Mechanism of GlvA from Bacillus subtilis: a detailed kinetic analysis of a 6-phospho-alpha-glucosidase from glycoside hydrolase family 4

GlvA, a 6-phospho-alpha-glucosidase from Bacillus subtilis assigned to glycoside hydrolase family 4, catalyzes the hydrolysis of maltose 6'-phosphate via a redox-elimination-addition mechanism requiring NAD+ as cofactor. In contrast to previous reports and consistent with the proposed mechanism, GlvA is only activated in the presence of the nicotinamide cofactor in its oxidized, and not the reduced NADH, form. Significantly, GlvA catalyzes the hydrolysis of both 6-phospho-alpha- and 6-phospho-beta-glucosides containing activated leaving groups such as p-nitrophenol and does so with retention and inversion, respectively, of anomeric configuration. Mechanistic details of the individual bond cleaving and forming steps were probed using a series of 6-phospho-alpha- and 6-phospho-beta-glucosides. Primary deuterium kinetic isotope effects (KIEs) were measured for both classes of substrates in which either the C2 or the C3 protons have been substituted with a deuterium, consistent with C-H bond cleavage at each center being partially rate-limiting. Kinetic parameters were also determined for 1-[2H]-substituted substrates, and depending on the substrates and the reaction conditions, the measurements of kcat and kcat/KM produced either no KIEs or inverse KIEs. In conjunction with results of Br?nsted analyses with both aryl 6-phospho-alpha- and beta-glucosides, the kinetic data suggest that GlvA utilizes an E1cb mechanism analogous to that proposed for the Thermotoga maritima BglT, a 6-phospho-beta-glucosidase in glycoside hydrolase family 4 (Yip, V.L.Y et al. (2006) Biochemistry 45, 571-580). The pattern of isotope effects measured and the observation of very similar kcat values for all substrates, including unactivated and natural substrates, indicate that the oxidation and deprotonation steps are rate-limiting steps in essentially all cases. This mechanism permits the cleavage of both alpha- and beta-glycosides within the same active site motif and, for activated substrates that do not require acid catalysis for cleavage, within the same active site, yielding the product sugar-6-phosphate in the same anomeric form in the two cases.     

NAD+ and metal-ion dependent hydrolysis by family 4 glycosidases: structural insight into specificity for phospho-beta-D-glucosides

The import of disaccharides by many bacteria is achieved through their simultaneous translocation and phosphorylation by the phosphoenolpyruvate-dependent phosphotransferase system (PEP-PTS). The imported phospho-disaccharides are, in some cases, subsequently hydrolyzed by members of the unusual glycoside hydrolase family GH4. The GH4 enzymes, occasionally found also in bacteria such as Thermotoga maritima that do not utilise a PEP-PTS system, require both NAD(+) and Mn(2+) for catalysis. A further curiosity of this family is that closely related enzymes may show specificity for either alpha-d- or beta-d-glycosides. Here, we present, for the first time, the three-dimensional structure (using single-wavelength anomalous dispersion methods, harnessing extensive non-crystallographic symmetry) of the 6-phospho-beta-glycosidase, BglT, from T.maritima in native and complexed (NAD(+) and Glc6P) forms. Comparison of the active-center structure with that of the 6-phospho-alpha-glucosidase GlvA from Bacillus subtilis reveals a striking degree of structural similarity that, in light of previous kinetic isotope effect data, allows the postulation of a common reaction mechanism for both alpha and beta-glycosidases. Given that the "chemistry" occurs primarily on the glycone sugar and features no nucleophilic attack on the intact disaccharide substrate, modulation of anomeric specificity for alpha and beta-linkages is accommodated through comparatively minor structural changes.     

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.     

Permeability of Rickettsia prowazekii to NAD.

Rickettsia prowazekii accumulated radioactivity from [adenine-2,8-3H]NAD but not from [nicotinamide-4-3H]NAD, which demonstrated that NAD was not taken up intact. Extracellular NAD was hydrolyzed by rickettsiae with the products of hydrolysis, nicotinamide mononucleotide and AMP, appearing in the incubation medium in a time- and temperature-dependent manner. The particulate (membrane) fraction contained 90% of this NAD pyrophosphatase activity. Rickettsiae which had accumulated radiolabel after incubation with [adenine-2,8-3H]NAD were extracted, and the intracellular composition was analyzed by chromatography. The cells contained labeled AMP, ADP, ATP, and NAD. The NAD-derived intracellular AMP was transported via a pathway distinct from and in addition to the previously described AMP translocase. Exogenous AMP (1 mM) inhibited uptake of radioactivity from [adenine-2,8-3H]NAD and hydrolysis of extracellular NAD. AMP increased the percentage of intracellular radiolabel present as NAD. Nicotinamide mononucleotide was not taken up by the rickettsiae, did not inhibit hydrolysis of extracellular NAD, and was not a good inhibitor of the uptake of radiolabel from [adenine-2,8-3H]NAD. Neither AMP nor ATP (both of which are transported) could support the synthesis of intracellular NAD. The presence of intracellular [adenine-2,8-3H]NAD within an organism in which intact NAD could not be transported suggested the resynthesis from AMP of [adenine-2,8-3H]NAD at the locus of NAD hydrolysis and translocation.     

Crystal structure of Thermotoga maritima alpha-glucosidase AglA defines a new clan of NAD+-dependent glycosidases

Glycoside hydrolase family 4 represents an unusual group of glucosidases with a requirement for NAD+, divalent metal cations, and reducing conditions. The family is also unique in its inclusion of both alpha- and beta-specific enzymes. The alpha-glucosidase A, AglA, from Thermotoga maritima is a typical glycoside hydrolase family 4 enzyme, requiring NAD+ and Mn2+ as well as strongly reducing conditions for activity. Here we present the crystal structure of the protein complexed with NAD+ and maltose, refined at a resolution of 1.9 A. The NAD+ is bound to a typical Rossman fold NAD+-binding site, and the nicotinamide moiety is localized close to the maltose substrate. Within the active site the conserved Cys-174 and surrounding histidines are positioned to play a role in the hydrolysis reaction. The electron density maps indicate that Cys-174 is oxidized to a sulfinic acid. Most likely, the strongly reducing conditions are necessary to reduce the oxidized cysteine side chain. Notably, the canonical set of catalytic acidic residues common to other glucosidases is not present in the active site. This, combined with a high structural homology to NAD-dependent dehydrogenases, suggests an unusual and possibly unique mechanism of action for a glycoside-hydrolyzing enzyme.     

Structural Enzymology of Cellvibrio japonicus Agd31B Protein Reveals α-Transglucosylase Activity in Glycoside Hydrolase Family 31

The metabolism of the storage polysaccharides glycogen and starch is of vital importance to organisms from all domains of life. In bacteria, utilization of these α-glucans requires the concerted action of a variety of enzymes, including glycoside hydrolases, glycoside phosphorylases, and transglycosylases. In particular, transglycosylases from glycoside hydrolase family 13 (GH13) and GH77 play well established roles in α-glucan side chain (de)branching, regulation of oligo- and polysaccharide chain length, and formation of cyclic dextrans. Here, we present the biochemical and tertiary structural characterization of a new type of bacterial 1,4-α-glucan 4-α-glucosyltransferase from GH31. Distinct from 1,4-α-glucan 6-α-glucosyltransferases (EC 2.4.1.24) and 4-α-glucanotransferases (EC 2.4.1.25), this enzyme strictly transferred one glucosyl residue from α(1→4)-glucans in disproportionation reactions. Substrate hydrolysis was undetectable for a series of malto-oligosaccharides except maltose for which transglycosylation nonetheless dominated across a range of substrate concentrations. Crystallographic analysis of the enzyme in free, acarbose-complexed, and trapped 5-fluoro-β-glucosyl-enzyme intermediate forms revealed extended substrate interactions across one negative and up to three positive subsites, thus providing structural rationalization for the unique, single monosaccharide transferase activity of the enzyme.     

β-Mannanase (Man26A) and α-galactosidase (Aga27A) synergism – A key factor for the hydrolysis of galactomannan substrates

This study investigated the behavior of mannan-degrading enzymes, specifically focusing on differences with respect to their substrate specificities and their synergistic associations with enzymes from different glycoside hydrolase (GH) families. Galactosidases from Cyamopsis tetragonolobus seeds (Aga27A, GH27) and Aspergillus niger (AglC, GH36) were evaluated for their abilities to synergistically interact with mannanases from Clostridium cellulovorans (ManA, GH5) and A. niger (Man26A, GH26) in hydrolysis of guar gum and locust bean gum. Among the mannanases, Man26A was more efficient at hydrolyzing both galactomannan substrates, while among the galactosidases; Aga27A was the most effective at removing galactose substituents on both galactomannan substrates and galactose-containing oligosaccharides. An optimal protein mass ratio of glycoside hydrolases required to maximize the release of both reducing sugar and galactose residues was determined. Clear synergistic enhancement of locust bean gum hydrolysis with respect to reducing sugar release was observed when both mannanases at 75% enzyme dosage were supplemented with 25% enzyme protein dosage of Aga27A. At a protein ratio of 75% Man26A to 25% Aga27A, the presence of Man26A significantly enhanced galactose release by 25% Aga27A (2.36 fold) with locust bean gum, compared to when Aga27A was used alone at 100% enzyme protein dosage. A dosage of Aga27A at 75% and ManA at 25% protein content liberated the highest reducing sugar release on guar gum hydrolysis. A dosage of Man26A and Aga27A at 75–25% protein content, respectively, liberated reducing sugar release equivalent to that when Man26A was used alone at 100% protein content. From the findings obtained in this study, it was observed that the GH family classification of an enzyme affects its substrate specificity and synergistic interactions with other glycoside hydrolases from different families (more so than its EC classification). The GH26 Man26A and GH27 Aga27A enzymes appeared to be more promising for applications that involve the hydrolysis of galactomannan containing biomass. This method of screening for maximal compatibility between various GH families can ultimately lead to a more rational development of tailored enzyme cocktails for lignocellulose hydrolysis.     

Elucidation of exo-beta-D-glucosaminidase activity of a family 9 glycoside hydrolase (PBPRA0520) from Photobacterium profundum SS9

A glycoside hydrolase (GH) gene from Photobacterium profundum SS9 (PBPRA0520) belonging to GH family 9 was expressed in Escherichia coli. The protein was expressed with the intact N-terminal sequence, suggesting that it is an intracellular enzyme. The recombinant protein showed hydrolytic activity toward chitobiose [(GlcN)(2)] and cellobiose (CG(2)) in various disaccharides. This protein also released 4-nitrophenol (PNP) from both 4-nitrophenyl-β-D-glucosaminide (GlcN-PNP) and 4-nitrophenyl-β-D-glucoside (Glc-PNP). The hydrolytic pattern observed in chitooligosaccharides and cellooligosaccharides suggested that the reaction proceeded from the nonreducing end in an exo-type manner. Time-dependent (1)H-nuclear magnetic resonance (NMR) analysis of the anomeric form of the enzymatic reaction products indicated that the protein is an inverting enzyme. k(cat)/K(m) of (GlcN)(2) hydrolysis was 14 times greater than that of CG(2) hydrolysis. These results suggested that the protein is an exo-β-D-glucosaminidase (EC 3.2.1.165) rather than a glucan 1,4-β-D-glucosidase (EC 3.2.1.74). Based on the results, we suggest that the function of conserved GH9 proteins in the chitin catabolic operon is to cleave a (GlcN)(2)-phosphate derivative by hydrolysis during intracellular chitooligosaccharide catabolism in Vibrionaceae.     

Crystal structure of Cel44A, a glycoside hydrolase family 44 endoglucanase from Clostridium thermocellum

The crystal structure of Cel44A, which is one of the enzymatic components of the cellulosome of Clostridium thermocellum, was solved at a resolution of 0.96 A. This enzyme belongs to glycoside hydrolase family (GH family) 44. The structure reveals that Cel44A consists of a TIM-like barrel domain and a beta-sandwich domain. The wild-type and the E186Q mutant structures complexed with substrates suggest that two glutamic acid residues, Glu(186) and Glu(359), are the active residues of the enzyme. Biochemical experiments were performed to confirm this idea. The structural features indicate that GH family 44 belongs to clan GH-A and that the reaction catalyzed by Cel44A is retaining type hydrolysis. The stereochemical course of hydrolysis was confirmed by a (1)H NMR experiment using the reduced cellooligosaccharide as a substrate.     

Improvement of corn stover bioconversion efficiency by using plant glycoside hydrolase

Plant cell wall is the most abundant substrate for bioethanol production, and plants also represent a key resource for glycoside hydrolase (GH). To exploit efficient way for bioethanol production with lower cellulase loading, the potential of plant GH for lignocellulose bioconversion was evaluated. The GH activity for cell wall proteins (CWPs) was detected from fresh corn stover (FCS), and the synergism of which with Trichoderma reesei cellulase was also observed. The properties for the GH of FCS make it a promising enzyme additive for lignocellulose biodegradation. To make use of the plant GH, novel technology for hydrolysis and ethanol fermentation was developed with corn stover as substrate. Taking steam-exploded corn stover as substrate for hydrolysis and ethanol fermentation, compared with T. reesei cellulase loaded alone, the final glucose and ethanol accumulation increased by 60% and 63% respectively with GH of FCS as an addition.     

Structural evidence for the evolution of xyloglucanase activity from xyloglucan endo-transglycosylases: biological implications for cell wall metabolism

High-resolution, three-dimensional structures of the archetypal glycoside hydrolase family 16 (GH16) endo-xyloglucanases Tm-NXG1 and Tm-NXG2 from nasturtium (Tropaeolum majus) have been solved by x-ray crystallography. Key structural features that modulate the relative rates of substrate hydrolysis to transglycosylation in the GH16 xyloglucan-active enzymes were identified by structure-function studies of the recombinantly expressed enzymes in comparison with data for the strict xyloglucan endo-transglycosylase Ptt-XET16-34 from hybrid aspen (Populus tremula x Populus tremuloides). Production of the loop deletion variant Tm-NXG1-DeltaYNIIG yielded an enzyme that was structurally similar to Ptt-XET16-34 and had a greatly increased transglycosylation:hydrolysis ratio. Comprehensive bioinformatic analyses of XTH gene products, together with detailed kinetic data, strongly suggest that xyloglucanase activity has evolved as a gain of function in an ancestral GH16 XET to meet specific biological requirements during seed germination, fruit ripening, and rapid wall expansion.     

Residues affecting hydrolysis of soy isoflavone glycosides, stability and catalytic properties of Thermotoga maritima β-glucosidase

The thermostable β-glucosidase A (TmBglA) from Thermotoga maritima is a promising biocatalyst for production of isoflavone aglycones. Use of enzymes with high specificity for soy isoflavone conjugates is however essential for efficient hydrolysis. The effect of the amino acids located in the aglycone binding pocket with non-conserved residues between specificity groups in family 1 glycoside hydrolase (GH1) was studied using wild-type TmBglA and 3 exchange mutants (M1-TmBglA, M2-TmBglA, M1M2-TmBglA). Three mutants were expressed in Escherichia coli, purified and characterized. They had shifts in both optimum temperature and thermal stability, and their narrowing pH-activity curve caused by removing the ionized side chain in mutation. All mutants demonstrated the decreased catalytic efficiency more effectively revealed with natural glycoside, salicin, than with artificial substrate, p-nitrophenyl-β-D-glucopyranoside, suggesting that these amino acids are the key residues to determine aglycone specificity. A lower hydrolysis of genistin and daidzin for M2-TmBglA than M1-TmBglA indicated that L400, A407 and E408 being preferable to V170, A171, V173, G174 and H180 residues of Tm-BglA could be essential for soy isoflavone glycoside binding and catalysis.     

Three-dimensional crystal structure and enzymic characterization of beta-mannanase Man5A from blue mussel Mytilus edulis

Endo-beta-1,4-d-mannanase is the key depolymerizing enzyme for beta-1,4-mannan polymers present in the cell walls of plants and some algae, as well as in some types of plant seeds. Endo-1,4-beta-mannanase from blue mussel Mytilus edulis (MeMan5A) belongs to the glycoside hydrolase (GH) family 5 enzymes. The MeMan5A structure has been determined to 1.6A resolution using the multiple-wavelength anomalous dispersion method at the selenium K edge with selenomethionyl MeMan5A expressed in the yeast Pichia pastoris. As expected for GH 5 enzymes, the structure showed a (betaalpha)(8)-barrel fold. An unusually large number of histidine side-chains are exposed on the surface, which may relate to its location within the crystalline style of the digestive tract of the mussel. Kinetic analysis of MeMan5A revealed that the enzyme requires at least six subsites for efficient hydrolysis. Mannotetraose (M4) and mannopentaose (M5) were shown to interact with subsites -3 to +1, and -3 to +2, respectively. A clear kinetic threshold was observed when going from M4 to M5, indicating that the +2 subsite provides important interaction in the hydrolysis of short oligomeric mannose substrates. The catalytic centre motif at subsite -1 found in superfamily GH clan A is, as expected, conserved in MeMan5A, but the architecture of the catalytic cleft differs significantly from other GH 5 enzyme structures. We therefore suggest that MeMan5A represents a new subfamily in GH 5.     

Synthesis of 3-amino-2,3,6-trideoxy-D-ribo-hexose hydrochloride.

The title sugar, the 5-epimer of daunosamine, was prepared in a sequence of high-yielding steps from methyl alpha-D-mannopyranoside (1). Conversion of 1 into methyl 3-acetamido-4-O-benzoyl-6-bromo-2,3,6-trideoxy-alpha-D-ribo-hexopyranoside (2), followed by reduction with hydrogen and Raney nickel, gave the 4-benzoate (3) of methyl 3-acetamido-2,3,6-trideoxy-alpha-D-ribo-hexopyranoside (4). Saponification of 3 gave 4 as an oil that gave a crystalline 4-acetate (8). N-Deacetylation of 4 was effected with barium hydroxide, and the resultant glycoside was hydrolyzed to give 3-amino-2,3,6-trideoxy-D-ribo-hexose hydrochloride (7). The 3-benzamido analogue (5) of 4 was prepared from 4 by N-deacetylation and subsequent benzoylation, and hydrolysis of 5 gave crystalline 3-benzamido-2,3,6-trideoxy-D-ribo-hexose (6). The crystalline 3-acetamido analogue (9) of 6 was obtained by acid hydrolysis of the glycoside 4.     

A structural and kinetic survey of GH5_4 endoglucanases reveals determinants of broad substrate specificity and opportunities for biomass hydrolysis

Broad-specificity glycoside hydrolases (GHs) contribute to plant biomass hydrolysis by degrading a diverse range of polysaccharides, making them useful catalysts for renewable energy and biocommodity production. Discovery of new GHs with improved kinetic parameters or more tolerant substrate-binding sites could increase the efficiency of renewable bioenergy production even further. GH5 has over 50 subfamilies exhibiting selectivities for reaction with β-(1,4)–linked oligo- and polysaccharides. Among these, subfamily 4 (GH5_4) contains numerous broad-selectivity endoglucanases that hydrolyze cellulose, xyloglucan, and mixed-linkage glucans. We previously surveyed the whole subfamily and found over 100 new broad-specificity endoglucanases, although the structural origins of broad specificity remained unclear. A mechanistic understanding of GH5_4 substrate specificity would help inform the best protein design strategies and the most appropriate industrial application of broad-specificity endoglucanases. Here we report structures of 10 new GH5_4 enzymes from cellulolytic microbes and characterize their substrate selectivity using normalized reducing sugar assays and MS. We found that GH5_4 enzymes have the highest catalytic efficiency for hydrolysis of xyloglucan, glucomannan, and soluble β-glucans, with opportunistic secondary reactions on cellulose, mannan, and xylan. The positions of key aromatic residues determine the overall reaction rate and breadth of substrate tolerance, and they contribute to differences in oligosaccharide cleavage patterns. Our new composite model identifies several critical structural features that confer broad specificity and may be readily engineered into existing industrial enzymes. We demonstrate that GH5_4 endoglucanases can have broad specificity without sacrificing high activity, making them a valuable addition to the biomass deconstruction toolset.     

Metabolism of Monoterpenes: Demonstration of (+)-Neomenthyl-beta-d-Glucoside as a Major Metabolite of (-)-Menthone in Peppermint (Mentha Piperita)

(-)-Menthone, the major monoterpene component of the essential oil of maturing peppermint (Mentha piperita L.) leaves (6 micromoles per leaf) is rapidly metabolized at the onset of flowering with a concomitant rise in the level of (-)-menthol (to about 2 micromoles per leaf). Exogenous (-)-[G-(3)H]menthone is converted into (-)-[(3)H]menthol as the major steam-volatile product in leaf discs in flowering peppermint (10% of incorporated tracer); however, the major portion of the incorporated tracer (86%) resided in the nonvolatile metabolites of (-)-[G-(3)H]menthone. Acid hydrolysis of the nonvolatile material released over half of the radioactivity to the steamvolatile fraction, and the major component of this fraction was identified as (+)-neomenthol by radiochromatographic analysis and by synthesis of crystalline derivatives, thus suggesting the presence of a neomenthyl glycoside. Thin layer chromatography, ion exchange chromatography, and gel permeation chromatography on Bio-Gel P-2 allowed the purification of the putative neomenthyl glycoside, and these results suggested that the glycoside contained a single, neutral sugar residue. Hydrolysis of the purified glycoside, followed by reduction of the resulting sugar moiety with NaB(3)H(4), generated a single labeled product that was subsequently identified as glucitol by radio gas-liquid chromatography of both the hexatrimethylsilyl ether and hexaacetate derivative, and by crystallization to constant specific radioactivity of both the alditol and the corresponding hexabenzoate. These results, along with studies on the hydrolysis of the glycoside by specific glycosidases, strongly suggest that (+)-neomenthyl-beta-d-glucoside is a major metabolite of (-)-menthone in flowering peppermint. This is the first report on the occurrence of a neomenthyl glycoside, and the first evidence implicating glycosylation as an early step in monoterpene catabolism.     

Function and structure studies of GH family 31 and 97 α-glycosidases

A huge number of glycoside hydrolases are classified into the glycoside hydrolase family (GH family) based on their amino-acid sequence similarity. The glycoside hydrolases acting on α-glucosidic linkage are in GH family 4, 13, 15, 31, 63, 97, and 122. This review deals mainly with findings on GH family 31 and 97 enzymes. Research on two GH family 31 enzymes is described: clarification of the substrate recognition of Escherichia coli α-xylosidase, and glycosynthase derived from Schizosaccharomyces pombe α-glucosidase. GH family 97 is an aberrant GH family, containing inverting and retaining glycoside hydrolases. The inverting enzyme in GH family 97 displays significant similarity to retaining α-glycosidases, including GH family 97 retaining α-glycosidase, but the inverting enzyme has no catalytic nucleophile residue. It appears that a catalytic nucleophile has been eliminated during the molecular evolution in the same way as a man-made nucleophile mutant enzyme, which catalyzes the inverting reaction, as in glycosynthase and chemical rescue.     

Antiviral isoflavonoid sulfate and steroidal glycosides from the fruits of Solanum torvum

The C-4 sulfated isoflavonoid, torvanol A (1), and the steroidal glycoside, torvoside H (3), together with the known glycoside, torvoside A (2), were isolated from a MeOH extract of Solanum torvum fruits. Upon enzymatic hydrolysis with beta-glucosidase, torvoside A (2) and torvoside H (3) yielded the corresponding acetal derivatives 4 and 5, respectively. Torvanol A (1), torvoside H (3) and compound 5 exhibited antiviral activity (herpes simplex virus type 1) with IC(50) values of 9.6, 23.2 and 17.4 microg/ml, respectively. Compounds 1-5 showed no cytotoxicity (at 50 microg/ml) against BC, KB and Vero cell lines.     

Mechanistic evaluation of MelA α-galactosidase from Citrobacter freundii: a family 4 glycosyl hydrolase in which oxidation is rate-limiting

The MelA gene from Citrobacter freundii, which encodes a glycosyl hydrolase family 4 (GH4) α-galactosidase, has been cloned and expressed in Escherichia coli. The recombinant enzyme catalyzes the hydrolysis of phenyl α-galactosides via a redox elimination-addition mechanism involving oxidation of the hydroxyl group at C-3 and elimination of phenol across the C-1-C-2 bond to give an enzyme-bound glycal intermediate. For optimal activity, the MelA enzyme requires two cofactors, NAD(+) and Mn(2+), and the addition of a reducing agent, such as mercaptoethanol. To delineate the mechanism of action for this GH4 enzyme, we measured leaving group effects, and the derived β(lg) values on V and V/K are indistinguishable from zero (-0.01 ± 0.02 and 0.02 ± 0.04, respectively). Deuterium kinetic isotope effects (KIEs) were measured for the weakly activated substrate phenyl α-D-galactopyranoside in which isotopic substitution was incorporated at C-1, C-2, or C-3. KIEs of 1.06 ± 0.07, 0.91 ± 0.04, and 1.02 ± 0.06 were measured on V for the 1-(2)H, 2-(2)H, and 3-(2)H isotopic substrates, respectively. The corresponding values on V/K were 1.13 ± 0.07, 1.74 ± 0.06, and 1.74 ± 0.05, respectively. To determine if the KIEs report on a single step or on a virtual transition state, we measured KIEs using doubly deuterated substrates. The measured (D)V/K KIEs for MelA-catalyzed hydrolysis of phenyl α-D-galactopyranoside on the dideuterated substrates, (D)V/K((3-D)/(2-D,3-D)) and (D)V/K((2-D)/(2-D,3-D)), are 1.71 ± 0.12 and 1.71 ± 0.13, respectively. In addition, the corresponding values on V, (D)V((3-D)/(2-D,3-D)) and (D)V((2-D)/(2-D,3-D)), are 0.91 ± 0.06 and 1.01 ± 0.06, respectively. These observations are consistent with oxidation at C-3, which occurs via the transfer of a hydride to the on-board NAD(+), being concerted with proton removal at C-2 and the fact that this step is the first irreversible step for the MelA α-galactosidase-catalyzed reactions of aryl substrates. In addition, the rate-limiting step for V(max) must come after this irreversible step in the reaction mechanism.     

Heterologous expression of glycoside hydrolase family 2 and 42 β-galactosidases of lactic acid bacteria in Lactococcus lactis

This study characterized a glycoside hydrolase family 42 (GH42) β-galactosidase of Lactobacillus acidophilus (LacA) and compared lactose hydrolysis, hydrolysis of oNPG, pNPG and pNPG-analogues and galactooligosaccharides (GOSs) formation to GH2 β-galactosidases of Streptococcus thermophilus (LacZ type), Lactobacillus plantarum and Leuconostoc mesenteroides subsp. cremoris (both LacLM type). Beta-galactosidases were heterologously expressed in Lactococcus lactis using a p170 derived promoter; experiments were performed with L. lactis crude cell extract (CCE). The novel GH42 β-galactosidase of Lb. acidophilus had lower activity on lactose, oNPG and pNPG but higher relative activity on pNP analogues compared to GH2 β-galactosidases, and did not transgalactosylate at high lactose concentrations. Temperature and pH optima for lactose hydrolysis varied between GH2 β-galactosidases. oNPG and pNPG were the preferred substrates for hydrolysis; in comparison, activity on pNPG-analogues was less than 1.5%. GH2 β-galactosidases formed structurally similar GOS with varying preferences.