Bacillus macerans cyclomaltodextrin glucanotransferase transglycosylation reactions with different molar ratios of D-glucose and cyclomaltohexaose

It was found that Bacillus macerans cyclomaltodextrin glucanotransferase (CGTase) reacts with cyclomaltohexaose (alpha-cyclodextrin, alpha-CD) to give a series of cyclomaltooligosaccharides (cyclomaltodextrins, CDs), having seven to more than 20 D-glucose residues and maltooligosaccharides (maltodextrins, MDs) from G5 to G12+. When D-glucose (Glc) was added to the alpha-CD at very low molar ratios (1:100) of Glc to alpha-CD, the predominant products (95%) were CDs, some of which were macrocyclic MDs with 20-60 D-glucose residues, along with MDs that also had high molecular weights, containing 10-75 D-glucose residues and gave a blue iodine-iodide color. As the molar ratio of Glc to alpha-CD was increased, the amount of CDs progressively decreased and MDs proportionately increased in the range of G2-G12. At 25 mM alpha-CD and Glc to alpha-CD molar ratio of 1:1, a 75% yield of MDs, G1-G12, each in approximately equal amounts, was obtained; and at 20 mM and a 5:1 ratio, a 97% yield of MDs, G2-G9, was obtained but in unequal amounts. At higher ratios (10:1), the CDs completely disappeared, and at very high ratios (50:1 to 100:1) only low-molecular-weight MDs, G2-G4, were formed.     

Addition of maltodextrins to the nonreducing-end of acarbose by reaction of acarbose with cyclomaltohexaose and cyclomaltodextrin glucanyltransferase

New kinds of acarbose analogues were synthesized by the reaction of acarbose with cyclomaltohexaose and cyclomaltodextrin glucanyltransferase (CGTase). Three major CGTase coupling products were separated and purified by Bio-Gel P2 gel-permeation chromatography. Digestion of the three products by beta-amylase and glucoamylase showed that they were composed of maltohexaose (G6), maltododecaose (G12), and maltooctadecaose (G18), respectively, attached to the nonreducing-end of acarbose. 13C NMR of the glucoamylase product (D-glucopyranosyl-acarbose) showed that the D-glucose moiety was attached alpha- to the C-4-OH group of the nonreducing-end cyclohexene ring of acarbose, indicating that the maltodextrins were attached alpha-(1-->4) to the nonreducing-end cyclohexene of acarbose.     

Optimized synthesis of specific sizes of maltodextrin glycosides by the coupling reactions of Bacillus macerans cyclomaltodextrin glucanyltransferase

Bacillus macerans cyclomaltodextrin glucanyltransferase (CGTase, EC 2.4.1.19), in reaction with cyclomaltohexaose and methyl alpha-D-glucopyranoside, methyl beta-D-glucopyranoside, phenyl alpha-D-glucopyranoside, and phenyl beta-D-glucopyranoside gave four kinds of maltodextrin glycosides. The reactions were optimized by using different ratios of the individual d-glucopyranosides to cyclomaltohexaose, from 0.5 to 5.0, to obtain the maximum molar percent yields of products, which were from 68.3% to 78.6%, depending on the particular D-glucopyranoside, and also to obtain different maltodextrin chain lengths. The lower ratios of 0.5-1.0 gave a wide range of sizes from d.p. 2-17 and higher. As the molar ratio was increased from 1.0 to 3.0, the larger sizes, d.p. 9-17, decreased, and the small and intermediate sizes, d.p. 2-8, increased; as the molar ratios were increased further from 3.0 to 5.0, the large sizes completely disappeared, the intermediate sizes, d.p. 4-8, decreased, and the small sizes, d.p. 2 and 3 became predominant. A comparison is made with the synthesis of maltodextrins by the reaction of CGTase with different molar ratios of d-glucose to cyclomaltohexaose.     

Enzymatic synthesis of two salicin analogues by reaction of salicyl alcohol with Bacillus macerans cyclomaltodextrin glucanyltransferase and Leuconostoc mesenteroides B-742CB dextransucrase

Beta-Salicin is a naturally occurring glycoside found in the bark of poplar and willow trees. Ancient man used it as an analgesic and antipyretic. It has a D-glucopyranose unit attached by a beta-linkage to the phenolic hydroxyl of salicyl alcohol. Two new salicin analogues have been enzymatically synthesized by transglycosylation reactions: (a) by the reaction of Bacillus macerans cyclomaltodextrin glucanyltransferase with cyclomaltohexaose and salicyl alcohol, followed by reactions with alpha amylase and glucoamylase to give D-glucopyranose attached by an alpha-linkage to the phenolic hydroxyl of salicyl alcohol as the major product, alpha-salicin; and (b) by the reaction of Leuconostoc mesenteroides B-742CB dextransucrase with sucrose and salicyl alcohol, followed by reactions with dextranase and glucoamylase to give alpha-d-glucopyranose attached to the primary alcohol hydroxyl of salicyl alcohol as the major product, alpha-isosalicin.     

Production of a novel transfructosylating enzyme from Bacillus macerans EG-6

A new bacterium producing a novel transfructosylating enzyme was isolated from soil and designated as Bacillus macerans EG-6. Various culture conditions for enzyme production were optimized in a flask culture. 1% (w/v) sucrose as a carbon source and a mixed nitrogen source (1% yeast extract, 1% polypeptone, and 0.5% ammonium chloride) gave the best enzyme production. Addition of phosphate and magnesium ion into the medium enhanced the enzyme yield. Optimum culture pH and temperature were 7.0 and 37?°C, respectively. Under optimal culture conditions, transfructosylating enzyme was rapidly produced in the early growth period, thereafter invertase activity was predominant as the culture proceeded. Using the culture filtrate, production of fructooligosaccharides from sucrose was preliminarily carried out. In a low sucrose concentration (200?g/l), transfructosylating activity competes with invertase activity in sucrose utilization. Subsequently, low fructooligosaccharide yield (20%) was achieved due to liberation of high amounts of glucose and fructose. The best oligosaccharide yield (43%) was achieved when 500?g/l sucrose was utilized.     

Catabolism of protocatechuate by Bacillus macerans.

An aerobic endospore-forming bacterium, tentatively identified as a strain (JJ-lb) of Bacillus macerans, was isolated by enrichment on 4-hydroxybenzoate (4HBA), using as an inoculum soil taken from a 50 degrees C Iadho hot spring. Enzymatic analyses of cells grown on succinate and 4HBA indicated that strain JJ-1b degrades 4HBA by way of the novel protocatechuate (PCA) 2,3-dioxygenase pathway. Purification of the PCA 2,3-dioxygenase by affinity chromatography allowed the first observation of the immediate ring fission product of PCA, namely, 5-carboxy-2-hydroxymuconic semialdehyde (CHMS; labmda max at pH 7.0 = 348 nm). An affinity column fraction was obtained that decarboxylated CHMS to 2-hydroxymuconic semialdehyde (HMS; lambdamax at pH 7.0 = 375 nm). Thus, conversion of PCA to HMS is accomplished in two steps, 2,3-fission of the PCA ring followed by enzymatic decarboxylation of the ring fission product, forming HMS.     

Catabolism of protocatechuate by Bacillus macerans.

An aerobic endospore-forming bacterium, tentatively identified as a strain (JJ-lb) of Bacillus macerans, was isolated by enrichment on 4-hydroxybenzoate (4HBA), using as an inoculum soil taken from a 50 degrees C Iadho hot spring. Enzymatic analyses of cells grown on succinate and 4HBA indicated that strain JJ-1b degrades 4HBA by way of the novel protocatechuate (PCA) 2,3-dioxygenase pathway. Purification of the PCA 2,3-dioxygenase by affinity chromatography allowed the first observation of the immediate ring fission product of PCA, namely, 5-carboxy-2-hydroxymuconic semialdehyde (CHMS; labmda max at pH 7.0 = 348 nm). An affinity column fraction was obtained that decarboxylated CHMS to 2-hydroxymuconic semialdehyde (HMS; lambdamax at pH 7.0 = 375 nm). Thus, conversion of PCA to HMS is accomplished in two steps, 2,3-fission of the PCA ring followed by enzymatic decarboxylation of the ring fission product, forming HMS.     

Trapping of an intermediate in the reaction catalyzed by 5-oxoprolinase

Bacterial 5-oxoprolinase is composed of two protein components: Component A, which catalyzes 5-oxoproline-dependent ATP-hydrolysis and Component B, which couples the hydrolysis of ATP with the decyclization of 5-oxoproline to form glutamate (Seddon, A. P., Li, L., and Meister, A. (1984) J. Biol. Chem. 259, 8091-8094). Studies on this unusual enzyme system have led to evidence that an intermediate is formed by Component A. Application of the isotope-trapping method demonstrated an activated 5-oxoproline intermediate, whose formation requires ATP, Mg2+, and Component A. The amount of ATP-dependent trapping was close to the number of enzyme active sites. The intermediate formed by Component A was shown to be reducible by potassium borohydride to proline in low yield; when Component B was added, the formation of proline was abolished. Treatment of reaction mixtures containing Component A, 5-oxoproline, and [gamma-32P] ATP with diazomethane led to appearance of a 32P-labeled compound (found on thin layer chromatography), whose formation was significantly reduced when Component B was present. The new compound, which is labile, breaks down to form dimethyl[32P]phosphate. The total amount of dimethyl[32P]phosphate formed after breakdown is close to the number of active sites of Component A. The data are consistent with the conclusion that a phosphorylated form of 5-oxoproline is formed by Component A and suggest that Component B is required for conversion of this intermediate to glutamate.     

Inhibition of thiaminase I from Bacillus thiaminolyticus. Evidence supporting a covalent 1,6-dihydropyrimidinyl-enzyme intermediate

Thiaminase I from Bacillus thiaminolyticus strain Matsukawa et Misawa is completely and irreversibly inhibited by treatment with 4-amino-6-chloro-2-methylpyrimidine. Inhibition is a time-dependent first-order process, exhibiting a half-time of 4 h at an inhibitor concentration of 5 mM. A specific active-site-directed inactivation is supported by protection of the enzymatic activity in the presence of the substrates thiamin and quinoline as well as by the observation that a stoichiometric amount of inorganic chloride is released during inactivation. 4-Amino-5-(anilinomethyl)-6-chloro-2-methylpyrimidine, which resembles the structure of the product of base exchange of thiamin with aniline, inactivates thiaminase approximately 2 orders of magnitude faster. Inactivation is again complete and irreversible and is a time-dependent first-order process, in this case exhibiting saturation at low inhibitor concentrations (KI = 96 microM). Enzyme inactivation can be explained as the result of displacement of chloride from the chloropyrimidine by a nucleophile at the enzyme active site. The inactivation suggests that the Zoltewicz-Kauffman model of bisulfite-catalyzed thiamin cleavage [Zoltewicz, J. A., & Kauffman, G. M. (1977) J. Am. Chem. Soc. 99, 3134-3142], which calls for the reversible nucleophilic addition of catalyst across the 1,6 double bond of thiamin's pyrimidine ring, may be applicable to thiaminase as well.     

Trapping of the thioacylglyceraldehyde-3-phosphate dehydrogenase intermediate from Bacillus stearothermophilus. Direct evidence for a flip-flop mechanism

The crystal structure of the thioacylenzyme intermediate of the phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus has been solved at 1.8A resolution. Formation of the intermediate was obtained by diffusion of the natural substrate within the crystal of the holoenzyme in the absence of inorganic phosphate. To define the soaking conditions suitable for the isolation and accumulation of the intermediate, a microspectrophotometric characterization of the reaction of GAPDH in single crystals was carried out, following NADH formation at 340 nm. When compared with the structure of the Michaelis complex (Didierjean, C., Corbier, C., Fatih, M., Favier, F., Boschi-Muller, S., Branlant, G., and Aubry, A. (2003) J. Biol. Chem. 278, 12968-12976) the 206-210 loop is shifted and now forms part of the so-called "new P(i)" site. The locations of both the O1 atom and the C3-phosphate group of the substrate are also changed. Altogether, the results provide evidence for the flipping of the C3-phosphate group occurring concomitantly or after the redox step.     

Trapping conformational intermediate states in the reaction center protein from photosynthetic bacteria

In protein, conformational changes are often crucial for function but not easy to observe. Two functionally relevant conformational intermediate states of photosynthetic reaction center protein (RCs) are trapped and characterized at low temperature. RCs frozen in the dark do not allow electron transfer from the reduced primary quinone, Q(A)(-), to the secondary quinone, Q(B). In contrast, RCs frozen under illumination in the product (P(+)Q(A)Q(B)(-)) state, with the oxidized electron donor, P(+), and reduced Q(B)(-), return to the ground state at cryogenic temperature in a conformation that allows a high yield of Q(B) reduction. Thus, RCs frozen under illumination are found to be trapped above the ground state in a conformation that allows product formation. When the temperature is raised above 120 K, the protein relaxes to an inactive conformation which is different from the RCs frozen in the dark. The activation energy for this change is 87 +/- 8 meV, and the active and inactive states differ in energy by only 16 +/- 3 meV. Thus, there are several conformational substates along the reaction coordinate with different transition temperatures. The ground state spectra of the RCs in active and inactive conformations report differences in the intraprotein electrostatic field, demonstrating that the dipole or charge distribution has changed. In addition, the electrochromic shift associated with the Q(A)(-) to Q(B) electron transfer at low temperature was characterized. The electron-transfer rate from Q(B)(-) to P(+) was measured at cryogenic temperature and is similar to the rate at room temperature, as expected for an exothermic, electron tunneling reaction in RCs.     

Trapping of an acyl-enzyme intermediate in a penicillin-binding protein (PBP)-catalyzed reaction

Class A penicillin-binding proteins (PBPs) catalyze the last two steps in the biosynthesis of peptidoglycan, a key component of the bacterial cell wall. Both reactions, glycosyl transfer (polymerization of glycan chains) and transpeptidation (cross-linking of stem peptides), are essential for peptidoglycan stability and for the cell division process, but remain poorly understood. The PBP-catalyzed transpeptidation reaction is the target of β-lactam antibiotics, but their vast employment worldwide has prompted the appearance of highly resistant strains, thus requiring concerted efforts towards an understanding of the transpeptidation reaction with the goal of developing better antibacterials. This goal, however, has been elusive, since PBP substrates are rapidly deacylated. In this work, we provide a structural snapshot of a “trapped” covalent intermediate of the reaction between a class A PBP with a pseudo-substrate, N-benzoyl-d-alanylmercaptoacetic acid thioester, which partly mimics the stem peptides contained within the natural, membrane-associated substrate, lipid II. The structure reveals that the d-alanyl moiety of the covalent intermediate (N-benzoyl-d-alanine) is stabilized in the cleft by a network of hydrogen bonds that place the carbonyl group in close proximity to the oxyanion hole, thus mimicking the spatial arrangement of β-lactam antibiotics within the PBP active site. This arrangement allows the target bond to be in optimal position for attack by the acceptor peptide and is similar to the structural disposition of β-lactam antibiotics with PBP clefts. This information yields a better understanding of PBP catalysis and could provide key insights into the design of novel PBP inhibitors.     

Trapping of a reaction intermediate by tetranitromethane during catalysis by ribulose biphosphate carboxylase

Illumination at 230 K of dithionite-reduced particles results in the appearance of an EPR detectable radical 13 G wide with g = 2.0033. This radical is formed in a ratio of 2.28 (±0.5)/P700. Investigation of the time course of formation shows two components are present. One (A₁) has g = 2.0051 and the other (A_og= 2.0024. Reduction of A₁ results in an increase in reaction centre triplet formation, subsequent reduction of A_o results in a decrease of triplet formation to the base level. We propose that these components function sequentially in the transfer of electrons from P700 to the iron—sulphur acceptors.     

The structure of high molecular weight dextrins obtained from potato starch by treatment with Bacillus macerans enzyme

Bacillus macerans enzyme (BME)-derived high molecular weight dextrins, which are by-products in the course of the industrial production of cylodextrins, were isolated and their chemical structures were characterized.Dextrin I was obtained in a yield of about 24% from BME-hydrolyzate (a mixture of dextrin and cylodextrins, 50% each) of potato starch by fractionation with an ultrafiltrator having a membrane of cut-off molecular weight 2.0 × 10⁴. Dextrin II was obtained in a yield of about 15% from BME-hydrolyzate (a mixture of dextrins and cyclodextrins, 70 : 30) of Dextrin I by the same method.Dextrin I and II consisted of dextrin having molecular weights over 20 × 10⁶ and dextrins having molecular weights 4 × 10³−1 × 10⁵ in the ratio of 80 : 12 and 66: 15, respectively.The results of hydrolysis by β-amylase and methylation analysis indicated that the average, exterior and interior chain lenghts of the dextrins having molecular weights over 20 × 10⁶ and 4 × 10³−1 × 10⁵ from Dextrin I were 16.5, 8.2 and 7.3, and 11.5, 6.9 and 3.6, respectively, than those from Dextrin II were 13.6, 4.7 and 9.9, and 10.4, 5.1 and 4.3, respectively.     

Analysis of the dynamic properties of Bacillus circulans xylanase upon formation of a covalent glycosyl-enzyme intermediate

NMR spectroscopy was used to search for mechanistically significant differences in the local mobility of the main-chain amides of Bacillus circulans xylanase (BCX) in its native and catalytically competent covalent glycosyl-enzyme intermediate states. 15N T1, T2, and 15N[1H] NOE values were measured for approximately 120 out of 178 peptide groups in both the apo form of the protein and in BCX covalently modified at position Glu78 with a mechanism-based 2-deoxy-2-fluoro-beta-xylobioside inactivator. Employing the model-free formalism of Lipari and Szabo, the measured relaxation parameters were used to calculate a global correlation time (tau(m)) for the protein in each form (9.2 +/- 0.2 ns for apo-BCX; 9.8 +/- 0.3 ns for the modified protein), as well as individual order parameters for the main-chain NH bond vectors. Average values of the order parameters for the protein in the apo and complexed forms were S2 = 0.86 +/- 0.04 and S2 = 0.91 +/- 0.04, respectively. No correlation is observed between these order parameters and the secondary structure, solvent accessibility, or hydrogen bonding patterns of amides in either form of the protein. These results demonstrate that the backbone of BCX is well ordered in both states and that formation of the glycosyl-enzyme intermediate leads to little change, in any, in the dynamic properties of BCX on the time scales sampled by 15N-NMR relaxation measurements.     

Ubiquitin manipulation by an E2 conjugating enzyme using a novel covalent intermediate

Degradation of misfolded and damaged proteins by the 26 S proteasome requires the substrate to be tagged with a polyubiquitin chain. Assembly of polyubiquitin chains and subsequent substrate labeling potentially involves three enzymes, an E1, E2, and E3. E2 proteins are key enzymes and form a thioester intermediate through their catalytic cysteine with the C-terminal glycine (Gly76) of ubiquitin. This thioester intermediate is easily hydrolyzed in vitro and has eluded structural characterization. To overcome this, we have engineered a novel ubiquitin-E2 disulfide-linked complex by mutating Gly76 to Cys76 in ubiquitin. Reaction of Ubc1, an E2 from Saccharomyces cerevisiae, with this mutant ubiquitin resulted in an ubiquitin-E2 disulfide that could be purified and was stable for several weeks. Chemical shift perturbation analysis of the disulfide ubiquitin-Ubc1 complex by NMR spectroscopy reveals an ubiquitin-Ubc1 interface similar to that for the ubiquitin-E2 thioester. In addition to the typical E2 catalytic domain, Ubc1 contains an ubiquitin-associated (UBA) domain, and we have utilized NMR spectroscopy to demonstrate that in this disulfide complex the UBA domain is freely accessible to non-covalently bind a second molecule of ubiquitin. The ability of the Ubc1 to bind two ubiquitin molecules suggests that the UBA domain does not interact with the thioester-bound ubiquitin during polyubiquitin chain formation. Thus, construction of this novel ubiquitin-E2 disulfide provides a method to characterize structurally the first step in polyubiquitin chain assembly by Ubc1 and its related class II enzymes.     

Chemical modification of cyclomaltodextrin glucanotransferase from Bacillus circulans var. alkalophilus.

Counting of integral numbers of cysteine residues of the reduced and denaturated form of cyclomaltodextrin glucanotransferase (CGTase) from Bacillus circulans var. alkalophilus (ATCC 21783) showed two cysteine residues per enzyme molecule. Titrations of the enzyme with 5,5'-dithiobis-(2-nitrobenzoic acid) led to the same result. No free SH-group was detected in denatured form of CGTase, indicating that the two cysteine residues are linked by one disulfide bridge. Cyclizing activity of the GdmCl-denaturated and reduced enzyme was 13% of that of the native one. Incubation of CGTase with diethylpyrocarbonate (DEP) showed a pseudo-first-order inhibition with second-order rate constant of 3.2 M-1 s-1. Reaction with hydroxylamine and spectroscopic studies implied that inactivation of CGTase by DEP is due to modification of one histidine residue concomitantly with a 50% decrease in the cyclizing activity (t1/2 = 10.8 min). The inhibition was partially reversible. CGTase was protected against inactivation by alpha- and beta-cyclodextrins suggesting that the modified histidine residue is at or near the active site. Conversion of starch with DEP-modified enzyme resulted in a decreased formation of cyclodextrins while the relative amount of reducing sugars increased. Preliminary results on modification of CGTase with other reagents, e.g., Woodward's reagent K, 2,3-butanedione and carbodiimide are included.     

Trapping of the substrate-derived acyl enzyme intermediate of purified penicillin-binding protein 1a of Escherichia coli

Purified penicillin-binding protein 1a of Escherichia coli formed an acyl enzyme intermediate with the highly reactive synthetic substrate diacetyl-L-lysyl-D-alanyl-D-lactate at acid pH, although in extremely low yields.