Stereochemical Course Of Hydrolysis Research Articles

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.

Identification and Molecular Cloning of a Novel Glycoside Hydrolase Family of Core 1 Type O-Glycan-specific Endo-α-N-acetylgalactosaminidase from Bifidobacterium longum

We found endo-alpha-N-acetylgalactosaminidase in most bifidobacterial strains, which are predominant bacteria in the human colon. This enzyme catalyzes the liberation of galactosyl beta1,3-N-acetyl-D-galactosamine (Galbeta1,3GalNAc) alpha-linked to serine or threonine residues from mucin-type glycoproteins. The gene (engBF) encoding the enzyme has been cloned from Bifidobacterium longum JCM 1217. The protein consisted of 1,966 amino acid residues, and the central domain (590-1381 amino acid residues) exhibited 31-53% identity to hypothetical proteins of several bacteria including Clostridium perfringens and Streptococcus pneumoniae. The recombinant protein expressed in Escherichia coli liberated Galbeta1,3GalNAc disaccharide from Galbeta1,3GalNAcalpha1pNP and asialofetuin, but did not release GalNAc, Galbeta1,3(GlcNAcbeta1,6)GalNAc, GlcNAcbeta1,3GalNAc, and Galbeta1,3GlcNAc from each p-nitrophenyl (pNP) substrate, and also did not release sialo-oligosaccharides from fetuin, indicating its strict substrate specificity for the Core 1-type structure. The stereochemical course of hydrolysis was determined by (1)H NMR and was found to be retention. Site-directed mutagenesis of a total of 22 conserved Asp and Glu residues suggested that Asp-682 and Asp-789 are critical residues for the catalytic activity of the enzyme. The enzyme also exhibited transglycosylation activity toward various mono- and disaccharides and 1-alkanols, demonstrating its potential to synthesize neoglycoconjugates. This is the first report for the isolation of a gene encoding endo-alpha-N-acetylgalactosaminidase from any organisms and for the establishment of a new glycoside hydrolase family (GH family 101).

Effects of endogenous endo-β-1,4-glucanase on cellulose biosynthesis in Acetobacter xylinum ATCC23769

Endo-β-1,4-glucanase (CMCax; EC 3.2.1.4) from Acetobacter xylinum ATCC23769 was expressed as a 6 × His-tagged fusion protein in Escherichia coli. The optimal temperature, pH, K m and V max of the purified His-tagged CMCax toward carboxymethyl cellulose were 50°C, 4.5, 20 mg/ml and 37.2 μM/min, respectively. The number of recognition residues of cello-oligosaccharide by this enzyme were five (cellopentaose) or longer, and the stereochemical course of hydrolysis was of the inverting type. Addition of a small amount (1.5 mg/l) of His-tagged CMCax into a culture medium enhanced cellulose production 1.2-fold. CMCax overproduction in A. xylinum also enhanced the yield of cellulose production. Transmission electron microscopic analysis revealed that the cellulose ribbons secreted from the CMCax overproducing strain were dispersed compared with those from the wild type strain in the same manner as by carboxymethyl cellulose addition. These results could suggest that CMCax from A. xylinum influences in cellulose ribbon assembly, which is considered to be a rate-determined process in cellulose synthesis.

Effects of Endogenous Endo-β-1,4-Glucanase on Cellulose Biosynthesis in Acetobacter xylinum ATCC23769

Endo-beta-1,4-glucanase (CMCax; EC 3.2.1.4) from Acetobacter xylinum ATCC23769 was expressed as a 6 x His-tagged fusion protein in Escherichia coli. The optimal temperature, pH, K(m) and V(max) of the purified His-tagged CMCax toward carboxymethyl cellulose were 50 degrees C, 4.5, 20 mg/ml and 37.2 microM/min, respectively. The number of recognition residues of cello-oligosaccharide by this enzyme were five (cellopentaose) or longer, and the stereochemical course of hydrolysis was of the inverting type. Addition of a small amount (1.5 mg/l) of His-tagged CMCax into a culture medium enhanced cellulose production 1.2-fold. CMCax overproduction in A. xylinum also enhanced the yield of cellulose production. Transmission electron microscopic analysis revealed that the cellulose ribbons secreted from the CMCax overproducing strain were dispersed compared with those from the wild type strain in the same manner as by carboxymethyl cellulose addition. These results could suggest that CMCax from A. xylinum influences in cellulose ribbon assembly, which is considered to be a rate-determined process in cellulose synthesis.

Substrate recognition by three family 13 yeast alpha-glucosidases.

Important hydrogen bonding interactions between substrate OH-groups in yeast alpha-glucosidases and oligo-1,6-glucosidase from glycoside hydrolase family 13 have been identified by measuring the rates of hydrolysis of methyl alpha-isomaltoside and its seven monodeoxygenated analogs. The transition-state stabilization energy, DeltaDeltaG, contributed by the individual OH-groups was calculated from the activities for the parent and the deoxy analogs, respectively, according to DeltaDeltaG = -RT ln[(Vmax/Km)analog/(Vmax/Km)parent]. This analysis of the energetics gave DeltaDeltaG values for all three enzymes ranging from 16.1 to 24.0 kJ.mol-1 for OH-2', -3', -4', and -6', i.e. the OH-groups of the nonreducing sugar ring. These OH-groups interact with enzyme via charged hydrogen bonds. In contrast, OH-2 and -3 of the reducing sugar contribute to transition-state stabilization, by 5.8 and 4.1 kJ.mol-1, respectively, suggesting that these groups participate in neutral hydrogen bonds. The OH-4 group is found to be unimportant in this respect and very little or no contribution is indicated for all OH-groups of the reducing-end ring of the two alpha-glucosidases, probably reflecting their exposure to bulk solvent. The stereochemical course of hydrolysis by these three members of the retaining family 13 was confirmed by directly monitoring isomaltose hydrolysis using 1H NMR spectroscopy. Kinetic analysis of the hydrolysis of methyl 6-S-ethyl-alpha-isomaltoside and its 6-R-diastereoisomer indicates that alpha-glucosidase has 200-fold higher specificity for the S-isomer. Substrate molecular recognition by these alpha-glucosidases are compared to earlier findings for the inverting, exo-acting glucoamylase from Aspergillus niger and a retaining alpha-glucosidase of glycoside hydrolase family 31, respectively.

Purification and characterization of α-galactosidase from a thermophilic fungus Thermomyces lanuginosus

An extracellular α-galactosidase was purified to electrophoretic homogeneity from a locust bean gum-spent culture fluid of a mannanolytic strain of the thermophilic fungus Thermomyces lanuginosus. Molecular mass of the enzyme is 57 kDa. The pure enzyme which has a glycoprotein nature, afforded several forms on IEF, indicating its microheterogeneity. Isoelectric point of the major form was 5.2. Enzyme is the most active against aryl α- d-galactosides but efficiently hydrolyzed α-glycosidically linked non-reducing terminal galactopyranosyl residues occurring in natural substrates such as melibiose, raffinose, stachyose, and fragments of galactomannan. In addition, the enzyme is able to catalyze efficient degalactosylation of polymeric galactomannans leading to precipitation of the polymers. Stereochemical course of hydrolysis of two substrates, 4-nitrophenyl α-galactopyranoside and galactosyl 1mannotriose, followed by 1H NMR spectroscopy, pointed out the α-anomer of d-galactose was the primary product of hydrolysis from which the β-anomer was formed by mutarotation. Hence the enzyme is a retaining glycosyl hydrolase. In accord with its retaining character the enzyme catalyzed transgalactosylation from 4-nitrophenyl α-galactopyranoside as a glycosyl donor. Amino acid sequence alignment of N-terminal and two internal sequences suggested that the enzyme is a member of family 27 of glycosyl hydrolases.

PPL-catalysed hydrolysis of 3,4-disubstituted β-lactams: Effect of chain length and stereochemistry on the enantioselectivity

3,4-Disubstituted β-lactam acetates 1a–15a have been subjected to PPL-catalysed hydrolysis. The stereochemical course of hydrolysis has been shown to depend upon the C3–C4 relative stereochemistry and the length of the chain bearing acetate functionality.

Stereochemical Course of Hydrolysis Catalysed by α- l-Rhamnosyl and α- d-Galacturonosyl Hydrolases from Aspergillus aculeatus

The stereochemical course of hydrolysis catalysed by four Aspergillus aculeatusenzymes acting on α- l-rhamnosyl and α- d-galacturonosyl linkages in the hairy regions of pectins has been determined using 1H-NMR. Exogalacturonase acts with inversion of anomeric configuration ( e→ a), shown by the initial release of β- d-Gal pA from the non-reducing end of polygalacturonic acid. Similarly, rhamnogalacturonan (RG) hydrolase also acts with inversion of anomeric configuration ( e→ a) during hydrolysis of α- d-Gal pA-(1→2)-α- l-Rha plinkages in RG, initially releasing oligosaccharides with β- d-Gal pA at the reducing end. This result is consistent with the recently solved crystal structure of this enzyme, as well as its classification based on amino acid sequence similarity into glycosyl hydrolase family 28. α- l-Rhamnosidase and RG-rhamnohydrolase also act with inversion of configuration ( a→ e), initially releasing β- l-Rha pfrom p-nitrophenyl α- l-rhamnopyranoside and RG oligosaccharides, respectively. Thus, all four enzymes examined are inverting hydrolases which probably catalyse hydrolysis via single displacement mechanisms.

Stereochemical course of hydrolysis catalyzed by arabinofuranosyl hydrolases

The stereochemical course of hydrolysis catalyzed by various enzymes acting on arabinofuranosyl linkages has been determined. 1H-NMR analysis of the action of endo-(1 → 5)-α- l- arabinanases from Aspergillus niger and Aspergillus aculeatus showed that both hydrolyze linear arabinan with inversion of configuration, and may therefore act via a single displacement mechanism. This is consistent with the A. niger enzyme's classification in glycosyl hydrolase family 43. The catalytic mechanisms of α- l-arabinofuranosidases from A. niger, A. aculeatus, Aspergillus awamori, Humicola insolens, Penicillium capsulatum and Bacillus subtilis were investigated using both 1H-NMR and high performance anion exchange chromatography to follow glycosyl transfer reactions to methanol. In all cases these enzymes catalyzed the reaction with retention of configuration, and therefore probably operate via double displacement hydrolytic mechanisms. From the results with arabinofuranosidase A and B from A. niger we predict that all members of glycosyl hydrolase family 51 and 54 catalyze hydrolysis with net retention of anomeric configuration. Similar studies with (1 → 4)-β- d- arabinoxylan arabinohydrolases from A. awamori, Trichoderma reesei and Bifidobacterium adolescentis only enabled their tentative classification as inverting enzymes on the basis of their lack of glycosyl transfer to methanol.

Stereochemical course of glucan hydrolysis by barley (1 → 3)- and (1 → 3,1 → 4)-β-glucanases

The stereochemical course of hydrolysis of Laminaria digitata laminarin and barley (1 → 3,1 → 4)-β-glucan by barley (1 → 3)-β-glucanase (E.C. 3.2.1.39) isoenzyme GII and (1 → 3,1 → 4)-β-glucanase (EC 3.2.1.73) isoenzyme EII, respectively, has been determined by 1H-NMR. Both enzymes catalyse hydrolysis with retention of anomeric configuration (e → e) and may therefore operate via a double displacement mechanism. We predict that all other members of Family 17 of β-glycosyl hydrolases also follow this stereochemical course of hydrolysis.

The Saccharomyces cerevisiae Processing α1,2-Mannosidase Is an Inverting Glycosidase

The α1,2-mannosidase from Saccharomyces cerevisiae, which removes one specific α1,2-linked mannose residue from Man 9GlcNAc 2, is a member of the Class 1 α1,2-mannosidase family conserved from yeast to mammals. Although Class 1 α1,2-mannosidases are essential for the maturation of N-linked oligosaccharides in mammalian cells, nothing is known about their mechanism of action. The availability of sufficient quantities of recombinant yeast α1,2-mannosidase and its homology with the mammalian enzymes make it a good model to study the catalytic mechanism of this family of α1,2-mannosidases. The stereochemical course of hydrolysis of Man 9GlcNAc by the yeast enzyme was followed by proton nuclear magnetic resonance spectroscopy. It was observed that β-D-mannose is released from the oligosaccharide substrate, thereby demonstrating that the enzyme is of the inverting type.

Stereochemistry, specificity and kinetics of the hydrolysis of reduced cellodextrins by nine cellulases.

The catalytic activity of nine enzymes (endoglucanases I-III, V, VI and cellobiohydrolases I and II from Humicola insolens; endoglucanases A and C from Bacillus lautus), representative of cellulase families A-C, H, J and K, has been investigated using a series of reduced cellooligosaccharides (cellotriitol to cellohexaitol) as substrates. For each enzyme, the specificity of cleavage was determined by analytical HPLC while the kinetic constants were obtained from a kinetic assay involving a cellobiose dehydrogenase purified from H. insolens as a coupled enzyme using 2,6-dichloroindophenol as the electron acceptor. These data were used to estimate the number of subsites in the enzymes. The stereochemical course of hydrolysis by seven enzymes, representing the six different families, was assessed using 1H-NMR. The enzymes belonging to families which had already been investigated (A-C), showed results in agreement with previous studies. The three other families (H, J and K), for which no mechanistic data was previously available, gave results which indicated that enzymes in group H had retaining-type activity and enzymes in groups J and K had inverting-type activity. The retaining endoglucanases I and III displayed a high glycosyl-transferase activity under the conditions used during the NMR experiments resulting in precipitates of higher oligomers.

Stereoselective hydrolysis catalyzed by related beta-1,4-glucanases and beta-1,4-xylanases.

Over 80 beta-1,4-glucanases and beta-1,4-xylanases can be classified into one of eight families on the basis of amino acid sequence similarities in their catalytic domains (Gilkes, N. R., Henrissat, B., Kilburn, D. G., Miller, R. C., Jr., and Warren, R. A. J. (1991) Microbiol. Rev. 55, 303-315). As a test of this classification, the stereochemical course of hydrolysis of 10 enzymes representative of five families has been determined using proton NMR. These data, together with published data for six additional enzymes, show that representatives of a given enzyme family have the same stereoselectivity: four families catalyze hydrolysis with retention of anomeric configuration, two with inversion. The results support the hypothesis that family members share a common general fold, active site topology, and catalytic mechanism.

Stereochemical course of hydrolysis and hydration reactions catalysed by cellobiohydrolases I and II from Trichoderma reesei

Cellobiohydrolase I from Trichoderma reesei catalyzes the hydrolysis of methyl β-D-cellotrioside ( K m = 48μM, k cat = 0.7 min −1) with release of the β-cellobiose (retention of configuration). The same enzyme catalyzes the ( trans-hydration of cellobial ( K m = 116 μM, K cat = 1.16 min −1) and lactal ( K m = 135 μM, k cat = 1.35 min −1), presumably with glycosyi oxo-carbonium ion mediation. Protonation of the double bond is from the direction opposite that assumed for methyl β-cellotrioside, but products formed from these prochiral substrates are again of β configuration. Cellobiohydrolase II from the same microrganism hydrolyzes methyl β-D-cellotetraoside ( K m = 4 μM, k cat = 112 min −1) with inversion of configuration to produce α-cellobiose. The other reaction product, methyl β-cellobioside, is in turn partly hydrolysed by Cellobiohydrolase II to form methyl β-D-glucoside and D-glucose, presumably the α-anomer. Reaction with cellobial is too slow to permit unequivocal determination of product configuration, but clear evidence is obtained that protonation occurs from the si-direction, again opposite that assumed for protonating glycosidic substrates. These results add substantially to the growing evidence that individual glycosidases create the anomeric configuration of their reaction products by means that are independent of substrate configuration.

Synthesis of (Rp, Rp)-P1, P4-Bis(5′-adenosyl)-1[17O, 18O2],4[17O, 18O2] tetraphosphate from (Sp, Sp)-P1, P4-Bis(5′-adenosyl)-1[thio-18O2], 4[thio-18O2]tetraphosphate with Retention at Phosphorus and the Stereochemical Course of Hydrolysis by the Unsymmetrical Ap4A Phosphodiesterase from Lupin Seeds

The three stereoisomers of P1,P4-bis(5'-adenosyl)-1,4-dithiotetraphosphate have been synthesized and their 31P NMR spectra investigated. The effect of temperature on the circular dichroic spectrum of the (Sp,Sp)-stereoisomer shows that unstacking of the molecule occurs as the temperature is raised. Treatment of the (Sp,Sp)-stereoisomer with cyanogen bromide in [18O]water leads to substitution of sulfur by 18O with predominant retention of configuration at P1 and P4. (Sp,Sp)-P1,P4-Bis(5'-adenosyl)-1[thio-18O2],4[thio-18O2]tetraphosphate was synthesized and on treatment with cyanogen bromide in [17O]water gave (Rp,Rp)-P1,P4-bis(5'-adenosyl)-1[17O,18O2],4[17O,18O2]tetraphosphate. Hydrolysis by unsymmetrical Ap4A phosphodiesterase from lupin seeds gave (Rp)-5'-[16O,17O,18O]AMP. The reaction therefore proceeds with inversion of configuration at phosphorus, indicating that the enzyme-catalyzed displacement by water occurs by a direct "in-line" mechanism.

Stereochemical course of the reaction catalyzed by the cyclic GMP phosphodiesterase from retinal rod outer segments.

The stereochemical course of hydrolysis catalyzed by the cyclic GMP phosphodiesterase from bovine retinal rod outer segments was determined. The Sp diastereomer of guanosine 3',5'-cyclic monophosphorothioate was hydrolyzed by cyclic GMP phosphodiesterase in H2(18)O to give [16O,18O]guanosine 5'-monophosphorothioate. This isotopomer was reacted with diphenyl phosphorochloridate to form the two diastereomers of P1-(5'-guanosyl) P2-(diphenyl) 1-thiodiphosphate. The 31P NMR spectrum of this mixture of diastereomers was identical to that obtained from [16O,18O]guanosine 5'-monophosphorothioate resulting from the hydrolysis of the Rp diastereomer of guanosine 5'-p-nitrophenyl phosphorothioate by snake venom phosphodiesterase. This finding indicates that the 18O is bridging in the Rp diastereomer of the P1-(5'-guanosyl) P2-(diphenyl) 1-thiodiphosphate and nonbridging in the Sp diastereomer. As the snake venom phosphodiesterase reaction is known to proceed with retention of configuration, it follows that hydrolysis by retinal rod cyclic GMP phosphodiesterase proceeds with inversion of configuration at the phosphorus atom.

Corrections - Stereochemical Course of Hydrolysis of DNA by Exonuclease I from Escherichia Coli

Corrections - Stereochemical Course of Hydrolysis of DNA by Exonuclease I from Escherichia Coli

Stereochemical course of hydrolysis of DNA by exonuclease I from Escherichia coli.

Exonuclease I has been purified from an overproducing strain of Escherichia coli K12 [Prasher, D. C., Conarro, L., & Kushner, S. R. (1983) J. Biol. Chem. 258, 6340-6343]. The enzyme hydrolyzes deoxyribonucleic acids that contain chiral phosphorothioate diester linkages, and the stereochemical course of the reaction is inversion of configuration at phosphorus. This result is most consistent with hydrolysis occurring via the direct attack of water on a phosphorothioate diester rather than through the intermediacy of a covalent nucleotidyl-enzyme intermediate. This finding represents the first example of a processive exonuclease whose stereochemical pathway has been determined.

The stereochemical course of hydrolysis catalysed by snake venom 5'-nucleotide phosphodiesterase.

Adenosine 5'-(S)-[16O,17O,18O]phosphate was pyrophosphorylated by the combined action of adenylate kinase and pyruvate kinase. The isotopomers of adenosine 5'-[alpha-16O,17O,18O]triphosphate were hydrolysed by venom 5'-nucleotide phosphodiesterase (Crotalus adamanteus) in H2(17)O. Analysis by 31P nuclear magnetic resonance spectroscopy of the resulting adenosine 5'-[16O,17O,18O]phosphate, after cyclization and esterification, showed that the hydrolysis occurs with retention of configuration at phosphorus. The most likely explanation of this observation is that the enzymic hydrolysis involves a double displacement at phosphorus with a covalent nucleotidyl--enzyme intermediate on the reaction pathway.

Positional Specificity of Lipoprotein Lipase

Abstract The stereochemical course of hydrolysis of radioactive triolein was determined using lipoprotein lipase of cow's milk and rat postheparin plasma. [3H]Glycerol trioleate or glycerol [1-14C]trioleate was emulsified with total egg yolk lipids to provide 0.2 to 2.5 µCi of 3H or 0.03 µCi of 14C per µm substrate. The hydrolysis products were isolated at 1 to 45 min and free fatty acids and the various positional isomers of mono- and diglycerides were resolved by thin layer chromatography. In a total yield of 0.03 to 0.16 µmole of diglyceride, 9 to 30% was 1,3- and 70 to 91%, 1,2-(2,3-)diglyceride. The 2,3-isomer was 73 to 96% of the latter. The yield of free fatty acids and monoglycerides varied with the length of incubation. In 15 of 17 determinations, the 1-(3-)isomer accounted for 51 to 87% of the monoglyceride. It is suggested that lipoprotein lipase attacks preferentially position 1 in sn-glycerides and follows it by hydrolysis of the positions 2 and 3. An intermediate formation of 2,3-diglycerides during lipoprotein lipase hydrolysis may be important in avoiding stimulation of triglyceride and phospholipid biosynthesis which proceed via 1,2-diglycerides.