sulfolobus solfataricus

B y MARCO MORACCI, MARIA CIARAMELLA, a n d M o s E ' R o s s I

Introduction

The hydrolysis of glycosidic bonds is crucial for several cell functions, such as energy uptake, cell wall expansion, and turnover of signaling mole- cules. Because the diversity of saccharides, there is a wide variety of glyco- sylhydrolases that have been classified in more than 74 families, based on amino acid sequence similarities. 1 However, despite the vast assortment of glycosylhydrolases, these enzymes follow only two mechanisms, both

I http://afmb.cnrs-mrs.fr/~pedro/CAZY/db.html

Copyright © 2001 by Academic Press All rights of reproduction in any form reserved.

METHODS IN ENZYMOLOGY, VOL. 330 0076-6879/00 $35.(10

202 SACCHAROLYTIC ENZYMES [ 1 II

involving two carboxyl groups in the active site. One involves the retention and the other the inversion of the anomeric configuration of the substrate. Interest in the structure and mechanisms of these enzymes has increased considerably in recent years because of the potential use of such biocatalysts including stable glycosidases isolated from thermophilic organisms. 2-4 In particular, the/3-glycosidase from Sulfolobus solfataricus (Ss/3-gly) has been studied extensively. This organism is an extremely thermoacidophilic arch- aeon that grows optimally in hot springs at 87 ° and pH 3.0; due to the absence of tools for molecular genetic studies in S. solfataricus, the physiological role of Ss/3-gly is currently unknown. Nevertheless, an increasing amount of structural and biochemical data is becoming available for this enzyme. This article provides a description of two purification methods for Ss/3-gly from recombinant Escherichia coli, the standard assay for its activity, as well as a summary of the functional and structural properties of the enzyme.

Assay

Assays for/3-glycosidase activity are extremely simple and can be per- formed either in liquid solutions or in situ; a wide range of synthetic sub- strates, exploiting either chromogenic or fluorogenic methods of detection, can be used, allowing high sensitivity and precise definition of the substrate specificity. Moreover, the activity of this class of enzymes can be assayed easily on disaccharides by coupling other enzymatic reactions, available in commonly used diagnostic kits, for detection of the monosaccharides pro- duced.

The standard assay for Ss/3-gly is performed at 65 ° in 1 ml of 5 mM 2- or 4-nitrophenyl-fl-D-galactopyranoside (2-/4-Np-/3-Gal) and 50 mM sodium phosphate buffer at pH 6.5. The pH value of the buffer is measured at 20°; although pH can change with temperature, sodium phosphate buffer is less affected by temperature than other systems. Assays are performed by following changes in absorbance with a spectrophotometer equipped with a circulating water bath; the temperature of the assay is measured accurately in the assay cuvette. Quartz cuvettes containing the standard assay mixtures are equilibrated at 65 ° and the temperature is kept constant during all activity measurements. After the addition of 5/zl of Ss/3-gly (0.1 mg/ml), the change at 405 nm is followed for 2 min. One unit of enzyme activity is defined as the amount of enzyme catalyzing the hydrolysis of 1/zmol of substrate over 1 min at 65 °, under the conditions described. The molar

2 A. Sunna, M. Moracci, M. Rossi, and G. Antranikian, Extremophiles 1, 2 (1997). 3 M. M. Bauer, L. E. Driskill, and R. M. Kelly, Curr. Opin. Biotechnol. 9, 141 (1998). 4 B. Henrissat and P. M. Coutinho, Methods Enzymol. 330 [10] (2001) (this volume).

[11] fl-GLYCOSIDASE FROM S. solfataricus 203

extinction coefficients of 2- and 4-nitrophenol measured at 405 nm, 65 ° in 50 mM sodium phosphate buffer, pH 6.5, are 1711 M -1 cm -1 and 9340 M -1 cm -1, respectively, and are used for calculating enzymatic activity. Most of the substrates used are stable to heat; however, a blank mixture containing all the reactants, except the enzyme, is always used to correct for spontane- ous hydrolysis. This method of assay can be adapted to all the Ss/3-gly preparations used, including those from Escherichia coli and S. solfataricus. However, when Ss/3-gly is expressed from E. coli, crude extracts have to be heated for at least 30 min at 65 ° before the assay in order to avoid interference by the endogenous proteins that aggregate during the assay.

When Ss/3-gly is assayed at different temperatures or in differnt buffer systems, the extinction coefficients of 2- and 4-nitrophenol groups have to be measured accurately, as they are sensitive to pH and temperature. Alternatively, the methods generally used for /3-glycosidase can be adapted:

In order to analyze the activity of Ss/3-gly on disaccharides, such as lactose, cellobiose, and laminaribiose, or on glucooligosaccharides, such as cellotriose and cellotetraose, the amount of D-glucose produced in the reaction can be determined by using a coupled assay for its quantitation (i.e., the glucose oxidase/peroxidase-coupled assay). For this purpose, several commercial diagnostic kits can be used and all give precise and reproduc- ible results.

Purification

/3-Glycosidase is expressed in S. solfataricus cells as about 0.1% of the total cell protein. The purification procedure has been modified 6 from the one described earlier 7 and includes several chromatographic steps that allow one to obtain homogeneous protein (specific activity of about 180 U/mg when assayed on 4-Np-Gal at 75 °) with good yields (44%). Both methods give samples of Ss/3-gly with a high degree of purity that can be crystallized successfully. 8 A complete description of the purification of Ss/3-gly from S. solfataricus has been reported previously. 6 However, the recovery from this source does not exceed 15 mg of pure protein per 500 g of frozen archaeal cells. For this reason, Ss/3-gly enzyme is routinely purified from E. coli cells, exploiting the fact that it is expressed efficiently as a soluble protein in

5 S. Hestrin, D. S. Feingold, and M. Sehramm, Methods EnzymoL 1, 241. 6 R. Nucci, M. Moracci, C. Vaccaro, N. Vespa, and M. Rossi, BiotechnoL AppL Biochem.

17, 239 (1993). 7 F. M. Pisani, R. Rella, C. A. Raia, C. Rozzo, R. Nucci, A. Gambacorta, M. De Rosa, and

M. Rossi, Eur. J. Biochem. 187, 321 (1990). 8 L. H. Pearl, A. M. Hemmings, R. Nucci, and M. Rossi. J. Mol. Biol. 229, 558 (1993).

204 SACCHAROLYTIC ENZYMES [ 11 ]

several mesophilic hosts while maintaining the features of the enzyme purified from the archaeon (see later).

Recombinant fl-Glycosidase

Recombinant Ss/3-gly has been cloned and expressed in E. coli, Sacchar- omyces cerevisiae, and Pichia pastoris. T M Ss/3-Gly proteins isolated from E. coli and S. cerevisiae are very similar in enzymatic activity, specificity, and stability to the enzyme purified from S. solfataricus. 1°'12

Bacterial Strains and Growth. We report here a modification of the expression/purification procedure of Ss/3-gly from E. coli described pre- viously, 1° which improves both the plasmid stability and the expression level of the enzyme. In order to remove a noncoding region of about 700 bp located downstream of the Ss/3-gly gene (lacS), NdeI and NcoI sites are added to the 5' and 3' ends of lacS, respectively, by polymerase chain reaction (PCR) amplification. The resulting fragment is cloned in the NcoI- NdeI sites of pET29 (Novagen, Madison, WI), which carries a kanamycin resitance gene, producing plasmid pETGIy29. In this new vector, lacS gene expression is driven by a T7 RNA polymerase-dependent promoter from its first natural codon. Expression is performed in E. coli strain BL21 (DE3), which contains a chromosomal copy of the T7 RNA polymerase gene under the control of an isopropylthio-/3-D-galactopyranoside (IPTG)-inducible lacUV promoter. In order to ensure the integrity of the pETGly29 plasmid in the recombination-competent BL21(DE3) strain, freshly transformed cells are always used instead of cryopreserved E. coli cells. For 2-liter scale preparations, one single colony selected on LB/kanamycin plates is inoculated in 50 ml of LB medium containing kanamycin (30/zg/ml) and is grown overnight at 37 ° with vigorous shaking. This culture is used to inoculate (at 1/100 dilution, v/v) four 2-liter flasks containing 500 ml each of the SB medium supplemented with kanamycin (30/zg/ml) and with 50 /zl of antifoam A (Sigma, St. Louis, MO). Cultures are incubated at 37 ° with vigorous shaking until the OD6o0 is 1.0-1.2, induced with 0.5 mMIPTG, and grown overnight. Kanamycin selection, instead of ampicillin, increases plasmid stability in large-scale cultures.

9 M. Moracci, A. La Volpe, J. F. Pulitzer, M. Rossi, and M. Ciaramella, J. Bacteriol. 174, 873 (1992).

10 M. Moracci, R. Nucci, F. Febbraio, C. Vaccaro, N. Vespa, F. La Cara, and M. Rossi, Enzyme Microb. Technol. 17, 992 (1995).

n S. Fusco, M. Moracci, I. Di Lernia, M. De Rosa, and M. Rossi, unpublished results (1998).

12 S. D'Auria, A. Morana, F. Febbraio, C. Vaccaro, M. De Rosa, and R. Nucci, Protein Exp. Purif 7, 299 (1996).

[11] fl-GLYCOSIDASE FROM S. solfataricus 205

Crude Extracts and Thermal Treatment. Cells collected by centrifugation at 4000g at 4 ° for 15 min and resuspended in 50 ml of 50 mM sodium phosphate, pH 6.8, are broken by three cycles of French Press (American Instrument Company, Travenol Laboratories Inc., Silver Spring, MD). The lysate is clarified by centrifugation at 17,000g at 4 ° for 30 min; DNA is removed by adding 0.5 mg of DNase I and MgSO4 (0.1 mM final concentra- tion) to the supernatant and incubating the sample at 37 ° for 30 min.

The extreme resistance to heat of Ss/3-gly allows one to perform a simple thermal treatment, which is crucial for both the speed and the yield of purification. To this aim, the sample is heated for 30 min at 75 ° and aggre- gated host proteins are eliminated by centrifugation at 27,000g at 4 ° for 30 min. Finally, the supernatant (90 ml) is filtered through 0.45-t~m filters (Millipore, Bedford, MA), After this step, more than 5000 total units of Ss/3-gly at the specific activity of about 26 U/mg can be recovered, as assayed under standard conditions (4-Np-Gal substrate at 65°). Because of the interference produced in the standard assay by endogenous proteins in the crude extract, which aggregate at 65 ° , it is not possible to estimate the yields and the purification fold after this step.

Hydrophobic Chromatography. (NH4)2SO4 (final concentration 1 M) is added to the sample obtained from the previous step, and the sample is loaded at a flow rate of 2 ml/min on a fast protein liquid chromatography (FPLC) Phenyl-Superose HR 10/10 column (Pharmacia Biotech, Uppsala, Sweden), equilibrated with 50 mM phosphate buffer, pH 6.8, 1 M (NH4)2SO 4 (running buffer). After washing, the column is eluted with a nonlinear gradient of running buffer and running buffer without (NH4)2804 and then with 50 ml of 50 mM phosphate buffer, pH 6.8. Enzyme activity is eluted as a broad peak after the gradient during the elution with running buffer. The active pool (around 120 ml) is dialyzed against 3 liters of 20 mM phosphate buffer, pH 7.0, for 24 hr at 4 ° and then concentrated by ultrafil- tration using a YM30 membrane (Amicon, Beverly, MA). This step usually produces high yields (>70%) and a purification fold of about 6.0 from the thermal treatment. The purity of the sample is checked by SDS-PAGE on continuous 7% polyacrylamide gels run at room temperature; at this step the enzyme is generally >95% homogeneous (specific activity above about 150 U/mg under the conditions described earlier). When Ss/3-gly is stored at 4 ° in 20 mM phosphate buffer, pH 7.0, and sodium azide 0.02%, no appreciable loss of activity can be detected even after several months. In contrast, the enzyme is stable at - 20 ° only if supplemented with 50% (v/v) glycerol, as freezing could inactivate homogeneous samples rapidly.

Size-Exclusion Chromatography. When a higher state of purity is re- quired, i.e., for crystallization studies, Ss/3-gly can be further purified by gel filtration using an FPLC Superdex 200 HG 26/60 column (Pharmacia

206 SACCHAROLYTIC ENZYMES [ 1 1 ]

Biotech). In this case, the enzyme solution (<5 ml) is applied to the column equilibrated previously with 20 mM phosphate buffer, pH 7.0, at a flow rate of 2 ml/min. Fractions containing/3-galactosidase activity are pooled and concentrated as described earlier.

Chimeric [3-Glycosidase

Mutations of Ss/3-gly residues involved in the activity and/or stability of the enzyme could hamper the normal protocol of purification. For these reasons, a different expression system in E. coli has been developed in which wild-type and mutant Ss/3-gly are expressed as fusions with the gluta- thione S-transferase (GST) protein from Schistosoma japonicum. This sys- tem allows the easy purification of proteins by affinity chromatography on glutathione Sepharose 4B matrix, following GST activity. The fused polypeptides can then be separated by thrombin cleavage.

Plasmid vectors pGEXGIy and pGEX-K-Gly are obtained by inserting the lacS gene into the BamHI site of pGEX-2T and pGEX-2TK (Pharmacia Biotech), respectively. Both resulting plasmids contain an open reading frame (ORF) coding for a polypeptide of around 80 kDa, consisting of GST (26 kDa) at the N terminus, and of Ss/3-gly (56 kDa) at the C terminus. In pGEX-K-GIy, the recognition sequence for the catalytic subunit of cAMP-dependent protein kinase (PK) is inserted between the GST and the Ssfl-gly portions (see later). The sequences at the junction of the poly- peptides deduced from the two plasmids are the following:

pGEXGly -Leu-Val-Pro-Arg-Gly-Ser-Met-Tyr-

pGEX-K-Gly -Leu-Val-Pro-Arg-Gly-Ser-Arg-Arg-Ala-Ser-Val-Met.Tyr-

The first two Ss/3-gly amino acids are in boldface type, the residues involved in the peptidic bond hydrolyzed by the thrombin protease are underlined, and the PK recognition sequence is in italics.

Bacterial Growth and Crude Extract. Expression is performed in E. coli strain BL21, harboring either pGEXGly or pGEX-K-Gly, starting from freshly transformed cells. For 2-liter scale preparations, one single colony selected on ampicillin is inoculated in 50 ml of LB medium containing ampicillin (50/zg/ml) and is grown overnight at 37 ° with vigorous shaking. This culture is used to inoculate (at 1/100 dilution, v/v) four 2-liter flasks containing LB medium (500 ml each), supplemented with ampicillin (50 /zg/ml) and antifoam A (50/zl each). Cell cultures are incubated at 37 ° with vigorous shaking until the OD600 is 0.6-0.8, induced by adding 0.5 mM IPTG, and grown overnight. Crude extract is prepared by collecting cells by centrifugation at 4000g at 4 ° for 15 min, resuspending the pellets

[11] ~-GLYCOSIDASE FROM S. solfataricus 207

in 50 ml of phosphate-buffered saline (PBS) (150 mM NaCI, 50 mM sodium phosphate buffer, pH 7.3) supplemented with Triton 0.1%, and by three cycles of French Press (American Instrument Company). The lysate is clarified by centrifugation at 17,000g for 30 min, diluted to 200 ml of PBS/ Triton 0.1%, and filtered through a 0.45-/zm filter (Millipore).

Glutathione Affinity Chromatography and Thrombin Treatment. The crude extract is slowly loaded overnight at 4 ° on a 2-ml bed volume of a gluthatione Sepharose 4B column (Pharmacia Biotech), equilibrated with PBS/Triton 0.1%, in order to allow the binding of GST to the matrix. The column is washed with 60 ml of PBS. The efficiency of binding is followed by a standard GST assay at 37 ° performed following the manufacturer's protocol (Pharmacia Biotech). The fusion protein is eluted from the column by the addition of 2 ml of 500 mM Tris-HC1, pH 8.0, supplemented with 10 mM reduced gluthatione, and incubation at room temperature (22-25 °) for 10 min; the eluate is collected and assayed for GST activity at 37 ° and for Ss/3-gly activity at 65 °. This procedure is repeated until all the fusion protein is eluted from the column; finally, active fractions are collected and stored at 4 ° . No accurate estimation of the yields and the purification fold from the crude extract can be performed after this step because of interference from aggregated E. coli proteins and because Ss/3-gly specific activity is reduced severely when the protein is fused to GST.

The active pool is then subjected to thrombin treatment in order to recover Ss/3-gly free of the fused GST polypeptide; to this aim, pooled fractions (about 50 ml) are incubated at 25 ° overnight with 50 units of thrombin solution prepared as suggested by the manifacturer (Pharmacia Biotech). The efficiency of thrombin cleavage is tested by SDS-PAGE.

Affinity chromatography samples obtained with pGEXGly vector, ana- lyzed by SDS-PAGE, reveal the expected 80-kDa band corresponding to the fusion; however, the fused polypepeptide is inefficiently cleaved by thrombin, suggesting that the thrombin recognition site in this chimera is unaccessible to the protease. 13 In contrast, the chimeric protein expressed from the pGEX-K-GIy vector, which contains five extra amino acids of the PK recognition sequence between the two portions, is cleaved efficiently and specifically by thrombin, suggesting that the PK recognition sequence works as a spacer, exposing the protease site. After this purification step, 1500 total units of Ss/3-gly (specific activity about 20 U/mg) are usually recovered, as assayed under standard conditions (4-Np-Gal substrate at

13 M. Moracci, L. Capalbo, M. De Rosa, R. La Montagna, A. Morana, R. Nucci, M. Ciaramella, and M. Rossi, in "Carbohydrate Bioengineering" (S. B. Petersen, B. Svenson, and S. Pedersen, eds.), p. 77. Elsevier, 1995.

208 SACCHAROLYTIC ENZYMES [ 111

65°). Only samples obtained from pGEX-K-GIy are, therefore, used for further purification steps.

Chromatography on Q-Sepharose. The sample obtained after thrombin treatment is loaded at a flow rate of 2 ml/min on an FPLC Q-Sepharose column (Pharmacia Biotech) (1.5 x 11 cm) equilibrated with 10 mM Tris- HC1 buffer, pH 8,0. The column is washed with 30 ml of the same buffer, Ss/3-gly is eluted after the first 12 ml, indicating that the enzyme did not bind the column. All the contaminants are eluted with a nonlinear gradient of 0-0.3m NaC1. This step usually produces high yields (about 80%) and a purification fold of about 5.0 from the affinity chromatography. After this step the sample is about 90% homogeneous by SDS-PAGE (specific activity above 130 U/mg) and can be stored as described earlier.

Ss/3-gly purified by this method should contain seven extra amino acids at its N terminus (see earlier discussion). However, only four extra amino acid residues are observed by direct N-terminal sequencing of samples obtained with this method, suggesting that the tail produced by thrombin cleavage is further hydrolyzed by contaminating proteases. This "long" form of Ss/3-gly shows the same features of the "normal" form; therefore, this procedure is used routinely for the production of Ss/3-gly mutants severely affected in their properties (see later).

Table I reports the comparison of the two purification methods de- scribed; it is clear that the expression system pETGly29/BL21(DE3) is far more efficient; however, the pGEX-K-Gly/BL21 expression system, although less efficient, allows purification of inactive and/or unstable mutant proteins, which are very difficult to purify using traditional methods.

Enzyme Function

Molecular mass determined by SDS-PAGE under reducing and nonre- ducing conditions, and by gel filtration chromatography, showed that Ssfl-

TABLE I COMPARATIVE Ss/3-GIy EXPRESSION IN E. coli

Cell Yield per Yield per cell Culture pellet culture volume (units/g)

Antibiotic volume yield (units/liter) a (g of protein/g Expression system resistance (liter) (g) (g of protein/liter) of cells)

pETGly29/BL21(DE3) Kanamicin 2 30 1912 127 12.5 0.8

pGEX-K-GLy/BL21 Ampicillin 2 20 666 66.6 5.2 0.52

Assays were performed at standard conditions using 4-Np-/3-D-Gal as substrate.

[1 I I fl-GLYCOSIDASE FROM S. solfataricus 2 0 9

T A B L E II KINETIC CONSTANTS OF S. solfataricus/3-GLYCOS~DASE ~

Km kcat kcat/ Km Substrate (mM) (sec -t) (sec q m M i)

4-Np-Fuc 1.0 994 994 4-Np-Glc 0.5 698 1396 2-Np-Glc 3.5 1898 542 4-Np-Gal 4.7 1313 279 2-Np-Gal 4.0 2146 536 4-Np-Xyl 4.0 366 41 Laminaribiose b,, 1.0 908 908 ~-Gentiobiose b.c 100 1360 14 Cellobiose b,c 30 1333 44 Cellotriose h 3.0 197 66 Cellotetraose b 1.7 584 343 Cellopentaose b 2.4 270 112 4-Np-Cellobioside b 0.3 503 1676

" A t 75 o.

h Hydrolysis products were detected as glucose, and Km values are shown as glucose equivalents.

' Kinetic values for these substrates were calculated taking into ac- count that two glucose equivalents were produced for every gluco- sidic bond hydrolyzed.

gly is a homotetrameric protein (240 kDa) with 56-kDa subunits. The enzyme is thermostable (half-life 48 hr at 85°), resistant to detergents and organic solvents, and shows maximal activity above 95 ° . One of the most interesting properties of Ss/3-gly is its wide substrate specificity, its ability to hydrolyze oligosaccharides, and its efficient synthesis of a-D-glycosides by transglycosylation. 14 For these reasons, the catalytic properties and the reaction mechanism have been studied extensively by classical enzymologi- cal methods and by site-directed mutagenesis.

Substrate Specificity

Kinetic constants of Ss/3-gly for several substrates are summarized in Table II: the enzyme is active on arylglycosides of galactose, fucose, glu- cose, and xylose and on di- and oligosaccharides derived from glucose. No hydrolysis occurred with 4-Np-t~-D-gluco- and galacto-pyranosides, 4-Np- /3-D-N-acetylglucosamine and galactosamine, 4-Np-/3- and a-L-fucopyrano- side, 4-Np-fl-o-glucuronic or galacturonic acid, or with maltose. Mannose,

14 A. Trincone, R. Improta, R. Nucci, M. Rossi, and Gambacorta, Biocatalysis 10, 195 (1994).

210 SACCHAROLYTIC ENZYMES [1 11

4-Np-a-L-arabinopyranoside, and 2-Np-/3-D-galactopyranoside 6-phosphate were susceptible to hydrolysis but with only limited efficiency. 6 Ssfl-Gly hydrolyzes arylglycosides very efficiently, showing that the kcat/Km values increase in the order of 4-Np-/3-o-Glc > Fuc > Gal. The hydrolysis of arylgalactosides is fast, but the kcat/Km values are smaller than those for arylglucosides. /3-Fucosidase activity is comparable to /3-glucosidase, whereas the hydrolysis of 4-Np-/3-D-xylopyranoside is much slower. It is interesting that Ss/3-gly shows, at high temperature, both catalytic efficiency and turnover number comparable to those of other/3-glycosidases from mesophilic sources.

The catalytic efficiency of hydrolysis of disaccharides and oligosaccha- rides is far lower than that of chromogenic substrates. Nevertheless, all three/3-1inked glucose dimers tested are substrates of the enzyme, with the order of preference being/31-3 >/31-4 >/31-6; laminaribiose is the disaccharide hydrolyzed with the highest efficiency.

Ss/3-Gly is able to hydrolyze oligosaccharides with good efficiency: k¢~t/ Km values are such that cellotetraose > cellotriose > cellobiose, whereas the hydrolysis of cellopentaose is slower. When incubating the enzyme with cellotetraose, the production of glucose and cellotriose is observed by thin- layer chromatography. Trace amounts of cellobiose are detected only after 5 min of incubation, probably as a result of hydrolysis of the initial product cellotriose. These results indicate that Ss/3-gly exhibits exoglucosidase activ- ity. This is further confirmed using 4-Np-/3-o-cellobioside as substrate: the production of both 4-nitrophenol and glucose could be detected, but while glucose release increases linearly from the onset of the reaction, the pres- ence of 4-nitrophenol could be detected only after several minutes. This indicated that Ss/3-gly exoglucosidase activity is specific toward the nonre- ducing end of the oligosaccharide substrate.

Analysis of Catalytic Machinery

Retaining glycosidases (Scheme 1) follow a double-displacement mecha- nism involving a glycosyl-enzyme intermediate. The carboxyl group in the active center functions as a general acid/base catalyst and the carboxylate as the nucleophile of the reaction. 15'16 The two carboxylic residues essential for catalysis in Ss/3-gly were determined from glutamic and aspartic acid residues conserved among all family 1 glycosylhydrolases members. The glutamic acid-206 and -387 residues of Ss/3-gly were found in two totally conserved motifs: Asn-Glu-Pro and Glu-Asn-Gly, corresponding to the general acid/base catalyst and the nucleophile, respectively, and identified

15 D. E. Koshland, Biol. Rev. 28, 416 (1953). 16 A. White, and D. R. Rose, Curt. Opin. Struct. Biol. 7, 645 (1997).

[1 11 /~-GLYCOSIDASE FROM S. solfataricus 211

Glu206 Glu206

o .o

Glu387 Glu387

SCHEME 1

OH

H OH

OH [~-D-GIc

previously in the active site of Agrobacterium /3-glucosidaseJ 7'18 These residues were changed to isosteric glutamine by site-directed mutagenesis; the resulting mutant proteins were expressed as fusions to GST as de- scribed previously.

The Glu387Gln mutant was completely inactive against all the substrates tested. 19 These data are consistent with the function of Glu387 as the attacking nucleophile. Moreover, this function has been further confirmed using active site-directed inhibitors 2° and inspection of the three-dimen- sional structure (see later discussion).

In contrast, the Glu206Gln mutation strongly affects, but does not com- pletely eliminate, the activity on 2/4-Np-/3-D-glycosides. 19 Kinetic constants determined for the Glu206Gln mutant at 65 ° are compared to the wild type in Table III. The mutant showed a 10- to 30-fold decrease in Km values and a 60-fold decrease of the kcat values on 2/4-Np-Glc, suggesting that the glucosyl-enzyme intermediate is accumulated during the reaction, kcat on 2/4-Np-Gal and 4-NpFuc are less affected. These results suggest that 2/4- Np-glucoside and 2/4-Np-galactoside substrates elicit different interactions with the Glu206 residue in the Ss/3-gly active site. Moreover, the pH sensitiv- ity of the reaction was affected by the mutation: whereas wild-type Ss/3- gly showed a typical bell-shaped curve, with maximum activity at pH 6.5, the Glu206Gln residual activity was apparently independent of pH. 19

These results strongly suggest that glutamic acid-206 in Ss/3-gly is the

17 S. G. Withers, R. A. J. Warren, I. P. Street, K. Rupitz, J. B. Kempton, and R. Aebersold, J. Am. Chem. Soc. 112, 5887 (1990).

18 Q. Wang, D. Trimbur, R. Graham, R. A. J. Warren, and S. G. Withers, Biochemistry 34, 14554 (1995).

19 M. Moracci, L. Capalbo, M. Ciaramella, and M. Rossi, Protein Eng. 9, 1191 (1996). 20 F. Febbraio, R. Barone, S. D'Auria, M. Rossi, R. Nucci, G. Piccialli, L. De Napoli, S. Orrh,

and P. Pucci, Biochemistry 36, 3068 (1997).

212 SACCHAROLYTIC ENZYMES [1 11

TABLE III KINETIC CONSTANTS OF HYDROLYSIS BY WILD-TYPE AND MUTANT E206Q a

Km (mM) kca t (sec -1)

Substrate Wild type Glu206Gln Wild type Glu206Gln

2-NpGal 0.95 ± 0.08 1.53 ± 0.1 537 ± 11 62 ± 1.4 4-NpGal 1.17 _ 0.06 4.79 _ 0.17 383 - 5 43 ___ 0.5 2-NpGlc 1.01 ___ 0.24 0.03 --- 0.006 457 --- 22 8.1 --- 0.13 4-NpGlc 0.30 ± 0.04 0.03 + 0.003 334 ± 10 6.1 ± 0.08 4-NpFuc 0.45 _+ 0.11 0.09 ___ 0.005 366 - 17 49 ± 0.5

a At 65 °.

general acid/base catalyst for the hydrolysis reaction. The studies reported so far on glycohydrolases mutated to influence general acid/base catalysis demonstrate that the kinetic parameters can be affected to different ex- tents. 1a,21,22 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 different degrees.

Enzyme Structure

Ssfl-Gly has been crystallized in its native, tetrameric form using multiple isomorphous replacement and has been refined at 2.6 A resolution. 23 The enzyme shows the classic (flo08 fold that has also been observed in other mesophilic members of the glycosylhydrolase family I crystallized so far: the cyanogenic fl-glucosidase from Trifolium repens, 24 the phospho-fl-glu- cosidase from Lactococcus lactis, 25 the fl-glucosidase from Bacillus polym- ixa, 26 a n d t h e m y r o s i n a s e f r o m Sinapis alba. 27 I n s p e c t i o n o f t h e Ssf l -g ly

t h r e e - d e m e n s i o n a l s t r u c t u r e g a v e ins igh t s i n t o b o t h t h e a r c h i t e c t u r e o f t h e

s u b s t r a t e b i n d i n g s i te a n d t h e pos s ib l e m o l e c u l a r d e t e r m i n a n t s o f t he r -

m a l s tabi l i ty .

21 E. Witt, R. Frank, and W. Hengstenberg, Protein Eng. 6, 913 (1993). 22 M. MacLeod, T. Lindhorst, S. G. Withers, and R. A. J. Warren, Biochemistry 33, 6371 (1994). 23 C. Aguilar, I. Sanderson, M. Moracci, M. Ciaramella, R. Nucci, M. Rossi, and L. H. Pearl,

Z Mol. Biol. 271, 789 (1997). 24 T. Barret, C. G. Suaresh, S. P. Tolley, E. J. Dodson, and M. A. Hughes, Structure 3, 951 (1995). 25 C. Weisman, G. Best, W. Hengstenberg, and G. E. Schultz, Structure 3, 961 (1995). 26 j. Sanz-Aparicio, J. A. Hermoso, M. Martinez-Ripoll, J. L. Lequerica, and J. Polaina, J.

Mol. Biol. 275, 491 (1998). 27 W. P. Burmeister, S. Cottaz, H. Driguez, R. Iori, S. Palmieri, and B. Henrissat, Structure

5, 663 (1997).

[11] /3-GLYCOSIDASE FROM S. solfataricus 213

Active Site and Substrate-Binding Tunnel

The Ss/3-gly (fla)8 fold is quite complex: the connections between/3 strands and a helices within/3or repeats at the top of the barrel are substan- tially elaborated and contain several loops with extra secondary structural elements. In particular, a deep indentation at the top of the barrel is con- nected to the outside via a radial channel, about 30 ,~ in length, which emerges between the helices of the fifth and sixth/3a units (Fig. 1). The opening is generated by a 45 ° kink in the helix of the fifth unit, occurring at an unusual sequence of aromatic residues (Trp-287, Trp-288, Phe-289, Phe-290). Because the tops of the barrel in the tetrameric structure are in contact, the loops provide a "roof" for the radial channel in each monomer,

Fie. 1. Substrate binding site of Ssfl-gly. Residues in the radial channel are shown with the Van der Waals radius of their atoms. The side chains of the catalytic diad Glu-206 and Glu-387 are shown as sticks.

214 SACCHAROLYTIC ENZYMES [1 I I

converting it into a tunnel. The radial channel in the monomer contains the catalytic residues Glu-206 and Glu-387 and a high concentration of other residues highly conserved in family 1 glycohydrolases and likely implicated in catalysis and/or substrate binding. Moreover, a cellohexaose chain could be modeled into the tunnel, with its nonreducing end and the scissile glycosidic bond in close proximity to the catalytic diad Glu-387-Glu 206. 23 These findings strongly suggest that this tunnel is the substrate binding site and support enzymological studies showing the exoglucosidase activity of Ss/3-gly.

The distance between the two carboxylate residues responsible of the catalytic activity is diagnostical for the assignment of the reaction mecha- nism in glycohydrolases: these two groups are, on average, 5.0 and 9.3 ,~ apart, in retainers and inverters, respectively. Analysis of the Ssfl-gly three- dimensional structure confirmed that this enzyme follows a retaining mecha- nism, as the distance between the CSgroups of Glu387 and Glu206 is 4.6 ,~.

Factors Producing Thermostability

The comparison of the Ss/3-gly amino acid sequence with those of ther- mophilic and mesophilic/3-glycosidases revealed that the enzyme shows a lower content of alanine and isoleucine and a higher content of tryptophan and asparagine than the mean of the mesophiles. 23 This suggests that most of the rules proposed to explain the molecular origins of the enhanced stability of enzymes from hyperthermophiles do not hold for this protein. In contrast, the Ss/3-gly three-dimensional structure revealed that two struc- tural features might contribute to thermal stabilization in this enzyme: a large number of both buried hydrophilic cavities and ionic groups involved in ion pairs. In Ssfl-gly, one buried solvent molecule on 11.4 amino acid residues is observed versus a 1 : 27 ratio in proteins in general. So far this feature has only been observed in Ssfl-gly, whereas the abundance of ion pairs arranged in large networks has been found in other enzymes from hyperthermophiles. 2s-3° The Ssfl-gly tetramer contains 524 charged groups; the majority of these (58%) are involved in ion pair interactions and around 60% of these interactions occur as part of multiple ion pair networks involv- ing at least three charged groups. The cyanogenic B-glycosidase from clover

18 M. Hennig, B. Dairmont, R. Sterner, K. Kirschner, and J. N. Jansonius, Structure 3, 1295 (1995).

29 K. S. P. Yip, T. J. Stillman, K. L. Britton, P. J. Artymiuk, P. J. Baker, S. E. Sedelnikova, P. C. Engel, A. Pasquo, R. Chiaraluce, V. Consalvi, R. Scandurra, and D. W. Rice, Structure 3, 1147 (1995).

30 A. Goldman, Structure 3, 1277 (1995).

[ 12] XYLOSE ISOMERASES FROM Thermotoga 215

has far less of its charged residues involved in ion pairs (41%) and the majority of them are isolated pairs. 24

Most of the networks in Ss/3-gly occur in noncontiguous positions on the surface of the molecule and spanning different domains and subunits; hence, they could act as cross-linkers between folded structural elements. It is interesting that arginines occur frequently in ion pair networks, presum- ably for their ability to form multidentate interactions; this might explain the higher number of arginines found in enzymes from hyperthermophiles. 31,32

These findings suggest that large networks of ion pairs are important for hyperthermostability in Ss/3-gly and provide a useful framework for direct experimental test, by site-directed mutagenesis, of the contribution of each interaction to the overall stability.

Acknowledgments

Work in the authors' laboratory was supported by EC project: "Biotechnology of Extrerno- philes" contract No. BIO2-CT93-0274 and by the EC FAIR project: "Enzymatic Lactose Valorization" contract No. 1048/96.

31 p. Argos, M. Rossmann, U. Grau, H. Zuber, G. Frank, and J. Tratschin, Biochemistry 25, 5698 (1979).

32 L. Men6ndez-Arias and P. Argos, J. Mol. BioL 206, 397 (1989).

[121 X y l o s e I s o m e r a s e s f r o m T h e r m o t o g a

By CLAIRE VIEILLE, DINLAKA SRIPRAPUNDH, ROBERT M. KELLY, and J. GREGORY ZEIKUS

Int roduct ion

D-Xylose ~ D-xylulose D-Glucose ~ D-fructose

Typically present in microorganisms that grow on xylose, xylose isom- erase (XI) (EC 5.3.1.5) converts xylose to xylulose, which is then phosphory- lated and enters the pentose-phosphate pathway. Because it also accepts glucose as substrate, XI is used extensively to isomerize glucose to fructose in the manufacture of high fructose corn syrup (HFCS). Performed at temperatures around 55-60 °, this isomerization process requires thermosta- ble enzymes. XIs have been characterized from a number of eubacterial sources and from rice and barley, but they have not been reported in

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