Insights into the reaction mechanism of glycosyl hydrolase family 49. Site-directed mutagenesis and substrate preference of isopullulanase.

Aspergillus niger isopullulanase (IPU) is the only pullulan-hydrolase in glycosyl hydrolase (GH) family 49 and does not hydrolyse dextran at all, while all other GH family 49 enzymes are dextran-hydrolysing enzymes. To investigate the common catalytic mechanism of GH family 49 enzymes, nine mutants were prepared to replace residues conserved among GH family 49 (four Trp, three Asp and two Glu). Homology modelling of IPU was also carried out based on the structure of Penicillium minioluteum dextranase, and the result showed that Asp353, Glu356, Asp372, Asp373 and Trp402, whose substitutions resulted in the reduction of activity for both pullulan and panose, were predicted to be located in the negatively numbered subsites. Three Asp-mutated enzymes, D353N, D372N and D373N, lost their activities, indicating that these residues are candidates for the catalytic residues of IPU. The W402F enzyme significantly reduced IPU activity, and the Km value was sixfold higher and the k0 value was 500-fold lower than those for the wild-type enzyme, suggesting that Trp402 is a residue participating in subsite -1. Trp31 and Glu273, whose substitutions caused a decrease in the activity for pullulan but not for panose, were predicted to be located in the interface between N-terminal and beta-helical domains. The substrate preference of the negatively numbered subsites of IPU resembles that of GH family 49 dextranases. These findings suggest that IPU and the GH family 49 dextranases have a similar catalytic mechanism in their negatively numbered subsites in spite of the difference of their substrate specificities.     

Mutational and Structural Analyses of Caldanaerobius polysaccharolyticus Man5B Reveal Novel Active Site Residues for Family 5 Glycoside Hydrolases

CpMan5B is a glycoside hydrolase (GH) family 5 enzyme exhibiting both β-1,4-mannosidic and β-1,4-glucosidic cleavage activities. To provide insight into the amino acid residues that contribute to catalysis and substrate specificity, we solved the structure of CpMan5B at 1.6 Å resolution. The structure revealed several active site residues (Y12, N92 and R196) in CpMan5B that are not present in the active sites of other structurally resolved GH5 enzymes. Residue R196 in GH5 enzymes is thought to be strictly conserved as a histidine that participates in an electron relay network with the catalytic glutamates, but we show that an arginine fulfills a functionally equivalent role and is found at this position in every enzyme in subfamily GH5_36, which includes CpMan5B. Residue N92 is required for full enzymatic activity and forms a novel bridge over the active site that is absent in other family 5 structures. Our data also reveal a role of Y12 in establishing the substrate preference for CpMan5B. Using these molecular determinants as a probe allowed us to identify Man5D from Caldicellulosiruptor bescii as a mannanase with minor endo-glucanase activity.     

Crystal structure of a β-fructofuranosidase with high transfructosylation activity from Aspergillus kawachii

β-Fructofuranosidases belonging to glycoside hydrolase family (GH) 32 are enzymes that hydrolyze sucrose. Some GH32 enzymes also catalyze transfructosylation to produce fructooligosaccharides. We found that Aspergillus kawachii IFO 4308 β-fructofuranosidase (AkFFase) produces fructooligosaccharides, mainly 1-kestose, from sucrose. We determined the crystal structure of AkFFase. AkFFase is composed of an N-terminal small component, a β-propeller catalytic domain, an α-helical linker, and a C-terminal β-sandwich, similar to other GH32 enzymes. AkFFase forms a dimer, and the dimerization pattern is different from those of other oligomeric GH32 enzymes. The complex structure of AkFFase with fructose unexpectedly showed that fructose binds both subsites ?1 and +1, despite the fact that the catalytic residues were not mutated. Fructose at subsite +1 interacts with Ile146 and Glu296 of AkFFase via direct hydrogen bonds.     

Single amino acid residue changes in subsite -1 of inulosucrase from Lactobacillus reuteri 121 strongly influence the size of products synthesized

Bacterial fructansucrase enzymes belong to glycoside hydrolase family 68 and catalyze transglycosylation reactions with sucrose, resulting in the synthesis of fructooligosaccharides and/or a fructan polymer. Significant differences in fructansucrase enzyme product specificities can be observed, i.e. in the type of polymer (levan or inulin) synthesized, and in the ratio of polymer versus fructooligosaccharide synthesis. The Lactobacillus reuteri 121 inulosucrase enzyme produces a diverse range of fructooligosaccharide molecules and a minor amount of inulin polymer [with beta(2-1) linkages]. The three-dimensional structure of levansucrase (SacB) of Bacillus subtilis revealed eight amino acid residues interacting with sucrose. Sequence alignments showed that six of these eight amino acid residues, including the catalytic triad (D272, E523 and D424, inulosucrase numbering), are completely conserved in glycoside hydrolase family 68. The other three completely conserved residues are located at the -1 subsite (W271, W340 and R423). Our aim was to investigate the roles of these conserved amino acid residues in inulosucrase mutant proteins with regard to activity and product profile. Inulosucrase mutants W340N and R423H were virtually inactive, confirming the essential role of these residues in the inulosucrase active site. Inulosucrase mutants R423K and W271N were less strongly affected in activity, and displayed an altered fructooligosaccharide product pattern from sucrose, synthesizing a much lower amount of oligosaccharide and significantly more polymer. Our data show that the -1 subsite is not only important for substrate recognition and catalysis, but also plays an important role in determining the size of the products synthesized.     

Friend or foe? Evolutionary history of glycoside hydrolase family 32 genes encoding for sucrolytic activity in fungi and its implications for plant-fungal symbioses

Background  

Many fungi are obligate biotrophs of plants, growing in live plant tissues, gaining direct access to recently photosynthesized carbon. Photosynthate within plants is transported from source to sink tissues as sucrose, which is hydrolyzed by plant glycosyl hydrolase family 32 enzymes (GH32) into its constituent monosaccharides to meet plant cellular demands. A number of plant pathogenic fungi also use GH32 enzymes to access plant-derived sucrose, but less is known about the sucrose utilization ability of mutualistic and commensal plant biotrophic fungi, such as mycorrhizal and endophytic fungi. The aim of this study was to explore the distribution and abundance of GH32 genes in fungi to understand how sucrose utilization is structured within and among major ecological guilds and evolutionary lineages. Using bioinformatic and PCR-based analyses, we tested for GH32 gene presence in all available fungal genomes and an additional 149 species representing a broad phylogenetic and ecological range of biotrophic fungi.     

A mutational analysis of the two motifs common to adenine methyltransferases.

All methyltransferases that use S-adenosyl methionine as the methyl group donor contain a sequence similar to (D/E/S)XFXGXG which has been postulated to form part of the cofactor binding site. In N6-adenine DNA methyltransferases there is a second motif, (D/N)PP(Y/F), which has been proposed to play a role similar to the catalytically essential PC motif conserved in all C5-cytosine DNA methyltransferases. We have made a series of amino acid changes in these two motifs in the EcoKI N6-adenine DNA methyltransferase. The mutant enzymes have been purified to homogeneity and characterized by physical biochemical methods. The first G is the most conserved residue in motif I. Changing this G to D completely abolished S-adenosyl methionine binding, but left enzyme structure and DNA target recognition unaltered, thus documenting the S-adenosyl methionine binding function of motif I in N6-adenine methyltransferases. Substitution of the N with D, or F with either G or C, in motif II abolished enzyme activity, but left S-adenosyl methionine and DNA binding unaltered. Changes of F to Y or W resulted in partial enzyme activity, implying that an aromatic residue is important for methylation. The substitution of W for F greatly enhanced UV-induced cross-linking between the enzyme and S-adenosyl methionine, suggesting that the aromatic residue is close in space to the methyl-group donor.     

Structural features of the Nostoc punctiforme debranching enzyme reveal the basis of its mechanism and substrate specificity

The debranching enzyme Nostoc punctiforme debranching enzyme (NPDE) from the cyanobacterium Nostoc punctiforme (PCC73102) hydrolyzes the α‐1,6 glycosidic linkages of malto‐oligosaccharides. Despite its high homology to cyclodextrin/pullulan (CD/PUL)‐hydrolyzing enzymes from glycosyl hydrolase 13 family (GH‐13), NPDE exhibits a unique catalytic preference for longer malto‐oligosaccharides (>G8), performing hydrolysis without the transgylcosylation or CD‐hydrolyzing activities of other GH‐13 enzymes. To investigate the molecular basis for the property of NPDE, we determined the structure of NPDE at 2.37‐Å resolution. NPDE lacks the typical N‐terminal domain of other CD/PUL‐hydrolyzing enzymes and forms an elongated dimer in a head‐to‐head configuration. The unique orientation of residues 25–55 in NPDE yields an extended substrate binding groove from the catalytic center to the dimeric interface. The substrate binding groove with a lengthy cavity beyond the ?1 subsite exhibits a suitable architecture for binding longer malto‐oligosaccharides (>G8). These structural results may provide a molecular basis for the substrate specificity and catalytic function of this cyanobacterial enzyme, distinguishing it from the classical neopullulanases and CD/PUL‐hydrolyzing enzymes. Proteins 2010. © 2009 Wiley‐Liss, Inc.     

Fructo-Oligosaccharide Synthesis by Mutant Versions of Saccharomyces cerevisiae Invertase

Efficient enzymatic synthesis of tailor-made prebiotic fructo-oligosaccharides (FOS) used in functional food formulation is a relevant biotechnological objective. We have engineered the Saccharomyces cerevisiae invertase (Suc2) to improve its transferase activity and to identify the enzymatic determinants for product specificity. Amino acid replacement (W19Y, N21S, N24S) within a conserved motif (β-fructosidase) specifically increased the synthesis of 6-kestose up to 10-fold. Mutants with lower substrate (sucrose) affinity produced FOS with longer half-lives. A mutation (P205V) adjacent to another conserved motif (EC) caused a 6-fold increment in 6-kestose yield. Docking studies with a Suc2 modeled structure defined a putative acceptor substrate binding subsite constituted by Trp 291 and Asn 228. Mutagenesis studies confirmed the implication of Asn 228 in directing the orientation of the sucrose molecule for the specific synthesis of β(2,6) linkages.     

Structural and functional analysis of a glycoside hydrolase family 97 enzyme from Bacteroides thetaiotaomicron

SusB, an 84-kDa alpha-glucoside hydrolase involved in the starch utilization system (sus) of Bacteroides thetaiotaomicron, belongs to glycoside hydrolase (GH) family 97. We have determined the enzymatic characteristics and the crystal structures in free and acarbose-bound form at 1.6A resolution. SusB hydrolyzes the alpha-glucosidic linkage, with inversion of anomeric configuration liberating the beta-anomer of glucose as the reaction product. The substrate specificity of SusB, hydrolyzing not only alpha-1,4-glucosidic linkages but also alpha-1,6-, alpha-1,3-, and alpha-1,2-glucosidic linkages, is clearly different from other well known glucoamylases belonging to GH15. The structure of SusB was solved by the single-wavelength anomalous diffraction method with sulfur atoms as anomalous scatterers using an in-house x-ray source. SusB includes three domains as follows: the N-terminal, catalytic, and C-terminal domains. The structure of the SusB-acarbose complex shows a constellation of carboxyl groups at the catalytic center; Glu532 is positioned to provide protonic assistance to leaving group departure, with Glu439 and Glu508 both positioned to provide base-catalyzed assistance for inverting nucleophilic attack by water. A structural comparison with other glycoside hydrolases revealed significant similarity between the catalytic domain of SusB and those of alpha-retaining glycoside hydrolases belonging to GH27, -36, and -31 despite the differences in catalytic mechanism. SusB and the other retaining enzymes appear to have diverged from a common ancestor and individually acquired the functional carboxyl groups during the process of evolution. Furthermore, sequence comparison of the active site based on the structure of SusB indicated that GH97 included both retaining and inverting enzymes.     

Polysaccharide synthesis of the levansucrase SacB from Bacillus megaterium is controlled by distinct surface motifs

Despite the widespread biological function of carbohydrates, the polysaccharide synthesis mechanisms of glycosyltransferases remain largely unexplored. Bacterial levansucrases (glycoside hydrolase family 68) synthesize high molecular weight, β-(2,6)-linked levan from sucrose by transfer of fructosyl units. The kinetic and biochemical characterization of Bacillus megaterium levansucrase SacB variants Y247A, Y247W, N252A, D257A, and K373A reveal novel surface motifs remote from the sucrose binding site with distinct influence on the polysaccharide product spectrum. The wild type activity (k(cat)) and substrate affinity (K(m)) are maintained. The structures of the SacB variants reveal clearly distinguishable subsites for polysaccharide synthesis as well as an intact active site architecture. These results lead to a new understanding of polysaccharide synthesis mechanisms. The identified surface motifs are discussed in the context of related glycosyltransferases.     

Structural and kinetic insights reveal that the amino acid pair Gln-228/Asn-254 modulates the transfructosylating specificity of Schwanniomyces occidentalis β-fructofuranosidase, an enzyme that produces prebiotics

Schwanniomyces occidentalis β-fructofuranosidase (Ffase) is a GH32 dimeric enzyme that releases fructose from the nonreducing end of various oligosaccharides and essential storage fructans such as inulin. It also catalyzes the transfer of a fructosyl unit to an acceptor producing 6-kestose and 1-kestose, prebiotics that stimulate the growth of bacteria beneficial for human health. We report here the crystal structure of inactivated Ffase complexed with fructosylnystose and inulin, which shows the intricate net of interactions keeping the substrate tightly bound at the active site. Up to five subsites were observed, the sugar unit located at subsite +3 being recognized by interaction with the β-sandwich domain of the adjacent subunit within the dimer. This explains the high activity observed against long substrates, giving the first experimental evidence of the direct role of a GH32 β-sandwich domain in substrate binding. Crucial residues were mutated and their hydrolase/transferase (H/T) activities were fully characterized, showing the involvement of the Gln-228/Asn-254 pair in modulating the H/T ratio and the type β(2-1)/β(2-6) linkage formation. We generated Ffase mutants with new transferase activity; among them, Q228V gives almost specifically 6-kestose, whereas N254T produces a broader spectrum product including also neokestose. A model for the mechanism of the Ffase transfructosylation reaction is proposed. The results contribute to an understanding of the molecular basis regulating specificity among GH-J clan members, which represent an interesting target for rational design of enzymes, showing redesigned activities to produce tailor-made fructooligosaccharides.     

Identification of the catalytic residues of bifunctional glycogen debranching enzyme

Eukaryotic glycogen debranching enzyme (GDE) possesses two different catalytic activities (oligo-1,4-->1,4-glucantransferase/amylo-1,6-glucosidase) on a single polypeptide chain. To elucidate the structure-function relationship of GDE, the catalytic residues of yeast GDE were determined by site-directed mutagenesis. Asp-535, Glu-564, and Asp-670 on the N-terminal half and Asp-1086 and Asp-1147 on the C-terminal half were chosen by the multiple sequence alignment or the comparison of hydrophobic cluster architectures among related enzymes. The five mutant enzymes, D535N, E564Q, D670N, D1086N, and D1147N were constructed. The mutant enzymes showed the same purification profiles as that of wild-type enzyme on beta-CD-Sepharose-6B affinity chromatography. All the mutant enzymes possessed either transferase activity or glucosidase activity. Three mutants, D535N, E564Q, and D670N, lost transferase activity but retained glucosidase activity. In contrast, D1086N and D1147N lost glucosidase activity but retained transferase activity. Furthermore, the kinetic parameters of each mutant enzyme exhibiting either the glucosidase activity or transferase activity did not vary markedly from the activities exhibited by the wild-type enzyme. These results strongly indicate that the two activities of GDE, transferase and glucosidase, are independent and located at different sites on the polypeptide chain.     

Molecular properties of the glucosaminidase AcmA from Lactococcus lactis MG1363: Mutational and biochemical analyses

The major autolysin AcmA of Lactococcus lactis ssp. cremoris MG1363 is a modular protein consisting of an N-terminal signal sequence, a central enzymatic region (glu_acma as a glucosaminidase), and a C-terminal cell-recognition domain (LysM123). glu_acma (about 160 amino acids) belongs to the glycoside hydrolase (GH) 73 family, and the two acidic residues E128 and D153 have been thought to be catalytically important. In this study, amino-acid substitution analysis of AcmA was first carried out in the Escherichia coli system. Point mutations E94A, E94Q, E128A, D153A, and Y191A markedly reduced cell-lytic activity (3.8%, 1.1%, 4.2%, 4.8%, and 2.4%, respectively), whereas E128Q and D153N retained significant residual activities (32.1% and 44.0%, respectively). On the other hand, Y191F and Y191W mutations retained high activities (66.2% and 46.0%, respectively). These results showed that E94 (rather than E128 and D153) and the aromatic residue Y191 probably play important roles in catalysis of AcmA. Together with mutational analysis of another GH73 glucoaminidase Glu_atlwm from the Staphylococcus warneri M autolysin Atl_WM, these results suggested that the GH73 members cleave a glycosidic bond via a substrate-assisted mechanism, as postulated in the GH20 members. AcmA and Glu_atlwm were purified from E. coli recombinant cells, and their enzymatic properties were studied.     

Structural elements to convert Escherichia coli alpha-xylosidase (YicI) into alpha-glucosidase

Escherichia coli YicI, a member of glycoside hydrolase family (GH) 31, is an alpha-xylosidase, although its amino-acid sequence displays approximately 30% identity with alpha-glucosidases. By comparing the amino-acid sequence of GH 31 enzymes and through structural comparison of the (beta/alpha)(8) barrels of GH 27 and GH 31 enzymes, the amino acids Phe277, Cys307, Phe308, Trp345, Lys414, and beta-->alpha loop 1 of (beta/alpha)(8) barrel of YicI have been identified as elements that might be important for YicI substrate specificity. In attempt to convert YicI into an alpha-glucosidase these elements have been targeted by site-directed mutagenesis. Two mutated YicI, short loop1-enzyme and C307I/F308D, showed higher alpha-glucosidase activity than wild-type YicI. C307I/F308D, which lost alpha-xylosidase activity, was converted into alpha-glucosidase.     

Extent and Origins of Functional Diversity in a Subfamily of Glycoside Hydrolases

Some glycoside hydrolases have broad specificity for hydrolysis of glycosidic bonds, potentially increasing their functional utility and flexibility in physiological and industrial applications. To deepen the understanding of the structural and evolutionary driving forces underlying specificity patterns in glycoside hydrolase family 5, we quantitatively screened the activity of the catalytic core domains from subfamily 4 (GH5_4) and closely related enzymes on four substrates: lichenan, xylan, mannan, and xyloglucan. Phylogenetic analysis revealed that GH5_4 consists of three major clades, and one of these clades, referred to here as clade 3, displayed average specific activities of 4.2 and 1.2 U/mg on lichenan and xylan, approximately 1 order of magnitude larger than the average for active enzymes in clades 1 and 2. Enzymes in clade 3 also more consistently met assay detection thresholds for reaction with all four substrates. We also identified a subfamily-wide positive correlation between lichenase and xylanase activities, as well as a weaker relationship between lichenase and xyloglucanase. To connect these results to structural features, we used the structure of CelE from Hungateiclostridium thermocellum (PDB 4IM4) as an example clade 3 enzyme with activities on all four substrates. Comparison of the sequence and structure of this enzyme with others throughout GH5_4 and neighboring subfamilies reveals at least two residues (H149 and W203) that are linked to strong activity across the substrates. Placing GH5_4 in context with other related subfamilies, we highlight several possibilities for the ongoing evolutionary specialization of GH5_4 enzymes.     

Chitobiose phosphorylase from Vibrio proteolyticus, a member of glycosyl transferase family 36, has a clan GH-L-like (alpha/alpha)(6) barrel fold

Vibrio proteolyticus chitobiose phosphorylase (ChBP) belongs to glycosyl transferase family 36 (GT-36), and catalyzes the reversible phosphorolysis of chitobiose into alpha-GlcNAc-1-phosphate and GlcNAc with inversion of the anomeric configuration. As the first known structures of a GT-36 enzyme, we determined the crystal structure of ChBP in a ternary complex with GlcNAc and SO(4). It is also the first structures of an inverting phosphorolytic enzyme in a complex with a sugar and a sulfate ion, and reveals a pseudo-ternary complex structure of enzyme-sugar-phosphate. ChBP comprises a beta sandwich domain and an (alpha/alpha)(6) barrel domain, constituting a distinctive structure among GT families. Instead, it shows significant structural similarity with glycoside hydrolase (GH) enzymes, glucoamylases (GH-15), and maltose phosphorylase (GH-65) in clan GH-L. The structural similarity reported here, together with distant sequence similarities between ChBP and GHs, led to the reclassification of family GT-36 into a novel GH family, namely GH-94.     

beta-fructosidase superfamily: homology with some alpha-L-arabinases and beta-D-xylosidases

Comparison of the amino acid sequences of four families of glycosyl hydrolases reveals that they are homologous and have several common conserved regions. Two of these families contain beta-fructosidases (glycosyl hydrolase families GH32 and GH68) and the other two include alpha-L-arabinases and beta-xylosidases (families GH43 and GH62). The latter two families are proposed to be grouped together with the former two into the beta-fructosidase (furanosidase) superfamily. Several ORFs can be considered as a fifth family of the superfamily on the basis of sequence similarity. It is shown for the first time that a glycosyl hydrolase superfamily can include enzymes with both inversion and retention mechanism of action. Composition of the active center for enzymes of the superfamily is discussed.     

Changes in the Catalytic Properties of Pyrococcus furiosus Thermostable Amylase by Mutagenesis of the Substrate Binding Sites

Pyrococcus furiosus thermostable amylase (TA) is a cyclodextrin (CD)-degrading enzyme with a high preference for CDs over maltooligosaccharides. In this study, we investigated the roles of four residues (His414, Gly415, Met439, and Asp440) in the function of P. furiosus TA by using site-directed mutagenesis and kinetic analysis. A variant form of P. furiosus TA containing two mutations (H414N and G415E) exhibited strongly enhanced α-(1,4)-transglycosylation activity, resulting in the production of a series of maltooligosaccharides that were longer than the initial substrates. In contrast, the variant enzymes with single mutations (H414N or G415E) showed a substrate preference similar to that of the wild-type enzyme. Other mutations (M439W and D440H) reversed the substrate preference of P. furiosus TA from CDs to maltooligosaccharides. Relative substrate preferences for maltoheptaose over β-CD, calculated by comparing k_cat/K_m ratios, of 1, 8, and 26 for wild-type P. furiosus TA, P. furiosus TA with D440H, and P. furiosus TA with M439W and D440H, respectively, were found. Our results suggest that His414, Gly415, Met439, and Asp440 play important roles in substrate recognition and transglycosylation. Therefore, this study provides information useful in engineering glycoside hydrolase family 13 enzymes.     

Structure and function of a family 10 beta-xylanase chimera of Streptomyces olivaceoviridis E-86 FXYN and Cellulomonas fimi Cex

The catalytic domain of xylanases belonging to glycoside hydrolase family 10 (GH10) can be divided into 22 modules (M1 to M22; Sato, Y., Niimura, Y., Yura, K., and Go, M. (1999) Gene (Amst.) 238, 93-101). Inspection of the crystal structure of a GH10 xylanase from Streptomyces olivaceoviridis E-86 (SoXyn10A) revealed that the catalytic domain of GH10 xylanases can be dissected into two parts, an N-terminal larger region and C-terminal smaller region, by the substrate binding cleft, corresponding to the module border between M14 and M15. It has been suggested that the topology of the substrate binding clefts of GH10 xylanases are not conserved (Charnock, S. J., Spurway, T. D., Xie, H., Beylot, M. H., Virden, R., Warren, R. A. J., Hazlewood, G. P., and Gilbert, H. J. (1998) J. Biol. Chem. 273, 32187-32199). To facilitate a greater understanding of the structure-function relationship of the substrate binding cleft of GH10 xylanases, a chimeric xylanase between SoXyn10A and Xyn10A from Cellulomonas fimi (CfXyn10A) was constructed, and the topology of the hybrid substrate binding cleft established. At the three-dimensional level, SoXyn10A and CfXyn10A appear to possess 5 subsites, with the amino acid residues comprising subsites -3 to +1 being well conserved, although the +2 subsites are quite different. Biochemical analyses of the chimeric enzyme along with SoXyn10A and CfXyn10A indicated that differences in the structure of subsite +2 influence bond cleavage frequencies and the catalytic efficiency of xylooligosaccharide hydrolysis. The hybrid enzyme constructed in this study displays fascinating biochemistry, with an interesting combination of properties from the parent enzymes, resulting in a low production of xylose.     

Molecular structure of the locus for sucrose utilization by Lactobacillus plantarum: comparison with Pediococcus pentosaceus

Structure of the sucrose utilization locus in a Lactobacillus plantarum type strain was studied using PCR and Southern hybridization. Restriction map analysis revealed its high similarity to the sequenced sucrose utilization locus of Pediococcus pentosaceus pSRQ1. The L. plantarum locus proved containing oppositely oriented scrA and the scrBRagl operon, but not agaS. The L. plantarum sucrase gene (scrB) was partly sequenced. A higher (98.6%) homology was revealed between scrB than between the 16S rRNA genes of L. plantarum and P. pentosaceus, suggesting horizontal transfer of the sucrose utilization locus between the genera of lactic acid bacteria. Amino acid sequence analysis showed that the ScrB proteins of the two species belong to a subfamily of glycosyl hydrolase family GH32 which includes various beta-fructosidases.