Structure-function relationship in cyclodextrin glycosyltransferase from Bacillus circulans DF 9R

Cyclodextrin glycosyltransferases (CGTases E.C.2.4.1.19) catalyze cyclomaltooligosaccharides (cyclodextrins) production, an important industrial process. We herein report structural features of Bacillus circulans DF 9R cyclodextrin glycosyltransferase including its sequence and several aspects of enzyme structure-function relationship. Protein ethoxyformylation, under our experimental conditions, indicated that only one out of the 13 enzyme histidines was modified leading to a drastic drop in cyclizing and hydrolytic activity. Besides, tryptic digestion of the ¹⁴C ethoxyformylated protein and studies of the peptide mixture showed that histidine 233 is the most reactive histidine residue. This is the first cyclodextrin glycosyltransferase with a known primary structure and a glutamine instead of glycine residue at position 179 in the highly conserved −6 subsite, shown to be involved in substrate binding. The presence of glycine at that position was considered as a requirement for such binding following the induced-fit mechanism already proposed. Moreover, the enzyme has all the features previously described for an α- or α/β-cyclodextrin producer.     

Three-dimensional structure of cyclodextrin glycosyltransferase from Bacillus circulans at 3.4 A resolution

Cyclodextrin glycosyltransferase (EC 2.4.1.19) from Bacillus circulans has been purified, crystallized and analyzed by X-ray diffraction. The enzyme is monomeric. SDS/polyacrylamide gel electrophoresis gave an Mr of 73,600(+/- 1000), corresponding to 670(+/- 10) amino acid residues. The structure of the crystalline enzyme has been elucidated at a resolution of 3.4 A, using multiple isomorphous replacement and solvent flattening for phase determination. The resulting electron density map allowed tracing of the polypeptide chain; 664 residue positions have been assigned. The chain fold has been subdivided into five domains. The N-terminal domain forms a (beta alpha)8-barrel, which contains the second domain of about 55 residues as an insert after the third beta-strand. The three remaining domains form almost exclusively beta-pleated sheet structures and consist of about 90, 80 and 95 residues. The chain fold of the three N-terminal domains of 492 residues resembles closely the two known structures of alpha-amylases. This geometric similarity corresponds to the observed amino acid sequence homology. On the basis of the sequence homology with alpha-amylases, the active center can be located. The fourth domain has an immunoglobulin fold and is far away from the active center, while the fifth domain participates in the formation of the broad depression at the active center. Accordingly, the cyclodextrin glycosyltransferase chain fold can be considered as an alpha-amylase chain fold with two additional domains.     

Succinylation of cyclodextrin glycosyltransferase from Thermoanaerobacter sp. 501 enhances its transferase activity using starch as donor

A simple modification procedure, the succinylation of amino groups, was suitable to increase the transferase (disproportionation) activity of cyclodextrin glycosyltransferase (CGTase) from Thermoanaerobacter sp. 501 using different linear oligosaccharides as acceptors. On the contrary, the synthesis of cyclodextrins (CDs), the coupling of CDs with oligosaccharides, and the hydrolysis of starch decreased after chemical modification. The degree of succinylation of amino groups (45%) was accurately determined by MALDI-TOF mass spectrometry. The formation of CDs under industrial conditions was analyzed for native and succinylated CGTases, showing similar selectivity to alpha-, beta-, gamma-CD. The acceptor reaction with D-glucose using soluble starch as glucosyl donor was studied at 60 degrees C and pH 5.5. Malto-oligosaccharides (MOS) production was notably higher using the semisynthetic enzyme at different ratios (w/w) starch:D-glucose. Thus, more than 90% of the initial starch was converted into MOS (G2-G7) in 48 h employing a ratio donor:acceptor 1:2 (w/w).     

Production of cyclodextrin from starch by cyclodextrin glycosyltransferase from Bacillus firmus and characterization of purified enzyme

During screening for cyclodextrin-forming microorganisms, an alkalophilic Bacillus sp, which produced high activity of cyclodextrin glycosyltransferase, was isolated and identified as Bacillus firmus. The crude enzyme transformed starch to mainly β-and γ-cyclodextrin. The purified enzyme had an optimum pH of 7.5–8.5 and its optimum temperature was 65°C, which is the highest optimum temperature as compared to other cyclodextrin glycosyltransferases except that produced by Bacillus amyloliquefaciens. Received 06 January 1997/ Accepted in revised form 20 March 1997     

The cyclization mechanism of cyclodextrin glycosyltransferase (CGTase) as revealed by a gamma-cyclodextrin-CGTase complex at 1.8-A resolution

The enzyme cyclodextrin glycosyltransferase is closely related to alpha-amylases but has the unique ability to produce cyclodextrins (circular alpha(1-->4)-linked glucoses) from starch. To characterize this specificity we determined a 1.8-A structure of an E257Q/D229N mutant cyclodextrin glycosyltransferase in complex with its product gamma-cyclodextrin, which reveals for the first time how cyclodextrin is competently bound. Across subsites -2, -1, and +1, the cyclodextrin ring binds in a twisted mode similar to linear sugars, giving rise to deformation of its circular symmetry. At subsites -3 and +2, the cyclodextrin binds in a manner different from linear sugars. Sequence comparisons and site-directed mutagenesis experiments support the conclusion that subsites -3 and +2 confer the cyclization activity in addition to subsite -6 and Tyr-195. On this basis, a role of the individual residues during the cyclization reaction cycle is proposed.     

Molecular imprinting of cyclodextrin glycosyltransferases from Paenibacillus sp. A11 and Bacillus macerans with gamma-cyclodextrin

Cyclodextrin glycosyltransferase catalyzes the formation of a mixture of cyclodextrins from starch by an intramolecular transglycosylation reaction. To manipulate the product specificity of the Paenibacillus sp. A11 and Bacillus macerans cyclodextrin glycosyltransferases towards the preferential formation of gamma-cyclodextrin (CD(8)), crosslinked imprinted proteins of both cyclodextrin glycosyltransferases were prepared by applying enzyme imprinting and immobilization methodologies. The crosslinked imprinted cyclodextrin glycosyltransferases obtained by imprinting with CD(8) showed pH and temperature optima similar to those of the native and immobilized cyclodextrin glycosyltransferases. However, the pH and temperature stability of the immobilized and crosslinked imprinted cyclodextrin glycosyltransferases were higher than those of the native cyclodextrin glycosyltransferases. When the catalytic activities of the native, immobilized and crosslinked imprinted cyclodextrin glycosyltransferases were compared, the efficiency of the crosslinked imprinted enzymes for CD(8) synthesis was increased 10-fold, whereas that for cyclodextrin hydrolysis was decreased. Comparison of the product ratios by high-performance anion exchange chromatography showed that the native cyclodextrin glycosyltransferases from Paenibacillus sp. A11 and Bacillus macerans produced CD(6) : CD(7) : CD(8) : > or = CD(9) ratios of 15 : 65 : 20 : 0 and 43 : 36 : 21 : 0 after 24 h of reaction at 40 degrees C with starch substrates. In contrast, the crosslinked imprinted cyclodextrin glycosyltransferases from Paenibacillus sp. A11 and Bacillus macerans produced cyclodextrin in ratios of 15 : 20 : 50 : 15 and 17 : 14 : 49 : 20, respectively. The size of the synthesis products formed by the crosslinked imprinted cyclodextrin glycosyltransferases was shifted towards CD(8) and > or = CD(9), and the overall cyclodextrin yield was increased by 12% compared to the native enzymes. The crosslinked imprinted cyclodextrin glycosyltransferases also showed higher stability in organic solvents, retaining 85% of their initial activity after five cycles of synthesis reactions.     

Catalytic center of cyclodextrin glycosyltransferase derived from X-ray structure analysis combined with site-directed mutagenesis.

An X-ray structure analysis of a crystal of mutant Asp229----Ala of cyclodextrin glycosyltransferase from Bacillus circulans (Ec 2.4.1.19) that had been shortly exposed to beta-cyclodextrin showed density corresponding to a maltose bound at the catalytic center. The crystal structure was refined to an R-factor of 18.7% at 2.5-A resolution. The catalytic center is defined by homology with the structurally known alpha-amylases and by the observation that mutants Asp229----Ala and Asp328----Ala are almost inactive. By model building, the density-defined maltose was extended to a full beta-cyclodextrin, which then indicated the general locations of seven subsites for glucosyl units. The catalytically competent residues Asp229, Glu257, and Asp328 are at the reducing end of the density-defined maltose. In the unligated wild-type structure, Glu257 and Asp328 form a 2.6-A hydrogen bond between their carboxylates in an arrangement that resembles those of the catalytically competent carboxylates in acid proteases. Presumably, the first catalytic step is an attack of the proton between Glu257 and Asp328 on the oxygen of the glycosidic bond.     

Cyclodextrin production by cyclodextrin glycosyltransferase from Bacillus circulans DF 9R

Cyclodextrins (CD) are cyclic oligosaccharides with multiple applications in the food, pharmaceutical, cosmetic, agricultural and chemical industries. In this work, the conditions used to produce CD with cyclodextrin glycosyltransferase from Bacillus circulans DF 9R were optimized using experimental designs. The developed method allowed the partial purification and concentration of the enzyme from the cultural broth and, subsequently, the CD production, using the same cassava starch as enzyme adsorbent and as substrate. Heat-treatment of raw starch at 70 degrees C for 15 min in the presence of adsorbed cyclodextrin glycosyltransferase allowed the starch liquefaction without enzyme inactivation. The optimum conditions for CD production were: 5% (w/v) cassava starch, 15 U of enzyme per gram of substrate, reaction temperature of 56 degrees C and pH 6.4. After 4h, the proportion of starch converted to CD reached 66% (w/w) and the weight ratio of alpha-CD:beta-CD:gamma-CD was 1.00:0.70:0.16.     

Cloning and nucleotide sequence of a thermostable cyclodextrin glycosyltransferase gene from Thermoanaerobactersp. ATCC 53627 and its expression in Escherichia coli

A gene, cgtA, encoding an extremely thermostable cyclodextrin glycosyltransferase (CGTase) was cloned from a thermophilic anaerobe, Thermoanaerobacter sp. ATCC 53627, and expressed in Escherichia coli. DNA and protein sequencing revealed that the mature enzyme of 683 amino acid residues (MW 75 kDa) was preceded by a signal peptide of 27 amino acid residues. The sequence of the Thermoanaerobacter CGTase was similar to sequences of Bacillus CGTases, with more than 58% identity, and very similar (89% identity) to a CGTase enzyme from Thermoanaerobacterium thermosulfurogenes.     

Structure of cyclodextrin glycosyltransferase refined at 2.0 A resolution.

The previously reported structural model of cyclodextrin glycosyltransferase (EC 2.4.1.19) from Bacillus circulans has been improved. For this purpose the known sequence was built into an electron density map established by multiple isomorphous replacement and subsequent solvent-flattening at 2.5 A resolution. The resulting model was refined at 2.0 A resolution using a simulated annealing refinement method. Based on 70,171 independent reflections in the range 7.0 to 2.0 A resolution, a final R-factor of 17.6% was obtained with a model obeying standard geometry within 0.013 A in bond lengths and 2.7 degrees in bond angles. The final model consists of all 684 amino acid residues, two calcium ions and 588 solvent molecules.     

Thermoanaerobacterium thermosulfurigenes cyclodextrin glycosyltransferase.

Cyclodextrin glycosyltransferase (CGTase) uses an alpha-retaining double displacement mechanism to catalyze three distinct transglycosylation reactions. To investigate these reactions as catalyzed by the CGTase from Thermoanaerobacterium thermosulfurigenes the enzyme was overproduced (8 mg.L(-1) culture) using Bacillus subtilis as a host. Detailed analysis revealed that the three reactions proceed via different kinetic mechanisms. The cyclization reaction (cyclodextrin formation from starch) is a one-substrate reaction, whereas the other two transglycosylation reactions are two-substrate reactions, which obey substituted enzyme mechanism kinetics (disproportionation reaction) or ternary complex mechanism kinetics (coupling reaction). Analysis of the effects of acarbose and cyclodextrins on the disproportionation reaction revealed that cyclodextrins are competitive inhibitors, whereas acarbose is a mixed type of inhibitor. Our results show that one molecule of acarbose binds either in the active site of the free enzyme, or at a secondary site of the enzyme-substrate complex. The mixed inhibition thus indicates the existence of a secondary sugar binding site near the active site of T. thermosulfurigenes CGTase.     

环糊精葡萄糖基转移酶高效异源表达研究进展

环糊精葡萄糖基转移酶(cyclodextringlycosyltransferase,CGTase)酶法合成环糊精是目前生产环糊精的主要方法。本文介绍了用于生产环糊精葡萄糖基转移酶的几种工程菌株:大肠杆菌、枯草芽孢杆菌以及毕赤酵母,其中大肠杆菌是目前应用最广泛的用于表达CGTase的表达系统。除此之外,本文还总结了高效表达环糊精葡萄糖基转移酶的有效策略:选择合适的表达载体、启动子以及信号肽,以及密码子优化和分子伴侣共表达,以期为在相关CGTase研究领域开展研究提供参考。     

Glyco-array technology for efficient monitoring of plant cell wall glycosyltransferase activities

The plant cell wall is a complex network of polysaccharides. The diversity in the linkage types connecting all monosaccharides within these polysaccharides would need a large set of glycosyltransferases to catalyze their formation. Development of a methodology that would allow monitoring of glycosyltransferase activities in an easy and high-throughput manner would help assign biochemical functions, and understand their roles in building this complex network. A microarray-based method was optimized for testing glycosyltransferases involved in plant wall biosynthesis using an α(1,2)fucosyltransferase involved in xyloglucan biosynthesis. The method is simple, sensitive, and easy to implement in any lab. Tamarind xyloglucan polymer and trimer, and a series of cello-oligosaccharides were immobilized on a thin-coated photo-activable glass slide. The slide with the attached sugars was then used to estimate the incorporation of [¹⁴C]Fuc onto xyloglucan polymer and trimer. [¹⁴C]-radiolabel incorporation is revealed with a standard phosphoimager scanner, after exposure of the glycochip to a phosphor screen and detection. The method proved to be sensitive enough to detect as low as 45 cpm/spot. Oriented anchoring of small oligosaccharides (trimer) was required for optimal transferase activities. The glycochip was also used to monitor and estimate xyloglucan fucosyltransferase activity in detergent-solubilized crude extracts from pea microsomes that are known to contain this enzyme activity. Our data indicate that the methodology can be used for efficient and rapid monitoring of glycosyltransferase activities involved in plant wall polysaccharides biosynthesis. Matthew Shipp and Ramya Nadella contributed equally to this work.     

Single amino acid mutations interchange the reaction specificities of cyclodextrin glycosyltransferase and the acarbose-modifying enzyme acarviosyl transferase

Acarviosyl transferase (ATase) from Actinoplanes sp. SE50/110 is a bacterial enzyme that transfers the acarviosyl moiety of the diabetic drug acarbose to sugar acceptors. The enzyme exhibits 42% sequence identity with cyclodextrin glycosyltransferases (CGTase), and both enzymes are members of the alpha-amylase family, a large clan of enzymes acting on starch and related compounds. ATase is virtually inactive on starch, however. In contrast, ATase is the only known enzyme to efficiently use acarbose as substrate (2 micromol min(-1) mg(-1)); acarbose is a strong inhibitor of CGTase and of most other alpha-amylase family enzymes. This distinct reaction specificity makes ATase an interesting enzyme to investigate the variation in reaction specificity of alpha-amylase family enzymes. Here we show that a G140H mutation in ATase, introducing the typical His of the conserved sequence region I of the alpha-amylase family, changed ATase into an enzyme with 4-alpha-glucanotransferase activity (3.4 micromol min(-1) mg(-1)). Moreover, this mutation introduced cyclodextrin-forming activity into ATase, converting 2% of starch into cyclodextrins. The opposite experiment, removing this typical His side chain in CGTase (H140A), introduced acarviosyl transferase activity in CGTase (0.25 micromol min(-1) mg(-1)).     

Identification of essential histidines in cyclodextrin glycosyltransferase isoform 1 from Paenibacillus sp. A11

The isoform 1 of cyclodextrin glycosyltransferase (CGTase, EC 2.4.1.19) from Paenibacillus sp. A11 was purified by a preparative gel electrophoresis. The importance of histidine, tryptophan, tyrosine, and carboxylic amino acids for isoform 1 activity is suggested by the modification of the isoform 1 with various group-specific reagents. Activity loss, when incubated with diethylpyrocarbonate (DEP), a histidine modifying reagent, could be protected by adding 25 mM methyl-beta-cyclodextrin substrate prior to the modification. Inactivation kinetics of isoform 1 with DEP resulted in second-order rate constants (k(inactivation)) of 29.5 M(-1)s(-1). The specificity of the DEP-modified reaction for the histidine residue was shown by the correlation between the loss of isoform activity and the increase in the absorbance at 246 nm of N-carbethoxyhistidine. The number of histidines that were modified by DEP in the absence and presence of a protective substrate was estimated from the increase in the absorbance using a specific extinction coefficient of N-carbethoxyhistidine of 3,200 M(-1)cm(-1). It was discovered that methyl-beta-CD protected per mole of isoform 1, two histidine residues from the modification by DEP. To localize essential histidines, the native, the DEP-modified, and the protected forms of isoform 1 were digested by trypsin. The resulting peptides were separated by HPLC. The peptides of interest were those with R(t) 11.34 and 40.93 min. The molecular masses of the two peptides were 5,732 and 2,540 daltons, respectively. When the data from the peptide analysis were checked with the sequence of CGTase, then His-140 and His-327 were identified as essential histidines in the active site of isoform 1.     

Studies of the mechanism of the cyclisation reaction catalysed by the wildtype and a truncated alpha-cyclodextrin glycosyltransferase from Klebsiella pneumoniae strain M 5 al, and the beta-cyclodextrin glycosyltransferase from Bacillus circulans strain 8

The actions of the wildtype and a truncated alpha-cyclodextrin glycosyltransferase from Klebsiella pneumoniae strain M 5 al on malto-oligosaccharides showed no significant differences, and there was marked dependence of the kinetic parameters on the chain lengths of the substrate. The action of the beta-cyclodextrin glycosyltransferase from Bacillus circulans was less dependent on the chain length of the substrate, but Vmax of the initial cyclisation with the longer malto-oligosaccharides was only 28% of that determined for the enzyme of K. pneumoniae. The rate parameters suggested that the active site of each enzyme spans nine glucosyl residues, and that the catalytic sites are situated between subsites three and four for the K. pneumoniae enzymes and between subsites two and three for the B. circulans enzyme. The molecular binding affinities and the affinities of the 9th subsite were calculated from the rate parameters. The primary and tertiary structures of alpha-amylases and cyclodextrin glycosyltransferases are compared in the context of the reaction mechanism of the latter enzymes.     

Sequence analysis of cyclodextrin glycosyltransferase from the alkaliphilic Bacillus agaradhaerens

The gene encoding an alkaline active cyclodextrin glycosyltransferase (CGTase) from the alkaliphilic B. agaradhaerens LS-3C was cloned and sequenced. It encodes a mature polypeptide of 679 amino acids with a molecular mass of 76488 Da. The deduced amino acid sequence of the mature CGTase revealed 99 and 95% identity to the CGTase sequences from the other B. agaradhaerens strains, DSM 8721^T and 9948, respectively. The next closest identity was of 59% with B. clarkii enzyme. CGTases from B. agaradhaerens, B. clarkii, and B. firmus/lentus formed a phylogenetically separated cluster from the other CGTases of Bacillus spp. origin. A number of usually conserved residues in the CGTases were found to be replaced in the sequence of B. agaradhaerens enzyme. The sequence analysis indicated the enzyme to be close to the so-called `intermediary enzymes' in the -amylase family.     

Specific alpha-cyclodextrin production by a novel thermostable cyclodextrin glycosyltransferase

Summary A novel bacterial isolate, Bacillus amyloliquefaciens strain AL 35, produced high yields of a cyclodextrin glycosyltransferase (CGT'ase) when grown in a submerged culture. The stability of CGT'ase to high temperature and alkaline pH enabled processing for cyclodextrin production to be carried out at 60° C and pH 9.0. Crude culture filtrates containing the CGT'ase could convert gelatinized starch substrates to predominatly -cyclodextrins (up to 95% of the total cyclodextrin yields).     

Optimization of cyclodextrin glycosyltransferase production from Klebsiella pneumoniae AS-22 in batch, fed-batch, and continuous cultures

Production of a novel cyclodextrin glycosyltransferase (CGTase) from Klebsiella pneumoniae AS-22 strain, which converts starch predominantly to alpha-CD at high conversion yields, in batch, fed-batch, and continuous cultures, is presented. In batch fermentations, optimization of different operating parameters such as temperature, pH, agitation speed, and carbon-source concentration resulted in more than 6-fold increase in CGTase activity. The enzyme production was further improved by two fed-batch approaches. First, using glucose-based feed to increase cell density, followed by starch-based feed to induce enzyme production, resulted in high cell density of 76 g dry cell weight/L, although the CGTase production was low. Using the second approach of a single dextrin-based feed, 20-fold higher CGTase was produced compared to that in batch fermentations with media containing tapioca starch. In continuous operation, more than 8-fold increase in volumetric CGTase productivity was obtained using dextrin-based media compared to that in batch culture using starch-based media.     

An approach for fluorometric determination of glycosyltransferase activities

A new strategy for the fluorometric determination of glycosyltransferase activities is reported. The method involves dansyl chloride derivatization of the reduced form (pNH₂phenyl) of a hydrophobic, aglycon moiety covalently linked to a number of acceptor substrates (pNO₂phenyl). Focusing on the Golgi enzyme core 2N-acetylglucosaminyltransferase, we found that synthesis and fractionation of the dansylated substrate derivative were rapid, easy and inexpensive. Additionally, the corresponding enzyme assay proved reproducible and very sensitive, as 0.4 pmol of reaction product were readily detected. This fluorometric approach appears therefore to be a valid tool for investigating the monitoring differential expression of glycosyltransferases exhibiting low levels of enzyme activity.Abbreviations T transferase - Gal D-N-galactose - GlcNAc D-N-acetylglucosamine - GalNAc D-N-acetylgalactosamine - HPLC high pressure liquid chromatography - UDP uridine diphosphate - TES 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid - pNp para-nitrophenyl - NMR nuclear magnetic resonance - DMSO dimethyl sulphoxide