Novel Members of Glycoside Hydrolase Family 13 Derived from Environmental DNA

Starch and pullulan-modifying enzymes of the α-amylase family (glycoside hydrolase family 13) have several industrial applications. To date, most of these enzymes have been derived from isolated organisms. To increase the number of members of this enzyme family, in particular of the thermophilic representatives, we have applied a consensus primer-based approach using DNA from enrichments from geothermal habitats. With this approach, we succeeded in isolating three new enzymes: a neopullulanase and two cyclodextrinases. Both cyclodextrinases displayed significant maltogenic amylase side activity, while one showed significant neopullulanase side activity. Specific motifs and domains that correlated with enzymatic activities were identified; e.g., the presence of the N domain was correlated with cyclodextrinase activity. The enzymes exhibited stability under thermophilic conditions and showed features appropriate for biotechnological applications.     

Analysis of mutations in cyclodextrin glucanotransferase from Bacillus stearothermophilus which affect cyclization characteristics and thermostability.

Cyclodextrin glucanotransferase (CGTase; EC 2.4.1.19) produces cyclodextrin from starch. The CGTase molecule is composed of four globular domains, A, B, C, and D. In order to gain better understanding of the amylolytic and cyclization mechanisms of CGTase, mutant CGTases were constructed from a CGTase gene (cgt1) of Bacillus stearothermophilus NO2. Cgt1-F191Y (Phe at position 191 was replaced by Tyr), Cgt1-F191Y-F255Y, Cgt1-W254V-F255I, Cgt1-W254V, and Cgt1-F255I were constructed for the analysis of the NH2-terminal region. It was revealed that amino acids surrounding a spiral amylose are important for cyclization characteristics and that hydrophobic amino acids just after the Glu catalytic site play an important role in the hydrolysis characteristics of the enzyme. Mutant CGTases Cgt1-T591F and Cgt1-W629F were also constructed to study the role of a second substrate-binding site in domain D, and it was suggested that substrate binding at both domains A and D stabilized the enzyme and optimized cyclodextrin production.     

Crystal structure of human glyoxalase I--evidence for gene duplication and 3D domain swapping.

The zinc metalloenzyme glyoxalase I catalyses the glutathione-dependent inactivation of toxic methylglyoxal. The structure of the dimeric human enzyme in complex with S-benzyl-glutathione has been determined by multiple isomorphous replacement (MIR) and refined at 2.2 A resolution. Each monomer consists of two domains. Despite only low sequence homology between them, these domains are structurally equivalent and appear to have arisen by a gene duplication. On the other hand, there is no structural homology to the 'glutathione binding domain' found in other glutathione-linked proteins. 3D domain swapping of the N- and C-terminal domains has resulted in the active site being situated in the dimer interface, with the inhibitor and essential zinc ion interacting with side chains from both subunits. Two structurally equivalent residues from each domain contribute to a square pyramidal coordination of the zinc ion, rarely seen in zinc enzymes. Comparison of glyoxalase I with other known structures shows the enzyme to belong to a new structural family which includes the Fe2+-dependent dihydroxybiphenyl dioxygenase and the bleomycin resistance protein. This structural family appears to allow members to form with or without domain swapping.     

Metabolism of cyclodextrins byKlebsiella oxytoca M5a1: purification and characterisation of a cytoplasmically located cyclodextrinase

It has been shown previously that the products of 11 genes are required for metabolism of starch byKlebsiella oxytoca via a novel pathway. An extracellular cyclodextrin glucanotransferase first degrades starch into -and -cyclodextrins; evidence then has been presented that the cyclodextrins are transported into the cytoplasma via a specific system and that they are metabolised inside the cell. To provide support for this model, we have analysed whetherKlebsiella oxytoca possesses a cytoplasmic enzyme able to linearise cyclodextrins. A possible candidate was the product of thecymH gene since it displays sequence similarity with cyclodextrinases from other organisms. ThecymH gene was overexpressed, and the CymH protein was purified. CymH is a monomer of 69 kDa molecular mass and hydrolysed cyclodextrins at an optimum pH of 7.0 and an optimum temperature of 23° C, respectively. The apparentK _m increased with increasing size of the cyclodextrins, but the reaction velocity decreased. Linear malto-oligosaccharides were also accepted as substrates, but were hydrolysed with a lower efficiency. Final products in each case were maltose and maltotriose. It was demonstrated by immunoblotting that CymH is located in the cytoplasm and that no signal peptide was cleaved off. SincecymH mutants were no longer able to grow on cyclodextrins, these results prove that cyclodextrins are degraded inside the cell, and they support the contention of the existence of a specific transport system.     

The X-ray crystallographic structure of Escherichia coli branching enzyme

Branching enzyme catalyzes the formation of alpha-1,6 branch points in either glycogen or starch. We report the 2.3-A crystal structure of glycogen branching enzyme from Escherichia coli. The enzyme consists of three major domains, an NH(2)-terminal seven-stranded beta-sandwich domain, a COOH-terminal domain, and a central alpha/beta-barrel domain containing the enzyme active site. While the central domain is similar to that of all the other amylase family enzymes, branching enzyme shares the structure of all three domains only with isoamylase. Oligosaccharide binding was modeled for branching enzyme using the enzyme-oligosaccharide complex structures of various alpha-amylases and cyclodextrin glucanotransferase and residues were implicated in oligosaccharide binding. While most of the oligosaccharides modeled well in the branching enzyme structure, an approximate 50 degrees rotation between two of the glucose units was required to avoid steric clashes with Trp(298) of branching enzyme. A similar rotation was observed in the mammalian alpha-amylase structure caused by an equivalent tryptophan residue in this structure. It appears that there are two binding modes for oligosaccharides in these structures depending on the identity and location of this aromatic residue.     

Relation between domain evolution, specificity, and taxonomy of the alpha-amylase family members containing a C-terminal starch-binding domain.

The alpha-amylase family (glycoside hydrolase family 13; GH 13) contains enzymes with approximately 30 specificities. Six types of enzyme from the family can possess a C-terminal starch-binding domain (SBD): alpha-amylase, maltotetraohydrolase, maltopentaohydrolase, maltogenic alpha-amylase, acarviose transferase, and cyclodextrin glucanotransferase (CGTase). Such enzymes are multidomain proteins and those that contain an SBD consist of four or five domains, the former enzymes being mainly hydrolases and the latter mainly transglycosidases. The individual domains are labelled A [the catalytic (beta/alpha)8-barrel], B, C, D and E (SBD), but D is lacking from the four-domain enzymes. Evolutionary trees were constructed for domains A, B, C and E and compared with the 'complete-sequence tree'. The trees for domains A and B and the complete-sequence tree were very similar and contain two main groups of enzymes, an amylase group and a CGTase group. The tree for domain C changed substantially, the separation between the amylase and CGTase groups being shortened, and a new border line being suggested to include the Klebsiella and Nostoc CGTases (both four-domain proteins) with the four-domain amylases. In the 'SBD tree' the border between hydrolases (mainly alpha-amylases) and transglycosidases (principally CGTases) was not readily defined, because maltogenic alpha-amylase, acarviose transferase, and the archaeal CGTase clustered together at a distance from the main CGTase cluster. Moreover the four-domain CGTases were rooted in the amylase group, reflecting sequence relationships for the SBD. It appears that with respect to the SBD, evolution in GH 13 shows a transition in the segment of the proteins C-terminal to the catalytic (beta/alpha)8-barrel(domain A).     

Characterization of an Archaeal Cyclodextrin Glucanotransferase with a Novel C-Terminal Domain

A gene encoding a cyclodextrin glucanotransferase (CGTase) from Thermococcus kodakaraensis KOD1 (CGT(Tk)) was identified and characterized. The gene (cgt(Tk)) encoded a protein of 713 amino acid residues harboring the four conserved regions found in all members of the alpha-amylase family. However, the C-terminal domain corresponding to domain E of previously known CGTases displayed a completely distinct primary structure. In order to elucidate the catalytic function of the gene product, the recombinant enzyme was purified by anion-exchange chromatography, and its enzymatic properties were investigated. The enzyme displayed significant starch-degrading activity (750 U/mg of protein) with an optimal temperature and pH of 80 degrees C and 5.5 to 6.0, respectively. The presence of Ca(2+) enhanced the enzyme activity and elevated the optimum temperature to 85 to 90 degrees C. With the addition of Ca(2+), the enzyme showed extreme thermostability, with almost no loss of enzymatic activity after 80 min at 85 degrees C, and a half-life of 20 min at 100 degrees C. CGT(Tk) could hydrolyze soluble starch and glycogen but failed to hydrolyze pullulan. Most importantly, although CGT(Tk) harbored a unique C-terminal domain, we found that the protein also exhibited significant CGTase activity, with beta-cyclodextrin as the main product. In order to identify the involvement, if any, of the C-terminal region in the CGTase activity, we analyzed a truncated protein (CGT(Tk)DeltaC) with 23 C-terminal amino acid residues deleted. CGT(Tk)DeltaC displayed similar properties in terms of starch-binding activity, substrate specificity, and thermostability, but unexpectedly showed higher starch-degrading activity than the parental CGT(Tk). In contrast, the cyclization activity of CGT(Tk)DeltaC was abolished. The results indicate that the presence of the structurally novel C-terminal domain is essential for CGT(Tk) to properly catalyze the cyclization reaction.     

Molecular cloning, DNA nucleotide sequencing, and expression in Bacillus subtilis cells of the Bacillus macerans cyclodextrin glucanotransferase gene.

The gene for cyclodextrin glucanotransferase from Bacillus macerans was cloned in an Escherichia coli bacteriophage, lambda D69, and was recloned in a Bacillus subtilis plasmid, pUB110. Starting from an ATG initiation codon, a unique reading frame was shown to extend for 2,142 base pairs (714 amino acids). The nucleotide sequence revealed that the enzyme is composed of two identical subunits.     

Structure and function of poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis T1.

Poly(3-hydroxybutyrate) (PHB) depolymerase from Alcaligenes faecalis T1 is composed of three domains: the catalytic (C) domain, the fibronectin type III-like (F) domain, and the substrate-binding (S) domain. We constructed domain deletion, inversion, chimera, and extra-F-domain mutants and examined their enzyme activity and PHB-binding ability. In addition, we performed substitution of 214Asp and 273His with glycine and aspartate, respectively, to examine their participation in a catalytic triad together with 139Ser. The mutant with both the F and S domains deleted and the trypsin-digested enzyme showed no PHB-hydrolyzing activity and less PHB-binding ability than that of the wild-type enzyme but retained D-(-)-3-hydroxybutyrate trimer-hydrolyzing activity at a level similar to that of the wild-type enzyme. The mutant with the F domain deleted and the mutant which had the order of the F and S domains inverted retained PHB-binding ability and trimer-hydrolyzing activity at levels similar to those of the wild-type enzyme but lost PHB-hydrolyzing activity. The chimera mutant, in which the F domain was substituted with a Thr-rich domain of PHB depolymerase A from Pseudomonas lemoignei, and the extra-F-domain mutant, with an additional F domain, retained trimer- and PHB-hydrolyzing activities and PHB-binding ability at levels similar to those of the wild-type enzyme. Two mutants (D214G and H273D) showed no enzymatic activity toward trimer and PHB, and they were not labeled with [3H]diisopropylfluorophosphate.     

Structure of the xylanase from Penicillium simplicissimum.

Despite its relatively low pH and temperature optimum, the xylanase from Penicillium simplicissimum performs exceedingly well under conditions of paper bleaching. We have purified and characterized this enzyme, which belongs to family 10 of glycosyl hydrolases. Its gene was cloned, and the sequence of the protein was deduced from the nucleotide sequence. The xylanase was crystallized from ammonium sulfate at pH 8.4, and X-ray data were collected at cryo-temperature to a crystallographic resolution of 1.75 A. The crystal structure was solved by molecular replacement using the catalytic domain of the Clostridium thermocellum xylanase as a search model, and refined to a residual of R = 20% (R(free) = 23%) for data between 10 and 1.75 A. The xylanase folds in an (alpha/beta)8 barrel (TIM-barrel), with additional helices and loops arranged at the "top" forming the active site cleft. In its overall shape, the P. simplicissimum xylanase structure is similar to other family 10 xylanases, but its active site cleft is much shallower and wider. This probably accounts for the differences in catalysis and in the mode of action of this enzyme. Three glycerol molecules were observed to bind within the active site groove, one of which interacts directly with the catalytic glutamate residues. It appears that they occupy putative xylose binding subsites.     

Lateral gene transfer of a dermonecrotic toxin between spiders and bacteria

MOTIVATION: Spiders in the genus Loxosceles, including the notoriously toxic brown recluse, cause severe necrotic skin lesions owing to the presence of a venom enzyme called sphingomyelinase D (SMaseD). This enzyme activity is unknown elsewhere in the animal kingdom but is shared with strains of pathogenic Corynebacteria that cause various illnesses in farm animals. The presence of the same toxic activity only in distantly related organisms poses an interesting and medically important question in molecular evolution. Results: We use superpositions of recently determined structures and sequence comparisons to infer that both bacterial and spider SMaseDs originated from a common, broadly conserved domain family, the glycerophosphoryl diester phosphodiesterases. We also identify a unique sequence/structure motif present in both SMaseDs but not in the ancestral family, supporting SMaseD origin through a single divergence event in either bacteria or spiders, followed by lateral gene transfer from one lineage to the other.     

Gene cloning and characterization of a novel alpha-amylase from alkaliphilic Alkalimonas amylolytica

A gene encoding an extracellular alpha-amylase (AmyA) was cloned from the alkaliphilic bacterium Alkalimonas amylolytica by enzymatic activity screening in Escherichia coli DH5alpha. The gene amyA consists of 1764 base pairs and was predicted to encode a 587-amino acid protein encompassing a 31-amino acid signal peptide. In addition, a 459-amino acid catalytic domain and a 97-amino acid starch-binding domain (SBD) were found. The SBD showed little similarity to other known SBDs; instead, it contains conserved amino acids typically belonging to the carbohydrate-binding module (CBM) family 20. AmyA could act on both granular and gelatinized starch. The catalytic domain of the enzyme showed little similarity to other known alpha-amylases. Rather, AmyA contains four characteristic conserved regions of glycoside hydrolase family 13. The recombinant enzyme was a liquefying enzyme with the highest activity at 50 degrees C and pH 9.5. The enzyme displayed a unique endo-product profile and action pattern on soluble starch to yield a series of malto-oligosaccharides ranging from maltose to maltoheptaose. The activity of the enzyme was enhanced by Co(2+), but not affected by 5 mM EDTA. Taken together, AmyA from A. amylolytica has potential to be used in paper, textile, detergent and other industries where starch needs to be degraded in an alkaline environment.     

Characterization of a novel endo-levanase and its gene from Bacillus sp. L7

A novel endo-levanase producing bacterium belonging to the Bacillus family has been isolated from soil. The enzyme was characterized and found to have no exo-β-fructofuranosidase activity. The endo-levanase gene was cloned and sequenced. Homology searches have shown that the C-terminal domain of the enzyme is homologous to a number of known β-fructofuranosidases, including exo-levanase from Bacillus subtilis and yeast invertases. The N-terminal region of the endo-levanase which is homologous to the C-terminal sequence of the B. subtilis levanase appears to be a levan-binding domain.     

Evolution and function of the sucrose-phosphate synthase gene families in wheat and other grasses

Suc-phosphate synthase (SPS) is a key regulatory enzyme in the pathway of Suc biosynthesis and has been linked to quantitative trait loci controlling plant growth and yield. In dicotyledonous plants there are three SPS gene families: A, B, and C. Here we report the finding of five families of SPS genes in wheat (Triticum aestivum) and other monocotyledonous plants from the family Poaceae (grasses). Three of these form separate subfamilies within the previously described A, B, and C gene families, but the other two form a novel and distinctive D family, which on present evidence is only found in the Poaceae. The D-type SPS proteins lack the phosphorylation sites associated with 14-3-3 protein binding and osmotic stress activation, and the linker region between the N-terminal catalytic glucosyltransferase domain and the C-terminal Suc-phosphatase-like domain is 80 to 90 amino acid residues shorter than in the A, B, or C types. The D family appears to have arisen after the divergence of mono- and dicotyledonous plants, with a later duplication event resulting in the two D-type subfamilies. Each of the SPS gene families in wheat showed different, but overlapping, spatial and temporal expression patterns, and in most organs at least two different SPS genes are expressed. Analysis of expressed sequence tags indicated similar expression patterns to wheat for each SPS gene family in barley (Hordeum vulgare) but not in more distantly related grasses. We identified an expressed sequence tag from rice (Oryza sativa) that appears to be derived from an endogenous antisense SPS gene, and this might account for the apparently low level of expression of the related OsSPS11 sense gene, adding to the already extensive list of mechanisms for regulating the activity of SPS in plants.     

Overexpression and characterization of a novel chitinase gene from a marine bacterium Pseudomonas sp. BK1

The chitinase A (ChiA)-coding gene of Pseudomonas sp. BK1, which was isolated from a marine red alga Porphyra dentata, was cloned and expressed in Escherichia coli. The structural gene consists of 1602 bp encoding a protein of 534 amino acids, with a predicted molecular weight of 55,370 Da. The deduced amino acid sequence of ChiA showed low identity (less than 32%) with other bacterial chitinases. The ChiA was composed of multiple domains, unlike the arrangement of domains in other bacterial chitinases. Recombinant ChiA overproduced as inclusion bodies was solubilized in the presence of 8 M urea, purified in a urea-denatured form and re-folded by removing urea. The purified enzyme showed maximum activity at pH 5.0 and 40 degrees C. It exhibited high activity towards glycol chitosan and glycol chitin, and lower activity towards colloidal chitin. The enzyme hydrolyzed the oligosaccharides from (GlcNAc)4 to (GlcNAc)6, but not GlcNAc to (GlcNAc)3. The results suggest that the ChiA is a novel enzyme, with different domain structure and action mode from bacterial family 18 chitinases.     

Action of Cyclodextrin Glycosyltransferase from Bacillus megaterium Strain No. 5 on Starch

The amounts of the cyclodextrins G6, G7 and G8 produced by the action of the enzyme from Bacillus megaterium (No. 5 enzyme) and Bacillus macerans amylase (BMA) on starch-¹⁴C (U) were determined by the calculation of radioactivity. Both fractions of No. 5 enzyme produced the cyclodextrin G6, G7 and G8 in the proportion of 1: 2.4: 1. On the other hand, BMA produced the cyclodextrin G6, G7 and G8 in the proportion of 2.7: 1:1. The cyclodextrin G6 and G8 which are smaller parts of the reaction products by both fractions of No. 5 enzyme were found to be produced directly from starch, not from the redecomposition of cyclodextrin G7. The ratio of the cyclodextrin G6, G7 and G8 were almost constant, regardless of the pH range of the reaction system.

By using the maltooligosaccharides terminated at the reducing end by radioactive glucose, the action of both fractions of No. 5 enzyme and BMA on the maltooligosaccharides were compared with each other. The results showed that both fractions of No. 5 enzyme acted on oligosaccharides larger than maltose, producing the radioactive glucose as the major product from each maltooligosaccharide (G2~G8). On the other hand, BMA acted on oligosaccharides larger than maltotriose, producing the radioactive maltose as the major product.     

Biochemical and genetic properties of Paenibacillus glycosyl hydrolase having chitosanase activity and discoidin domain

Cells of "Paenibacillus fukuinensis" D2 produced chitosanase into surrounding medium, in the presence of colloidal chitosan or glucosamine. The gene of this enzyme was cloned, sequenced, and subjected to site-directed mutation and deletion analyses. The nucleotide sequence indicated that the chitosanase was composed of 797 amino acids and its molecular weight was 85,610. Unlike conventional family 46 chitosanases, the enzyme has family 8 glycosyl hydrolase catalytic domain, at the amino-terminal side, and discoidin domain at the carboxyl-terminal region. Expression of the cloned gene in Escherichia coli revealed beta-1,4-glucanase function, besides chitosanase activity. Analyses by zymography and immunoblotting suggested that the active enzyme was, after removal of signal peptide, produced from inactive 81-kDa form by proteolysis at the carboxyl-terminal region. Replacements of Glu(115) and Asp(176), highly conserved residues in the family 8 glycosylase region, with Gln and Asn caused simultaneous loss of chitosanase and glucanase activities, suggesting that these residues formed part of the catalytic site. Truncation experiments demonstrated indispensability of an amino-terminal region spanning 425 residues adjacent to the signal peptide.     

Bioinformatics Characterization of Potential New Beta-Glucuronidase from <Emphasis Type="Italic">Streptococcus</Emphasis> <Emphasis Type="Italic">equi</Emphasis> subsp. <Emphasis Type="Italic">zooepidemicus</Emphasis>

Recently, the gene coding for a new beta-glucuronidase enzyme has been identified and cloned from Streptococcus equi subsp. zooepidemicus. This is another report of a beta-glucuronidase gene cloned from bacterial species. The ORF Finder analysis of a sequenced DNA (EMBL, AJ890474) revealed a presence of 1,785 bp large ORF potentially coding for a 594 aa protein. Three protein families in (Pfam) domains were identified using the Conserved Domain Database (CDD) analysis: Pfam 02836, glycosyl hydrolases family 2, triose phosphate isomerase (TIM) barrel domain; Pfam 02837, glycosyl hydrolases family 2, sugar binding domain; and Pfam 00703, glycosyl hydrolases family 2, immunoglobulin-like beta-sandwich domain. To gain more insight into the enzymatic activity, the domains were used to generate a bootstrapped unrooted distance tree using ClustalX. The calculated distances for two domains, TIM barrel domain, and sugar-binding domain were comparable and exhibited similarity pattern based on function and thus being in accordance with recently published works confirming beta-glucuronidase activity of the enzyme. The calculated distances and the tree arrangement in the case of centrally positioned immonoglobulin-like beta-sandwich domain were somewhat higher when compared to other two domains but clustering with other beta-glucuronidases was rather clear. Nine proteins, including beta-glucuronidases, beta-galactosidase, and mannosidase were selected for multiple alignment and subsequent distance tree creation.     

A novel beta-N-acetylglucosaminidase of Clostridium paraputrificum M-21 with high activity on chitobiose

A beta- N-acetylglucosaminidase gene ( nag3A) from Clostridium paraputrificum M-21 was cloned in Escherichia coli. The nag3A gene consists of an open reading frame of 1,239-bp, encoding 413 amino acids with a deduced molecular weight of 45,531 Da. Nag3A is a single domain enzyme containing a family 3 glycoside hydrolase catalytic domain. Nag3A was purified from recombinant E. coli and characterized. The enzyme hydrolyzed chitooligomers such as di- N-acetylchitobiose, tri- N-acetylchitotriose, tetra- N-acetylchitotetraose, penta- N-acetylchitopentaose, hexa- N-acetylchitohexaose, ball-milled chitin, and synthetic substrates such as 4-methylumbelliferyl N-acetyl beta- D-glucosaminide [4-MU-(GlcNAc)], but had no activity at all against p-nitrophenyl-beta- D-glucoside, p-nitrophenyl-beta- D-xyloside, or p-nitrophenyl-beta- D-galactosamine. The enzyme was optimally active at 50 degrees C and pH 7.0, and the apparent K(m) and V(max) values for 4-MU-(GlcNAc) were 7.9 micro M and 21.8 micro mol min(-1) mg protein(-1), respectively. SDS-PAGE, zymogram, and immunological analyses suggested that this enzyme is induced by ball-milled chitin.