Structure activity studies with xenobiotic substrates using carboxylesterases isolated from Arabidopsis thaliana

Carboxylesterases (CXEs) catalyse the hydrolysis of xenobiotics and natural products radically altering their biological activities. Whereas the substrate selectivity of animal CXEs, such as porcine liver esterase (PLE) have been well studied, the respective enzymes in plants have yet to be defined and their activities determined. Using Arabidopsis thaliana (At) as a source, five representative members of the alpha/beta hydrolase AtCXE family of proteins have been cloned, expressed and the purified recombinant proteins assayed for esterase activity with xenobiotic substrates. Two members, AtCXE5 and AtCXE18 were found to be active carboxylesterases, though AtCXE5 proved to be highly unstable as a soluble protein. AtCXE18 and the previously characterised S-formylglutathione hydrolase from Arabidopsis (AtSFGH) were assayed against a series of esters based on methylumbelliferone in which the acyl moiety was varied with respect to size and conformation. The same series was used to assay crude esterase preparation from Arabidopsis plants and the results compared with those obtained with the commonly used PLE. With straight chain esters, AtCXE18 behaved like PLE, but the Arabidopsis hydrolases proved less tolerant of branched chain acyl components than the mammalian enzyme. While none of the enzyme preparations accurately reflected all the activities determined with crude Arabidopsis protein extracts, the plant enzymes proved more useful than PLE in predicting the hydrolysis of the more sterically constrained esters.     

Isolation and characterization of Xenopus soluble epoxide hydrolase

Soluble epoxide hydrolase (sEH) contributes to cell growth, but the contribution of sEH to embryonic development is not well understood. In this study, Xenopus sEH cDNA was isolated from embryos of Xenopus laevis. The Xenopus sEH was expressed in Escherichia coli and was purified. The epoxide hydrolase and phosphatase activities of purified sEH were investigated. The Xenopus sEH did not show phosphatase activity toward 4-methylumbelliferyl phosphate or several lysophosphatidic acids although it had EH activity. The amino acid sequence of Xenopus sEH was compared with that reported previously. We found amino acid substitutions of the 29th Thr to Asn and the 146th Arg to His and prepared a sEH mutant (N29T/H146R), designed as mutant 1. Neither wild-type sEH nor mutant 1 had phosphatase activity. Additional substitution of the 11th Gly with Asp was found by comparison with human sEH which has phosphatase activity, but the Xenopus sEH mutant G11D prepared as mutant 2 did not have phosphatase activity. The epoxide hydrolase activity of sEH seemed to be similar to that of human sEH, while Xenopus sEH did not have phosphatase activity toward several substrates that human sEH metabolizes.     

Novel role for pectin methylesterase in Arabidopsis: A new function showing ribosome-inactivating protein (RIP) activity

Ribosome-inactivating proteins (RIPs, EC 3.2.2.22) are plant enzymes that can inhibit the translation process by removing single adenine residues of the large rRNA. These enzymes are known to function in defense against pathogens, but their biological role is unknown, partly due to the absence of work on RIPs in a model plant. In this study, we purified a protein showing RIP activity from Arabidopsis thaliana by employing chromatography separations coupled with an enzymatic activity. Based on N-terminal and internal amino acid sequencing, the RIP purified was identified as a mature form of pectin methylesterase (PME, At1g11580). The purified native protein showed both PME and RIP activity. PME catalyzes pectin deesterification, releasing acid pectin and methanol, which cause cell wall changes. We expressed the full-length and mature form of cDNA clones into an expression vector and transformed it in Escherichia coli for protein expression. The recombinant PME proteins (full-length and mature) expressed in E. coli did not show either PME or RIP activity, suggesting that post-translational modifications are important for these enzymatic activities. This study demonstrates a new function for an old enzyme identified in a model plant and discusses the possible role of a protein's conformational changes corresponding to its dual enzymatic activity.     

Crystal structures and proposed structural/functional classification of three protozoan proteins from the isochorismatase superfamily

We have determined the crystal structures of three homologous proteins from the pathogenic protozoans Leishmania donovani, Leishmania major, and Trypanosoma cruzi. We propose that these proteins represent a new subfamily within the isochorismatase superfamily (CDD classification cd004310). Their overall fold and key active site residues are structurally homologous both to the biochemically well-characterized N-carbamoylsarcosine-amidohydrolase, a cysteine hydrolase, and to the phenazine biosynthesis protein PHZD (isochorismase), an aspartyl hydrolase. All three proteins are annotated as mitochondrial-associated ribonuclease Mar1, based on a previous characterization of the homologous protein from L. tarentolae. This would constitute a new enzymatic activity for this structural superfamily, but this is not strongly supported by the observed structures. In these protozoan proteins, the extended active site is formed by inter-subunit association within a tetramer, which implies a distinct evolutionary history and substrate specificity from the previously characterized members of the isochorismatase superfamily. The characterization of the active site is supported crystallographically by the presence of an unidentified ligand bound at the active site cysteine of the T. cruzi structure.     

A central role for gamma-glutamyl hydrolases in plant folate homeostasis

Most cellular folates carry a short poly-γ-glutamate tail, and this tail is believed to affect their efficacy and stability. The tail can be removed by γ-glutamyl hydrolase (GGH; EC 3.4.19.9), a vacuolar enzyme whose role in folate homeostasis remains unclear. In order to probe the function of GGH, we modulated its level of expression and subcellular location in Arabidopsis plants and tomato fruit. Three-fold overexpression of GGH in vacuoles caused extensive deglutamylation of folate polyglutamates and lowered the total folate content by approximately 40% in Arabidopsis and tomato. No such effects were seen when GGH was overexpressed to a similar extent in the cytosol. Ablation of either of the major Arabidopsis GGH genes (AtGGH1 and AtGGH2) alone did not significantly affect folate status. However, a combination of ablation of one gene plus RNA interference (RNAi)-mediated suppression of the other (which lowered total GGH activity by 99%) increased total folate content by 34%. The excess folate accumulated as polyglutamate derivatives in the vacuole. Taken together, these results suggest a model in which: (i) folates continuously enter the vacuole as polyglutamates, accumulate there, are hydrolyzed by GGH, and exit as monoglutamates; and (ii) GGH consequently has an important influence on polyglutamyl tail length and hence on folate stability and cellular folate content.     

A novel surface autolysin of Listeria monocytogenes serotype 4b, IspC, contains a 23-residue N-terminal signal peptide being processed in E. coli

The 86-kDa protein IspC of 774 amino acids in Listeria monocytogenes serotype 4b has been recently identified as the target of humoral immune response to listerial infection and as a novel surface autolysin. A signal peptide is predicted at the N-terminal end of IspC, but no biochemical data has been shown to confirm the presence of the cleavage site of a signal peptidase. To address this and prepare sufficient amount of the protein for biochemical and structural characterization, we present a strategy for efficient expression and purification of IspC and analyze the purified protein by N-terminal sequencing and mass spectrometry. Expression of IspC in Escherichia coli using a pET30a-based expression construct was efficiently improved by incubating the culture at 37 degrees C for 2h followed by 4 degrees C for 16-18 h. The recombinant product rIspC remained as a soluble form in the cellular extract and was purified to electrophorectic homogeneity by the combination of metal chelate affinity chromatography with cation-exchange chromatography. The IspC was shown to contain a 23-residue N-terminal signal peptide being processed between Thr 23 and Thr 24 in E. coli, resulting in an 84-kDa mature protein. The highly purified form of rIspC from this study, exhibiting both peptidoglycan hydrolase activity and immunogenicity as previously reported, would facilitate further biochemical, structural, and functional studies of this autolysin.     

Cloning,partial purification and in vivo developmental profile of expression of the juvenile hormone epoxide hydrolase of Ctenocephalides felis

cDNAs encoding two different epoxide hydrolases (nCfEH1 and nCfEH2) were cloned from a cDNA library prepared from the wandering larval stage of the cat flea, Ctenocephalides felis. Predicted translations of the open reading frames indicated the clones encoded proteins of 464 (CfEH1) and 465 (CfEH2) amino acids. These proteins have a predicted molecular weight of 53 kDa and a putative 22 amino acid N-terminal hydrophobic membrane anchor. The amino acid sequences are 77% identical, and both are homologous to previously isolated epoxide hydrolases from Manduca sexta, Trichoplusia ni, and Rattus norvegicus. Purification of native juvenile hormone epoxide hydrolase (JHEH) from unfed adult cat fleas generated a partially pure protein that hydrolyzed juvenile hormone III to juvenile hormone III-diol. The amino terminal sequence of this;50-kDa protein is identical to the deduced amino terminus of the protein encoded by the nCfEH1 clone. Affinity-purified rabbit polyclonal antibodies raised against Escherichia coli-expressed HisCfEH1 recognized a approximately 50-kDa protein present in the partially purified fraction containing JHEH activity. Immunohistochemistry experiments using the same affinity-purified rabbit polyclonal antibodies localized the epoxide hydrolase in developing oocytes, fat body, and midgut epithelium of the adult flea. The presence of JHEH in various flea life stages and tissues was assessed by Northern blot and enzymatic activity assays. JHEH mRNA expression remained relatively constant throughout the different flea larval stages and was slightly elevated in the unfed adult flea. JHEH enzymatic activity was highest in the late larval, pupal, and adult stages. In all stages and tissues examined, JHEH activity was significantly lower than juvenile hormone esterase (JHE) activity, the other enzyme responsible for JH catalysis.     

Unique regulation of the active site of the serine esterase S-formylglutathione hydrolase

S-Formylglutathione hydrolases (SFGHs) are highly conserved thioesterases present in prokaryotes and eukaryotes, and form part of the formaldehyde detoxification pathway, as well as functioning as xenobiotic-hydrolysing carboxyesterases. As defined by their sensitivity to covalent modification, SFGHs behave as cysteine hydrolases, being inactivated by thiol alkylating agents, while being insensitive to inhibition by organophosphates such as paraoxon. As such, the enzyme has been classified as an esterase D in animals, plants and microbes. While SFGHs do contain a conserved cysteine residue that has been implicated in catalysis, sequence analysis also reveals the classic catalytic triad of a serine hydrolase. Using a combination of selective protein modification and X-ray crystallography, AtSFGH from Arabidopsis thaliana has been shown to be a serine hydrolase rather than a cysteine hydrolase. Uniquely, the conserved reactive cysteine (Cys59) previously implicated in catalysis lies in close proximity to the serine hydrolase triad, serving a gate-keeping function in comprehensively regulating access to the active site. Thus, any covalent modification of Cys59 inhibited all hydrolase activities of the enzyme. When isolated from Escherichia coli, a major proportion of recombinant AtSFGH was recovered with the Cys59 forming a mixed disulfide with glutathione. Reversible disulfide formation with glutathione could be demonstrated to regulate hydrolase activity in vitro. The importance of Cys59 in regulating AtSFGH in planta was demonstrated in transient expression assays in Arabidopsis protoplasts. As determined by fluorescence microscopy, the Cys59Ser mutant enzyme was shown to rapidly hydrolyse 4-methylumbelliferyl acetate in paraoxon-treated cells, while the native enzyme was found to be inactive. Our results clarify the classification of AtSFGHs as hydrolases and suggest that the regulatory and conserved cysteine provides an unusual redox-sensitive regulation to an enzyme functioning in both primary and xenobiotic metabolism in prokaryotes and eukaryotes.     

Skl, a novel choline-binding N-acetylmuramoyl-L-alanine amidase of Streptococcus mitis SK137 containing a CHAP domain

The skl gene from Streptococcus mitis SK137 encodes a peptidoglycan hydrolase (Skl) that has been purified and biochemically characterized. Analysis of the degradation products obtained by digestion of pneumococcal cell walls with Skl revealed that this enzyme is an N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28), showing optimum activity at 30 degrees C and at a pH of 6.5. Skl is a unique member of the choline-binding family of proteins since it contains a cysteine, histidine-dependent amidohydrolases/peptidases (CHAP) domain. The CHAP domain of Skl showed homology to lysins of unknown especificity from a variety of streptococcal prophages. Skl represents the first characterized member of a new subfamily of CHAP-containing choline-binding proteins.     

A novel glycoside hydrolase family 105: the structure of family 105 unsaturated rhamnogalacturonyl hydrolase complexed with a disaccharide in comparison with family 88 enzyme complexed with the disaccharide

YteR, a hypothetical protein with unknown functions, is derived from Bacillus subtilis strain 168 and has an overall structure similar to that of bacterial unsaturated glucuronyl hydrolase (UGL), although it exhibits little amino acid sequence identity with UGL. UGL releases unsaturated glucuronic acid from glycosaminoglycan treated with glycosaminoglycan lyases. The amino acid sequence of YteR shows a significant homology (26% identity) with the hypothetical protein YesR also from B. subtilis strain 168. To clarify the intrinsic functions of YteR and YesR, both proteins were overexpressed in Escherichia coli, purified, and characterized. Based on their gene arrangements in genome and enzyme properties, YteR and YesR were found to constitute a novel enzyme activity, "unsaturated rhamnogalacturonyl hydrolase," classified as new glycoside hydrolase family 105. This enzyme acts specifically on unsaturated rhamnogalacturonan (RG) obtained from RG type-I treated with RG lyases and releases an unsaturated galacturonic acid. The crystal structure of YteR complexed with unsaturated chondroitin disaccharide (UGL substrate) was obtained and compared to the structure of UGL complexed with the same disaccharide. The UGL substrate is sterically hindered with the active pocket of YteR. The protruding loop of YteR prevents the UGL substrate from being bound effectively. The most likely candidate catalytic residues for general acid/base are Asp143 in YteR and Asp135 in YesR. This is supported by three-dimensional structural and site-directed mutagenesis studies. These findings provide molecular insights into novel enzyme catalysis and sequential reaction mechanisms involved in RG-I depolymerization by bacteria.     

Purification and Characterization of Porcine Skeletal Muscle Aminopeptidase T,a Novel Metallopeptidase Homologous to Leukotriene A4 Hydrolase

A novel aminopeptidase, Aminopeptidase T (APase T), was purified from porcine skeletal muscle following successive column chromatography: twice on DEAE-cellulose, hydroxyapatite, and Sephacryl S-200 HR using Leu-β-naphthylamide (LeuNap) as a substrate. The molecular mass of the enzyme was 69 kDa on SDS–PAGE. The optimum pH towards LeuNap of the enzyme was about 7. The enzyme activity was strongly inhibited by bestatin and was negatively affected by ethylenediaminetetraacetic acid (EDTA). Chlorine-activated APase T liberated Leu, Ala, Met, Pro, and Arg from Nap derivatives. The APase T gene consisted of an ORF of 1,836 bp encoding a protein of 611 amino acid residues. The APase T was highly homologous to bovine, human, and mouse Leukotriene A₄ hydrolase (LTA₄H), a bifunctional enzyme which exhibits APase and epoxide hydrolase activity.     

Purification and characterization of bile salt hydrolase from Bacteroides fragilis subsp. fragilis.

A high-molecular-weight (250 000) bile salt hydrolase (cholylglycine hydrolase, EC 3.5.-.-) was isolated and purified 128-fold from the "spheroplast lysate" fraction prepared from Bacteroids fragilis subsp. fragilis ATCC 25285. The intact enzyme had a molecular weight of approx. 250 000 as determined by gel infiltration chromatography. One major protein band, corresponding to a molecular weight of 32 500, was observed on 7% sodium dodecyl sulfate polyacrylamide gel electrophoresis of pooled fractions from DEAE-cellulose column chromatography (128-fold purified). The pH optimum for the 64-fold purified enzyme isolated from Bio-Gel A 1.5 M chromatography was 4.2 and bile salt hydrolase activity measured in intact cell suspensions had a pH optimum of 4.5. Substrate specificity studies indicated that taurine and glycine conjugates of cholic acid, chenodeoxycholic acid and deoxycholic acid were readily hydrolyzed; however, lithocholic acid conjugates were not hydrolyzed. Substrate saturation kinetics were biphasic with an intermediate plateau (0.2--0.3 mM) and a complete loss of enzymatic activity was observed at high concentration for certain substrates. The presence or absence of 7-alpha-hydroxysteroid dehydrogenase was absolutely correlated with that of bile salt hydrolase activity in six to ten strains and subspecies of B. fragilis.     

Nonflowering plants possess a unique folate-dependent phenylalanine hydroxylase that is localized in chloroplasts

Tetrahydropterin-dependent aromatic amino acid hydroxylases (AAHs) are known from animals and microbes but not plants. A survey of genomes and ESTs revealed AAH-like sequences in gymnosperms, mosses, and algae. Analysis of full-length AAH cDNAs from Pinus taeda, Physcomitrella patens, and Chlamydomonas reinhardtii indicated that the encoded proteins form a distinct clade within the AAH family. These proteins were shown to have Phe hydroxylase activity by functional complementation of an Escherichia coli Tyr auxotroph and by enzyme assays. The P. taeda and P. patens AAHs were specific for Phe, required iron, showed Michaelian kinetics, and were active as monomers. Uniquely, they preferred 10-formyltetrahydrofolate to any physiological tetrahydropterin as cofactor and, consistent with preferring a folate cofactor, retained activity in complementation tests with tetrahydropterin-depleted E. coli host strains. Targeting assays in Arabidopsis thaliana mesophyll protoplasts using green fluorescent protein fusions, and import assays with purified Pisum sativum chloroplasts, indicated chloroplastic localization. Targeting assays further indicated that pterin-4a-carbinolamine dehydratase, which regenerates the AAH cofactor, is also chloroplastic. Ablating the single AAH gene in P. patens caused accumulation of Phe and caffeic acid esters. These data show that nonflowering plants have functional plastidial AAHs, establish an unprecedented electron donor role for a folate, and uncover a novel link between folate and aromatic metabolism.     

Detoxification of the phytoalexin brassinin by isolates of Leptosphaeria maculans pathogenic on brown mustard involves an inducible hydrolase

Brassinin is a phytoalexin produced by plants from the family Brassicaceae that displays antifungal activity against a number of pathogens of Brassica species, including Leptosphaeria maculans (Desm.) Ces. et de Not. [asexual stage Phoma lingam (Tode ex Fr.) Desm.] and L. biglobosa. The interaction of a group of isolates of L. maculans virulent on brown mustard (Brassica juncea) with brassinin was investigated. The metabolic pathway for degradation of brassinin, the substrate selectivity of the putative detoxifying hydrolase, as well as the antifungal activity of metabolites and analogs of brassinin are reported. Brassinin hydrolase activity was detectable only in cell-free homogenates resulting from cultures induced with brassinin, N'-methylbrassinin, or camalexin. The phytoalexin camalexin was a substantially stronger inhibitor of these isolates than brassinin, causing complete growth inhibition at 0.5mM.     

Functional genomic analysis of Arabidopsis thaliana glycoside hydrolase family 35

Catalysing the hydrolysis of terminal beta-galactosyl residues from carbohydrates, galactolipids, and glycoproteins, glycoside hydrolase family 35 (beta-galactosidases; BGALs) are widely distributed in plants and believed to play many key roles, including modification of cell wall components. Completion of the Arabidopsis thaliana genome sequencing project has, for the first time, allowed an examination of the total number, gene structure, and evolutionary patterns of all Family 35 members in a representative (model) angiosperm. Reiterative database searches established a multigene family of 17 members (designated BGAL1-BGAL17). Using these genes as query sequences, BLAST and Hidden Markov Model searches identified BGAL genes among 22 other eukaryotes, whose genomic sequences are known. The Arabidopsis (n=17) and rice (n=15) BGAL families were much larger than those of Chlamydomonas, fungi, and animals (n=0-4), and a lineage-specific expansion of BGAL genes apparently occurred after divergence of the Arabidopsis and rice lineages. All plant BGAL genes, with the exception of Arabidopsis BGAL17 and rice Os 9633.m04334, form a monophyletic group. Arabidopsis BGAL expression levels are much higher in mature leaves, roots, flowers, and siliques but are lower in young seedlings. BGAL8, BGAL11, BGAL13, BGAL14, and BGAL16 are expressed only in flowers. Catalytically active BGAL4 was produced in the E. coli and baculoviral expression systems, purified to electrophoretic homogeneity, and partially characterized. The purified enzyme hydrolyzed p- and o-nitrophenyl-beta-d-galactosides. It also cleaved beta-(1,3)-, beta-(1,4)-, and beta-(1,6)-linked galactobiosides and galactotriosides, showing a marked preference for beta-(1,3)- and beta-(1,4)-linkages.     

ADP-ribosylarginine hydrolases

ADP-ribosylation is a reversible post-translational modification of proteins involving the addition of the ADP-ribose moiety of NAD to an acceptor protein or amino acid. NAD: arginine ADP-ribosyltransferase, purified from numerous animal tissues, catalyzes the transfer of ADP-ribose to an arginine residue in proteins. The reverse reaction, catalyzed by ADP-ribosylarginine hydrolase, removes ADP-ribose, regenerating free arginine. An ADP-ribosylarginine hydrolase, purified extensively from turkey erythrocytes, was a 39-kDa monomeric protein under denaturing and non-denaturing conditions, and was activated by Mg²⁺ and dithiothreitol. The ADP-ribose moiety was critical for substrate recognition; the enzyme hydrolyzed ADP-ribosylarginine and (2-phospho-ADP-ribosyl)arginine but not phosphoribosylarginine or ribosylarginine. The hydrolase cDNA was cloned from rat and subsequently from mouse and human brain. The rat hydrolase gene contained a 1086-base pair open reading frame, with deduced amino acid sequences identical to those obtained by amino terminal sequencing of the protein or of HPLC-purified tryptic peptides. Deduced amino acid sequences from the mouse and human hydrolase cDNAs were 94% and 83% identical, respectively to the rat. Anti-rat brain hydrolase polyclonal antibodies reacted with turkey erythrocyte, mouse and bovine brain hydrolase. The rat hydrolase, expressed inE. coli, demonstrated enhanced activity in the presence of Mg²⁺ and thiol, whereas the recombinant human hydrolase was stimulated by Mg²⁺ but was thiol-independent. In the rat and mouse enzymes, there are five cysteines in identical positions; four of the cysteines are conserved in the human hydrolase. Replacement of cysteine 108 in the rat hydrolase (not present in the human enzyme) resulted in a thiol-independent hydrolase without altering specific activity. Rabbit anti-rat brain hydrolase antibodies reacted on immunoblot with the wild-type rat hydrolase and only weakly with the mutant hydrolase. There was no immunoreactivity with either the wild-type or mutant human enzyme. Cysteine 108 in the rat and mouse hydrolase may be responsible in part for thiol-dependence as wall as antibody recognition. Based on these studies, the mammalian and avian ADP-ribosylarginine hydrolases exhibit considerable conservation in structure and function.     

X-ray structure of potato epoxide hydrolase sheds light on substrate specificity in plant enzymes

Epoxide hydrolases catalyze the conversion of epoxides to diols. The known functions of such enzymes include detoxification of xenobiotics, drug metabolism, synthesis of signaling compounds, and intermediary metabolism. In plants, epoxide hydrolases are thought to participate in general defense systems. In the present study, we report the first structure of a plant epoxide hydrolase, one of the four homologous enzymes found in potato. The structure was solved by molecular replacement and refined to a resolution of 1.95 A. Analysis of the structure allows a better understanding of the observed substrate specificities and activity. Further, comparisons with mammalian and fungal epoxide hydrolase structures reported earlier show the basis of differing substrate specificities in the various epoxide hydrolase subfamilies. Most plant enzymes, like the potato epoxide hydrolase, are expected to be monomers with a preference for substrates with long lipid-like substituents of the epoxide ring. The significance of these results in the context of biological roles and industrial applications is discussed.     

Crystal structures and mutagenesis of sucrose hydrolase from Xanthomonas axonopodis pv. glycines: insight into the exclusively hydrolytic amylosucrase fold

Neisseria polysaccharea amylosucrase (NpAS), a transglucosidase of glycoside hydrolase family 13, is a hydrolase and glucosyltransferase that catalyzes the synthesis of amylose-like polymer from a sucrose substrate. Recently, an NpAS homolog from Xanthomonas axonopodis pv. glycines was identified as a member of the newly defined carbohydrate utilization locus that regulates the utilization of plant sucrose in phytopathogenic bacteria. Interestingly, this enzyme is exclusively a hydrolase and not a glucosyltransferase; it is thus known as sucrose hydrolase (SUH). Here, we elucidated the novel functional features of SUH using X-ray crystallography and site-directed mutagenesis. Four different crystal structures of SUH, including the SUH-Tris and the SUH-sucrose and SUH-glucose complexes, represent structural snapshots along the catalytic reaction coordinate. These structures show that SUH is distinctly different from NpAS in that ligand-induced conformational changes in SUH cause the formation of a pocket-shaped active site and in that SUH lacks the three arginine residues found in the NpAS active site that appear to be crucial for NpAS glucosyltransferase activity. Mutation of SUH to insert these arginines failed to confer glucosyltransferase activity, providing evidence that its enzymatic activity is limited to sucrose hydrolysis by its pocket-shaped active site and the identity of residues in the vicinity of the active site.