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Isomaltulose Synthase (PalI) of Klebsiella sp. LX3

2003; Elsevier BV; Volume: 278; Issue: 37 Linguagem: Inglês

10.1074/jbc.m302616200

1083-351X

Daohai Zhang, Nan Li, Shee‐Mei Lok, Lian‐Hui Zhang, Kunchithapadam Swaminathan,

Enzyme Structure and Function

Isomaltulose synthase from Klebsiella sp. LX3 (PalI, EC 5.4.99.11) catalyzes the isomerization of sucrose to produce isomaltulose (α-d-glucosylpyranosyl-1,6-d-fructofuranose) and trehalulose (α-d-glucosylpyranosyl-1,1-d-fructofuranose). The PalI structure, solved at 2.2-Å resolution with an R-factor of 19.4% and R free of 24.2%, consists of three domains: an N-terminal catalytic (β/α)8 domain, a subdomain between Nβ3 and Nα3, and a C-terminal domain having seven β-strands. The active site architecture of PalI is identical to that of other glycoside hydrolase family 13 members, suggesting a similar mechanism in substrate binding and hydrolysis. However, a unique RLDRD motif in the proximity of the active site has been identified and shown biochemically to be responsible for sucrose isomerization. A two-step reaction mechanism for hydrolysis and isomerization, which occurs in the same pocket is proposed based on both the structural and biochemical data. Selected C-terminal truncations have been shown to reduce and even abolish the enzyme activity, consistent with the predicted role of the C-terminal residues in the maintenance of enzyme conformation and active site topology. Isomaltulose synthase from Klebsiella sp. LX3 (PalI, EC 5.4.99.11) catalyzes the isomerization of sucrose to produce isomaltulose (α-d-glucosylpyranosyl-1,6-d-fructofuranose) and trehalulose (α-d-glucosylpyranosyl-1,1-d-fructofuranose). The PalI structure, solved at 2.2-Å resolution with an R-factor of 19.4% and R free of 24.2%, consists of three domains: an N-terminal catalytic (β/α)8 domain, a subdomain between Nβ3 and Nα3, and a C-terminal domain having seven β-strands. The active site architecture of PalI is identical to that of other glycoside hydrolase family 13 members, suggesting a similar mechanism in substrate binding and hydrolysis. However, a unique RLDRD motif in the proximity of the active site has been identified and shown biochemically to be responsible for sucrose isomerization. A two-step reaction mechanism for hydrolysis and isomerization, which occurs in the same pocket is proposed based on both the structural and biochemical data. Selected C-terminal truncations have been shown to reduce and even abolish the enzyme activity, consistent with the predicted role of the C-terminal residues in the maintenance of enzyme conformation and active site topology. Isomaltulose synthase (PalI), also known as sucrose isomerase (EC 5.4.99.11), catalyzes the isomerization of sucrose to produce isomaltulose (α-d-glucosylpyranosyl-1,6-d-fructofuranose) and trehalulose (α-d-glucosylpyranosyl-1,1-d-fructofuranose) as the main products with residual amounts of glucose and fructose (1Huang J.H. Hsu L.H. Su Y.C. J. Ind. Microbiol. Biotechnol. 1998; 21: 22-27Crossref Scopus (61) Google Scholar, 2Veronese T. Perlot P. Enzyme Microb. Technol. 1999; 24: 263-269Crossref Scopus (54) Google Scholar), as shown in Scheme 1. Isomaltulose and trehalulose, two functional isomers of sucrose, have been suggested as non-cariogenic alternatives to sucrose and are widely used in health products and the food industry (3Baer A. Wiss. Technol. 1989; 22: 46-53Google Scholar). The isomaltulose synthase activity has been reported in a range of bacterial species (1Huang J.H. Hsu L.H. Su Y.C. J. Ind. Microbiol. Biotechnol. 1998; 21: 22-27Crossref Scopus (61) Google Scholar, 4Miyata Y. Sugitani T. Tsuyuki K. Ebashi T. Nakajima Y. Biosci. Biotechnol. Biochem. 1992; 54: 1680-1681Crossref Scopus (34) Google Scholar, 5Mattes R. Klein K. Schiwech H. Kunz M. Munir M. U. S. Patent 5. 1998; 786: 140Google Scholar, 6Zhang D.H. Li X.Z. Zhang L.H. Appl. Environ. Microbiol. 2002; 68: 2676-2682Crossref PubMed Scopus (51) Google Scholar). The ratio of the enzyme products varies, from mainly isomaltulose (75–85%) to predominantly trehalulose (∼90%), depending on the bacterial strain (1Huang J.H. Hsu L.H. Su Y.C. J. Ind. Microbiol. Biotechnol. 1998; 21: 22-27Crossref Scopus (61) Google Scholar, 4Miyata Y. Sugitani T. Tsuyuki K. Ebashi T. Nakajima Y. Biosci. Biotechnol. Biochem. 1992; 54: 1680-1681Crossref Scopus (34) Google Scholar, 5Mattes R. Klein K. Schiwech H. Kunz M. Munir M. U. S. Patent 5. 1998; 786: 140Google Scholar, 6Zhang D.H. Li X.Z. Zhang L.H. Appl. Environ. Microbiol. 2002; 68: 2676-2682Crossref PubMed Scopus (51) Google Scholar, 7Nagai-Miyata J. Tsuyuki K. Sugitani T. Ebashi T. Nakajima Y. Biosci. Biotechnol. Biochem. 1993; 57: 2049-2053Crossref Scopus (4) Google Scholar). The molecular mechanism of isomaltulose synthase that controls sucrose isomerization has not been fully characterized, except the recent prediction by Veronese and Perlot (2Veronese T. Perlot P. Enzyme Microb. Technol. 1999; 24: 263-269Crossref Scopus (54) Google Scholar, 8Veronese T. Perlot P. FEBS Lett. 1998; 441: 348-352Crossref PubMed Scopus (30) Google Scholar), in which sucrose binding, hydrolysis, and isomerization depend on the charges provided by residues in a closed shell. To understand the mechanism of sucrose isomerization at the molecular level and identify the key amino acids involved in the enzyme reaction, we recently cloned the palI gene encoding isomaltulose synthase (PalI) from the bacterial isolate Klebsiella sp. LX3 (6Zhang D.H. Li X.Z. Zhang L.H. Appl. Environ. Microbiol. 2002; 68: 2676-2682Crossref PubMed Scopus (51) Google Scholar). Sequence alignment and secondary structure prediction revealed that PalI is a novel member of glycoside hydrolase family 13. Family 13 contains enzymes that act on starch such as α-amylase and cyclodextrin glycosyltransferase (CGTase) as well as enzymes specific for the cleavage of other glycosidic linkage such as α-1,6- and α-1,1-bonds (9Davies G. Henrissat B. Structure. 1995; 3: 853-859Abstract Full Text Full Text PDF PubMed Scopus (1579) Google Scholar). The basic structural characteristics of this family of enzymes is that the catalytic core domain contains a (β/α)8-fold. The potential catalytic triad (Asp241, Glu295, and Asp369) and two histidine residues (His145 and His368) in PalI are highly conserved in α-amylase and glycosyltransferase (6Zhang D.H. Li X.Z. Zhang L.H. Appl. Environ. Microbiol. 2002; 68: 2676-2682Crossref PubMed Scopus (51) Google Scholar). These residues, as found in oligo-1,6-glucosidase from Bacillus cereus (Asp199, Glu255, Asp329, His103, and His328) (10Watanabe K. Hata Y. Kizaki H. Katsube Y. Suzuki Y. J. Mol. Biol. 1997; 269: 142-153Crossref PubMed Scopus (237) Google Scholar) and in amylosucrase from Neisseria polysacchareais (Asp286, Glu328, Asp393, His187, and His392) (11Skov L.K. Mirza O. Henriksen A. de Montalk G.P. Remaud-Simeon M. Sarcabal P. Willemot R.M. Monsan P. Gajhede M. J. Biol. Chem. 2001; 276: 25273-25278Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar), form a catalytic pocket that binds the substrate and hydrolyzes the glycosidic bond. The similarity of the active site architecture strongly suggests that PalI adopts the same molecular mechanism for hydrolysis of the glycosidic bond and formation of the glucosyl-enzyme complex. The reaction mechanism occurs via a general acid catalysis, as do those of all glucoside hydrolases (12Koshland D.E. Biol. Rev. Camb. Philos. Soc. 1953; 28: 416-436Crossref Scopus (794) Google Scholar). In addition, PalI adopts the same mechanistic scheme in the formation of enzyme-substrate intermediate. As shown in Scheme 2, the glycosidic bond is protonated by a proton donor and the anomeric carbon of the glucose moiety is attacked by the nucleophilic acid, simultaneously, leading to the formation of the covalently linked enzyme-substrate intermediate. The glucosyl moiety can be transferred to a water molecule for hydrolysis and to the fructose moiety for sucrose isomers synthesis. In the isomerization step, the structure of fructose determines the balance in the formation of two sucrose isomers, isomaltulose and trehalulose (8Veronese T. Perlot P. FEBS Lett. 1998; 441: 348-352Crossref PubMed Scopus (30) Google Scholar). To understand the specific structural features required for isomerization in PalI, the PalI protein was overexpressed, purified, and crystallized (13Li N. Zhang D.H. Zhang L.H. Swaminathan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 150-151Crossref PubMed Scopus (4) Google Scholar). Here we describe, at 2.2-Å resolution, the crystal structure of PalI and the potential function of an RLDRD motif, at the structural level, in determining the mechanism of sucrose hydrolysis and isomerization. The function of the C-terminal domain in the enzymatic activity is also reported. Based on these data, we present a mechanism to explain the sucrose isomerization process. We believe this paper is the first structure-function report on PalI, a sucrose isomerase that converts sucrose to isomaltulose and trehalulose simultaneously. Strains, Plasmids, and Site-directed Mutagenesis—Escherichia coli DH5α were used as host cells for plasmid propagation and protein overexpression. Site-directed mutation of palI was performed by using the QuikChange™ site-directed mutagenesis kit (Stratagene). The double-stranded DNA vector pGEK (6Zhang D.H. Li X.Z. Zhang L.H. Appl. Environ. Microbiol. 2002; 68: 2676-2682Crossref PubMed Scopus (51) Google Scholar) was used as the template, and two synthetic oligonucleotides containing the desired mutation were used as primers. Five mutants, PalI:D241A, PalI:E295A, PalI:D369A, PalI: H145A, and PalI:H368A, were created in which the indicated residues were replaced by Ala. For the C-terminal deletion mutants PalI:Δ587, PalI:Δ572 and PalI:Δ545, a stop codon was separately introduced after the indicated amino acid positions. All point mutations (Table I) were confirmed by DNA sequencing, using the dideoxy chain termination procedure. The digestion of DNA with restriction endonucleases, agarose gel electrophoresis, and transformation of E. coli DH5α were carried out according to standard procedures.Table ISpecific activity of PalI mutantsProteinDescriptionSpecific activityunits/mgPalINative protein330.5 ± 2.56PalI:H145AHis145 replaced by Ala2.15 ± 0.28PalI:H368AHis368 replaced by Ala8.85 ± 0.65PalI:D241AAsp241 replaced by Ala0.66 ± 0.44PalI:E295AGlu295 replaced by Ala0PalI:D369AAsp369 replaced by Ala4.1 ± 0.21C-terminal deletionPalI:Δ587C termini after Leu587 deleted305.05 ± 3.45PalI:Δ572C termini after Asp572 deleted32.95 ± 1.71PalI:Δ545C termini after Tyr545 deleted0Enzyme activity (units/mg) was assayed as described previously (6Zhang D.H. Li X.Z. Zhang L.H. Appl. Environ. Microbiol. 2002; 68: 2676-2682Crossref PubMed Scopus (51) Google Scholar). Briefly, 2 μg of purified protein was mixed with 400 μl of 0.1 m citratephosphate buffer, pH 6.5, containing sucrose (40 g l-1) and incubated at 35 °C for 15 min with gentle agitation. One unit of enzyme is defined as the amount of protein that form 1 μmol of reducing sugar (with isomaltulose as standard) per min under the conditions specified. Open table in a new tab Enzyme activity (units/mg) was assayed as described previously (6Zhang D.H. Li X.Z. Zhang L.H. Appl. Environ. Microbiol. 2002; 68: 2676-2682Crossref PubMed Scopus (51) Google Scholar). Briefly, 2 μg of purified protein was mixed with 400 μl of 0.1 m citratephosphate buffer, pH 6.5, containing sucrose (40 g l-1) and incubated at 35 °C for 15 min with gentle agitation. One unit of enzyme is defined as the amount of protein that form 1 μmol of reducing sugar (with isomaltulose as standard) per min under the conditions specified. Enzyme Purification and Assays—Overexpression and purification of enzymes (PalI and its mutant versions) were carried out as described previously (6Zhang D.H. Li X.Z. Zhang L.H. Appl. Environ. Microbiol. 2002; 68: 2676-2682Crossref PubMed Scopus (51) Google Scholar). Further purification was performed by gel filtration chromatography with a HiPrep Sephacryl S-200, 16/60 column (Amersham Biosciences) at a flow rate of 0.5 ml min–1 with a buffer of 150 mm NaCl, 10 mm Hepes, pH 7.5, and 1 mm dithiothreitol. The purified enzymes were further examined by sodium dodecyl sulfate-polyacrylamide (10%) gel electrophoresis (SDS-PAGE). The enzyme activity was assayed as described previously (6Zhang D.H. Li X.Z. Zhang L.H. Appl. Environ. Microbiol. 2002; 68: 2676-2682Crossref PubMed Scopus (51) Google Scholar). One unit of enzyme is defined as the amount of enzyme required to catalyze the formation of 1 μmol of isomaltulose in 1 min under assay conditions. The data presented are the means of three individual experiments. Crystallization and Data Collection—Crystallization of wild type PalI (residues 29–598) was performed as described by Li et al. (13Li N. Zhang D.H. Zhang L.H. Swaminathan K. Acta Crystallogr. Sect. D Biol. Crystallogr. 2003; 59: 150-151Crossref PubMed Scopus (4) Google Scholar), using the hanging drop vapor diffusion method at 22 °C. Crystals that were 1.2 mm in length were cut to an optimum size, treated with a cryo-protectant (reservoir solution with 4% increased precipitant and supplemented with 10% glycerol) for 3 min, and flash-cooled in liquid nitrogen. The diffraction data were collected at Spring8, Japan (beam-line BL40B2, ADSC Quantum4 CCD detector, –173 °C). The data were indexed, processed, and scaled using the programs DENZO and SCALEPACK (14Otwinowski Z. Minor W. Methods Enzymol. 1997; 276: 307-326Crossref Scopus (38253) Google Scholar). Structure Determination and Refinement—The primary amino acid sequence of PalI shows 47% identity and 65% similarity to that of oligo-1,6-glucosidase (6Zhang D.H. Li X.Z. Zhang L.H. Appl. Environ. Microbiol. 2002; 68: 2676-2682Crossref PubMed Scopus (51) Google Scholar). Moreover, these two proteins have the highest structural similarity using the DALI server (15Holm L. Sander C. Trends Biochem. Sci. 1995; 20: 478-480Abstract Full Text PDF PubMed Scopus (1268) Google Scholar). Consequently, the structure of PalI was determined by the method of molecular replacement by the MOLREP program (16Collaborative Computing Project Number 4Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 760-763Crossref PubMed Scopus (19668) Google Scholar) using oligo-1,6-glucosidase as the search model. The asymmetric unit contains one protein molecule. The resulting electron density map was solvent flattened using PHASES (17Furey W. Swaminathan S. Methods Enzymol. 1997; 277: 590-620Crossref PubMed Scopus (255) Google Scholar) and model building was carried out with the help of O (18Jones T.A. Zou J.Y. Cowan S.W. Kjeldgaard M. Acta Crystallogr. Sect. A. 1991; 47: 110-119Crossref PubMed Scopus (12999) Google Scholar). Electron density for the first 15 N-terminal amino acids was not observed in the map. The structure was refined with the CNS program suite (19Brünger A.T. Adams P.D. Clore G.M. Gros W.L. Grosse-Kunstleve R.W. Jiang J.S. Kuszewski J. Nilges M. Pannu N.S. Read R.J. Rice L.M. Simonson T. Warren G.L. Acta Crystallogr. Sect. D Biol. Crystallogr. 1998; 54: 905-921Crossref PubMed Scopus (16919) Google Scholar) and the geometry of the molecule was checked with PROCHECK (20Laskowski R. MacArthur M. Moss D. Thornton J. J. Appl. Crystallogr. 1993; 26: 283-291Crossref Google Scholar). No residue was found to lie in the disallowed region. All drawings were prepared with MOLSCRIPT (21Kraulis P.J.J. Appl. Crystallogr. 1991; 24: 946-950Crossref Google Scholar) and RASTER 3D (22Merritt E.A. Murphy M.E.P. Acta Crystallogr. Sect. D Biol. Crystallogr. 1994; 50: 869-873Crossref PubMed Scopus (2854) Google Scholar) programs. Table II shows the data and refinement statistics.Table IIData collection and refinement statisticsData collectionWavelength (Å)1.0Unit-cell parameters (Å)a = 59.24, b = 94.15, c = 111.29Space groupP212121Resolution range (Å)20-2.2Total no. of reflections199,659Total no. of unique reflections29,756Redundancy6.1Completeness (%)aValues in parentheses are for highest resolution shell (2.28-2.20 Å)99.2 (96.6)R sym (%)aValues in parentheses are for highest resolution shell (2.28-2.20 Å),bR sym = ΣjΣi | 〈Ii 〉 - Ii | /Σl Ii5.3 (26.0)RefinementR-factor (%)19.4R free (%)24.2Root mean square deviation fromideal valuesBond length (Å)0.006Bond angle (°)1.30Average temperature factor37.5(Å2)No. of protein atoms4648No. of water molecules303a Values in parentheses are for highest resolution shell (2.28-2.20 Å)b R sym = ΣjΣi | 〈Ii 〉 - Ii | /Σl Ii Open table in a new tab Coordinates—The atomic coordinates and structure factors for PalI have been deposited at the Protein Data Bank (accession code 1M53). Overall Structure—As shown in Fig. 1A, the PalI molecule consists of three domains: the N-terminal catalytic (β/α)8 domain (residues 43–146 and 216–521, colored blue), a subdomain (residues 147–215, magenta), and the C-terminal domain (residues 522–598, red), as reported for oligo-1,6-glucosidase (Fig. 1B) and amylosucrase (Fig. 1C). The (β/α)8 domain, as the main body of the structure, is sandwiched between the subdomain and C-terminal domain. It consists of the well characterized (β/α)8 barrel with eight alternating β-strands (Nβ1–Nβ8) and α-helices (Nα1–Nα8). This domain contains the residues involved in catalysis and substrate-binding. The cavity contains the acidic residues (Asp241, Glu295, and Asp369: PalI numbering) that are highly conserved in members of α-amylase family 13. The active site is shown in Fig. 2A as an example of the quality of the 2Fo – Fc electron density. This active site cleft is surrounded by a loop (residues 321–340), forming a pocket with the dimensions of 20 × 20 × 25 Å, large enough to accommodate a sucrose molecule. The overall surface around the active site pocket of PalI is highly negatively charged (data not shown). This highly negative character of the (β/α)8 barrel domain is important for sugar-protein interactions. The subdomain, which is inserted between Nβ3 and Nα3, consists of two α-helices and three anti-parallel β-strands (Fig. 1A) and has no known function either in PalI or in other family 13 members. Only one strong salt bridge (Lys248Nζ... Asp211Oδ2 with a distance of 2.6 Å) connects the subdomain and the N-terminal domain. The C-terminal domain is made of two antiparallel β-sheets. Five β-strands (Cβ1, Cβ2, Cβ3, Cβ5, and Cβ7) form the larger β-sheet, and two strands (Cβ4 and Cβ6) form the smaller one. The six loop segments that are present between the pairs of adjacent β-strands in the C-terminal domain are named as Clp1–Clp6, respectively. A network of salt bridges and hydrogen bonds between the two C-terminal β-sheets as well as the N- and C-terminal domains ensure the conformational stability of the structure in general and the active pocket in particular.Fig. 2Catalytic pocket, isomerization region, and N-C termini interactions in Pal I. A, the 2Fo – Fc electron density map at the catalytic pocket is drawn at the 2.5 σ level. The five conserved residues that participate in substrate binding and hydrolysis are labeled in green, and the five residues that are involved in the isomerization of sucrose are labeled in red. B, the superimposition of PalI on the amylosucrase-sucrose complex based on the atoms of the five conserved hydrolysis residues. PalI is blue, amylosucrase is gray, and sucrose is magenta. The residues in amylosucrase that interact with sucrose and their corresponding residues in PalI are shown. Note the deviation of the helix from the substrate in amylosucrase and the approach of the substrate by the RLDRD motif residues in PalI. Arg333 of PalI occupies the position of Arg446 in amylosucrase. C, the N- and C-terminal interactions. Hydrogen bonds are represented by dashed lines. The truncation positions of the mutants PalI:Δ587, PalI:Δ572, and PalI:Δ545 are indicated by the symbol ×. For clarity, only the residues that form important salt bridges and hydrogen bonds that are disrupted by the truncations are shown.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Active Site Architecture—Structural alignment results from the DALI server (15Holm L. Sander C. Trends Biochem. Sci. 1995; 20: 478-480Abstract Full Text PDF PubMed Scopus (1268) Google Scholar) show that the tertiary structures of PalI and oligo-1,6-glucosidase can be aligned in four parts (residues 43–257, 260–381, 383–566, and 576–598) with 46.4% sequence identity. A total of 544 Cα atoms could be superimposed with root mean square deviations in the range 0.46–1.69 Å. The alignment between PalI and amylosucrase is very poor, resulting in several shorter fragments. However, superimposition of the five conserved amino acids (His141, Asp241, Glu295, Asp369, and His368 in PalI) in the substrate-binding pocket of PalI, oligo-1,6-glucosidase, and amylosucrase (Fig. 2B; oligo-1,6-glucosidase data not shown) shows a high degree of structural similarity of the active site architecture with a root mean square deviation of 0.41 Å between PalI and oligo-1,6-glucosidase and 0.89 Å between PalI and amylosucrase. To verify the importance of these conserved residues, the five residues (His141, Asp241, Glu295, Asp369, and His368) were replaced individually by Ala, and the created mutant PalI proteins were purified (Table I). With the specific activity of the wild type enzyme (335.3 units mg–1) defined as 100%, the remaining activity of the mutants PalI:D241A, PalI:E295A, PalI:D369A, PalI:H145A, and PalI:H368A is only 0.2, 0, 1.23, 0.65, and 2.68% of the native PalI, respectively, strongly suggesting that these conserved residues are essential for PalI activity. The substrate recognition scheme and binding sites have been identified in CGTase (23Uitdehaag J.C.M. Mosi R. Kalk K.H. van der Veen B.A. Dijkhuizen L. Withers S.G. Dijkstra B.W. Nat. Struct. Biol. 1999; 6: 432-436Crossref PubMed Scopus (362) Google Scholar), TAKA-amylase with substrate analogs (24Brzozowski A.M. Davies G.J. Biochemistry. 1997; 36: 10837-10845Crossref PubMed Scopus (193) Google Scholar), amylosucrase with d-glucose, and mutated amylosucrase with sucrose (25Mirza O. Skov L.K. Remaud-Simeon M. de Montalk G.P. Albenne C. Monsan P. Gajhede M. Biochemistry. 2001; 40: 9032-9039Crossref PubMed Scopus (79) Google Scholar, 26Skov L.K. Mirza O. Sprogoe D. Dar I. Remaud-Simeon M. Albenne C. Monsan P. Gajhede M. J. Biol. Chem. 2002; 277: 47741-47747Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). Structural comparison of PalI with amylosucrase is of particular interest as these two enzymes use sucrose as their sole substrate. The superimposition of the structure of PalI with the complex of amylosucrase and sucrose (Fig. 2B) clearly indicates that the active site pocket in PalI is closely similar with that of amylosucrase and also suitable for containing one sucrose molecule. In PalI, Glu295 (equivalent to Glu328 in amylosucrase) acts as the general acid catalyst to protonate the oxygen of the glycosidic linkage for substrate hydrolysis; Asp241 (Asp286 in amylosucrase), the attacking nucleophile, forms a bond with C1 to form β-glucosylenzyme intermediate, whereas Asp369 (Asp393 in amylosucrase) forms hydrogen bonds to O2 and O3. Arg239 in PalI forms a salt bridge to Oδ1 of Asp241, which is essential for the correct positioning of the nucleophile. Similarly, His145 forms a hydrogen bond to O6 and His368 to O2, as is the case for the equivalent residues His187 and His392 in amylosucrase (11Skov L.K. Mirza O. Henriksen A. de Montalk G.P. Remaud-Simeon M. Sarcabal P. Willemot R.M. Monsan P. Gajhede M. J. Biol. Chem. 2001; 276: 25273-25278Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 25Mirza O. Skov L.K. Remaud-Simeon M. de Montalk G.P. Albenne C. Monsan P. Gajhede M. Biochemistry. 2001; 40: 9032-9039Crossref PubMed Scopus (79) Google Scholar). In addition, a salt bridge between Asp102 and Arg456 is formed in PalI. The equivalent salt bridges between Asp144 and Arg509 in amylosucrase (25Mirza O. Skov L.K. Remaud-Simeon M. de Montalk G.P. Albenne C. Monsan P. Gajhede M. Biochemistry. 2001; 40: 9032-9039Crossref PubMed Scopus (79) Google Scholar) and Asp60 and Arg415 in oligo-1,6-glucosidase (10Watanabe K. Hata Y. Kizaki H. Katsube Y. Suzuki Y. J. Mol. Biol. 1997; 269: 142-153Crossref PubMed Scopus (237) Google Scholar) have been reported. One notable feature in amylosucrase, when compared with TAKA-amylase, is that the +1 subsite is modified from Lys to Ala289 (amylosucrase numbering), providing the specificity of amylosucrase for the furanosyl ring of sucrose (11Skov L.K. Mirza O. Henriksen A. de Montalk G.P. Remaud-Simeon M. Sarcabal P. Willemot R.M. Monsan P. Gajhede M. J. Biol. Chem. 2001; 276: 25273-25278Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). The residues in PalI at the equivalent positions follow those of amylosucrase, mainly with Ala244, implying similar modifications of PalI at the +1 subsite to accommodate sucrose as the major substrate. Evidently, PalI adopts a mechanism similar to that in amylosucrase for sucrose binding, hydrolysis, and formation of covalent intermediate. A Motif Influencing Sucrose Isomerization—The hydrolysis of sucrose by PalI constitutes only a minor part of the reactions mediating the synthesis of sucrose isoforms (6Zhang D.H. Li X.Z. Zhang L.H. Appl. Environ. Microbiol. 2002; 68: 2676-2682Crossref PubMed Scopus (51) Google Scholar). Amylosucrase catalyzes the transfer of a d-glucopyranosyl moiety in the active site cleft to an acceptor molecule in a ravine formed by its domain B′ that plays the pivotal role in transferase reaction (11Skov L.K. Mirza O. Henriksen A. de Montalk G.P. Remaud-Simeon M. Sarcabal P. Willemot R.M. Monsan P. Gajhede M. J. Biol. Chem. 2001; 276: 25273-25278Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 26Skov L.K. Mirza O. Sprogoe D. Dar I. Remaud-Simeon M. Albenne C. Monsan P. Gajhede M. J. Biol. Chem. 2002; 277: 47741-47747Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar). In PalI, however, breakage of α-1,2-linkage in sucrose and formation of α-1,6- and α-1,1-linkages occur in the same pocket. To elucidate the mechanism of isomerization of PalI, the crucial structural features that interact with fructofuranose at the active site cleft and determine the change of fructofuranose to fructopyranose must be determined. This is because the conversion of fructofuranose to fructopyranose is the key step for trehalulose formation (8Veronese T. Perlot P. FEBS Lett. 1998; 441: 348-352Crossref PubMed Scopus (30) Google Scholar). Two residues in amylosucrase, Asp394 and Arg446, directly interact with the fructosyl ring of sucrose through hydrogen bonds (25Mirza O. Skov L.K. Remaud-Simeon M. de Montalk G.P. Albenne C. Monsan P. Gajhede M. Biochemistry. 2001; 40: 9032-9039Crossref PubMed Scopus (79) Google Scholar). The equivalent residues in PalI are Asn370 and Arg333, respectively. Amylosucrase and oligo-1,6-glucosidase significantly differ from PalI in that PalI contains a flexible loop region from Phe321 to Ser340 against a more rigid α-helical structure in oligo-1,6-glucosidase and amylosucrase (Fig. 2B). The unique 325RLDRD329 sequence of PalI is located in this loop adjacent to the active site cleft (Fig. 2, A and B). Not surprisingly, the RLDRD motif is not present in oligo-1,6-glucosidase and amylosucrase, as these two enzymes are functionally different from PalI. Notably, all known isomaltulose synthases contain the RLDRD sequence at equivalent regions (5Mattes R. Klein K. Schiwech H. Kunz M. Munir M. U. S. Patent 5. 1998; 786: 140Google Scholar, 27Bornke F. Hajirezaei M. Sonnewwald U. J. Bacteriol. 2001; 183: 2425-2430Crossref PubMed Scopus (50) Google Scholar). This specific region has also been identified by sequence alignment (28Zhang D.H. Li N. Swaminathan K. Zhang L.H. FEBS Lett. 2003; 534: 151-155Crossref PubMed Scopus (24) Google Scholar). The crystal structural analysis further indicates its unique location and possible interactions with the substrate. The role of the charged residues in this motif was investigated by creating the mutant versions of PalI and analyzing the relative amount of glucose, fructose, isomaltulose, and trehalulose synthesized (28Zhang D.H. Li N. Swaminathan K. Zhang L.H. FEBS Lett. 2003; 534: 151-155Crossref PubMed Scopus (24) Google Scholar). The isomaltulose content is decreased and the trehalulose content is increased in all of the PalI mutants, compared with the native PalI. However, mutation of Asp327, Arg328, and Asp329 did not significantly affect the ratio of sucrose hydrolysis to sucrose isomerization activity (≅0.06), but resulted in a 13–25-fold increase in trehalulose production. Only the mutation of residue Arg325 enhanced both sucrose hydrolysis and trehalulose formation. Evidently, the charge distributions in the isomaltulose synthase motif influence the stability of glucose and fructose binding to the enzyme and α-1,1- and α-1,6-glucosidic bond formation. As such, the unique location of the 325RLDRD329 motif highlights its importance in the isomerization process and therefore in the control of PalI product specificity. Mechanistic Implication of Sucrose Isomerization—Comparison of PalI with the amylosucrase-sucrose complex structure should reveal the mechanism of interactions between the side chains of active site residues and the glucosyl or the fructosyl moiety. In PalI, as shown in Scheme 2, A, Glu295 interacts with a bound sucrose molecule by protonating the glycosidic bond. It, therefore, serves to activate Asp241 to nucleophilically attack C1 to form the β-glucosyl-enzyme intermediate, as described for other members of glycosyl hydrolase family 13 (9Davies G. Henrissat B. Structure. 1995; 3: 853-859Abstract Full Text Full Text PDF PubMed Scopus (1579) Google Scholar, 10Watanabe K. Hata Y. Kizaki H. Katsube Y. Suzuki Y. J. Mol. Biol. 1997; 269: 142-153Crossref PubMed Scopus (237) Google Scholar, 11Skov L.K. Mirza O. Henriksen A. de Montalk G.P. Remaud-Simeon M. Sarcabal P. Willemot R.M. Monsan P. Gajhede M. J. Biol. Chem. 2001; 276: 25273-25278Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar). The residues that most probably interact with glucosyl moiety at –1 subsite include the conserved active residues, Asp369, Arg239, His145, and His368 and the salt bridge residues, Asp102 and Arg456. These remote glucose-binding subsites may prevent the release of glucose and thus confer less hydrolase activity on PalI. The mechanism of sucrose isomerization is speculative, although the biochemical functions of the motif 325RLDRD329 provide molecular evidence that the isomerization of sucrose is controlled by the charged residues in the proximity of the active site cleft. Based on our data, we propose that enzyme-bound sucrose interacts with the conserved residues of the active site and the isomaltulose synthase motif. The fructofuranose conformation of the fructose moiety is tightly preserved by the charged residues of the RLDRD motif so that isomaltulose is the main isomer synthesized (Scheme 2, B). Disruption of the charge distribution balance by mutations of the 325RLDRD329 motif or the pH changes enhances the tautomerization of fructofuranose to fructopyranose (6Zhang D.H. Li X.Z. Zhang L.H. Appl. Environ. Microbiol. 2002; 68: 2676-2682Crossref PubMed Scopus (51) Google Scholar), thereby forming the sucrose isomer, trehalulose (Scheme 2, C). Molecular dynamics analysis suggests that the fructose moiety bound to the mutant enzyme displays variable conformation (29Immel S. Lichtenthaler F.W. Liebigs Ann. 1995; : 1925-1937Crossref Scopus (84) Google Scholar). This electrostatic shift in the active site pocket also appears to cause the movement of 6′-OH group toward C2′ to form fructopyranose as well as the rotation of the C1′-OH toward C1 of glucosyl ring. Although direct evidence for the proposed mechanism is not yet available, further structural studies of PalI-substrate complex would allow us to present a complete isomerization scheme. C-terminal Domain—The C-terminal domain of PalI interacts with the N-terminal domain by forming salt bridges and hydrogen bonds (Fig. 2C). All hydrogen bonds between the two domains are clustered in two regions. In the first region, residues of Nα6 and the loops formed by residues 398–401 and 515–521 in the N-terminal domain make hydrogen bonds with residues in Cβ1-Clp1-Cβ2 segment. The second region involves residues 379–384 and 497–501 in the N-terminal domain and Clp3 and Clp5 in the C-terminal domain. An interdomain salt bridge (Arg381Nη2... Glu553Oϵ1), which is not present in oligo-1,6-glucosidase (10Watanabe K. Hata Y. Kizaki H. Katsube Y. Suzuki Y. J. Mol. Biol. 1997; 269: 142-153Crossref PubMed Scopus (237) Google Scholar), connects Clp3 in the C-terminal domain and the loop after the active site in the N-terminal domain (Fig. 2C). To understand the influence of the C-terminal domain on PalI functions, we have constructed three C-terminal deletion mutants, PalI:Δ587, PalI:Δ572, and PalI:Δ545, which are truncated after the indicated residue. The truncated PalI mutants were overexpressed, purified, and the remaining activity has been assayed. As shown in Table I, the mutant PalI:Δ587, created by the deletion of Clp6 and Cβ7, reduces the enzyme activity to 92.3% of the native PalI. Further deletion of Cβ5 and C1p5 in PalI:Δ572 leads to about 90% loss of activity, and deletion of Cβ3-Cβ7 in PalI:Δ545 completely abolishes the enzyme activity. C-terminal deletions interrupt the interdomain interactions and may cause significant changes in the structure of the N-terminal domain. The preceding discussion is based on the comparison of the PalI structure with oligo-1,6-glucosidase and amylosucrase, together with biochemical analysis of a series of mutant PalI proteins. The unique RLDRD motif in the loop region participates in sucrose isomerization and thus influences product specificity. The protein-substrate complex structure should allow us to elucidate the real interaction of identified key residues with substrate and the true mechanism of sucrose isomerization. We thank K. Miura (Spring8 in Japan) for data collection and M. James (University of Alberta) and D. Voet (University of Pennsylvania) for advice and revision of the manuscript.