Structural characterization of linear isomalto-/malto-oligomer products synthesized by the novel GTFB 4,6-α-glucanotransferase enzyme from Lactobacillus reuteri 121

Recently, a novel glucansucrase (GS)-like gene (gtfB) was isolated from the probiotic bacterium Lactobacillus reuteri 121 and expressed in Escherichia coli. The purified recombinant GTFB enzyme was characterized and turned out to be inactive with sucrose, the natural GS substrate. Instead, GTFB acted on malto-oligosaccharides (MOSs), thereby yielding elongated gluco-oligomers/polymers containing besides (α1?→?4) also (α1?→?6) glycosidic linkages, and it was classified as a 4,6-α-glucanotransferase. To gain more insight into its reaction specificity, incubations of the GTFB enzyme with a series of MOSs and their corresponding alditols [degree of polymerization, DP2(-ol)-DP7(-ol)] were carried out, and (purified) products were structurally analyzed with matrix-assisted laser desorption ionization time-of-flight mass spectrometry and one-/two-dimensional (1)H and (13)C nuclear magnetic resonance spectroscopy. With each of the tested malto-oligomers, the GTFB enzyme yielded series of novel linear isomalto-/malto-oligomers, in the case of DP7 up to DP >35.     

Crystal Structure and Characterization of the Glycoside Hydrolase Family 62 α-l-Arabinofuranosidase from Streptomyces coelicolor

α-l-Arabinofuranosidase, which belongs to the glycoside hydrolase family 62 (GH62), hydrolyzes arabinoxylan but not arabinan or arabinogalactan. The crystal structures of several α-l-arabinofuranosidases have been determined, although the structures, catalytic mechanisms, and substrate specificities of GH62 enzymes remain unclear. To evaluate the substrate specificity of a GH62 enzyme, we determined the crystal structure of α-l-arabinofuranosidase, which comprises a carbohydrate-binding module family 13 domain at its N terminus and a catalytic domain at its C terminus, from Streptomyces coelicolor. The catalytic domain was a five-bladed β-propeller consisting of five radially oriented anti-parallel β-sheets. Sugar complex structures with l-arabinose, xylotriose, and xylohexaose revealed five subsites in the catalytic cleft and an l-arabinose-binding pocket at the bottom of the cleft. The entire structure of this GH62 family enzyme was very similar to that of glycoside hydrolase 43 family enzymes, and the catalytically important acidic residues found in family 43 enzymes were conserved in GH62. Mutagenesis studies revealed that Asp²⁰² and Glu³⁶¹ were catalytic residues, and Trp²⁷⁰, Tyr⁴⁶¹, and Asn⁴⁶² were involved in the substrate-binding site for discriminating the substrate structures. In particular, hydrogen bonding between Asn⁴⁶² and xylose at the nonreducing end subsite +2 was important for the higher activity of substituted arabinofuranosyl residues than that for terminal arabinofuranoses.     

Structural and Biochemical Analyses of Glycoside Hydrolase Family 26 β-Mannanase from a Symbiotic Protist of the Termite Reticulitermes speratus

Termites and their symbiotic protists have established a prominent dual lignocellulolytic system, which can be applied to the biorefinery process. One of the major components of lignocellulose from conifers is glucomannan, which comprises a heterogeneous combination of β-1,4-linked mannose and glucose. Mannanases are known to hydrolyze the internal linkage of the glucomannan backbone, but the specific mechanism by which they recognize and accommodate heteropolysaccharides is currently unclear. Here, we report biochemical and structural analyses of glycoside hydrolase family 26 mannanase C (RsMan26C) from a symbiotic protist of the termite Reticulitermes speratus. RsMan26C was characterized based on its catalytic efficiency toward glucomannan, compared with pure mannan. The crystal structure of RsMan26C complexed with gluco-manno-oligosaccharide(s) explained its specificities for glucose and mannose at subsites −5 and −2, respectively, in addition to accommodation of both glucose and mannose at subsites −3 and −4. RsMan26C has a long open cleft with a hydrophobic platform of Trp⁹⁴ at subsite −5, facilitating enzyme binding to polysaccharides. Notably, a unique oxidized Met⁸⁵ specifically interacts with the equatorial O-2 of glucose at subsite −3. Our results collectively indicate that specific recognition and accommodation of glucose at the distal negative subsites confers efficient degradation of the heteropolysaccharide by mannanase.     

4,6-α-Glucanotransferase activity occurs more widespread in Lactobacillus strains and constitutes a separate GH70 subfamily

Family 70 glycoside hydrolase glucansucrase enzymes exclusively occur in lactic acid bacteria and synthesize a wide range of α-d-glucan (abbreviated as α-glucan) oligo- and polysaccharides. Of the 47 characterized GH70 enzymes, 46 use sucrose as glucose donor. A single GH70 enzyme was recently found to be inactive with sucrose and to utilize maltooligosaccharides [(1→4)-α-d-glucooligosaccharides] as glucose donor substrates for α-glucan synthesis, acting as a 4,6-α-glucanotransferase (4,6-αGT) enzyme. Here, we report the characterization of two further GH70 4,6-αGT enzymes, i.e., from Lactobacillus reuteri strains DSM 20016 and ML1, which use maltooligosaccharides as glucose donor. Both enzymes cleave α1→4 glycosidic linkages and add the released glucose moieties one by one to the non-reducing end of growing linear α-glucan chains via α1→6 glycosidic linkages (α1→4 to α1→6 transfer activity). In this way, they convert pure maltooligosaccharide substrates into linear α-glucan product mixtures with about 50% α1→6 glycosidic bonds (isomalto/maltooligosaccharides). These new α-glucan products may provide an exciting type of carbohydrate for the food industry. The results show that 4,6-αGTs occur more widespread in family GH70 and can be considered as a GH70 subfamily. Sequence analysis allowed identification of amino acid residues in acceptor substrate binding subsites +1 and +2, differing between GH70 GTF and 4,6-αGT enzymes.     

Crystal Structure of an Exo-1,5-α-l-arabinofuranosidase from Streptomyces avermitilis Provides Insights into the Mechanism of Substrate Discrimination between Exo- and Endo-type Enzymes in Glycoside Hydrolase Family 43

Exo-1,5-α-l-arabinofuranosidases belonging to glycoside hydrolase family 43 have strict substrate specificity. These enzymes hydrolyze only the α-1,5-linkages of linear arabinan and arabino-oligosaccharides in an exo-acting manner. The enzyme from Streptomyces avermitilis contains a core catalytic domain belonging to glycoside hydrolase family 43 and a C-terminal arabinan binding module belonging to carbohydrate binding module family 42. We determined the crystal structure of intact exo-1,5-α-l-arabinofuranosidase. The catalytic module is composed of a 5-bladed β-propeller topologically identical to the other family 43 enzymes. The arabinan binding module had three similar subdomains assembled against one another around a pseudo-3-fold axis, forming a β-trefoil-fold. A sugar complex structure with α-1,5-l-arabinofuranotriose revealed three subsites in the catalytic domain, and a sugar complex structure with α-l-arabinofuranosyl azide revealed three arabinose-binding sites in the carbohydrate binding module. A mutagenesis study revealed that substrate specificity was regulated by residues Asn-159, Tyr-192, and Leu-289 located at the aglycon side of the substrate-binding pocket. The exo-acting manner of the enzyme was attributed to the strict pocket structure of subsite −1, formed by the flexible loop region Tyr-281–Arg-294 and the side chain of Tyr-40, which occupied the positions corresponding to the catalytic glycon cleft of GH43 endo-acting enzymes.     

Gene Cloning and Heterologous Expression of Glycoside Hydrolase Family 55 β-1,3-Glucanase from the Basidiomycete Phanerochaete Chrysosporium

The basidiomycete Phanerochaete chrysosporium produces several β-1,3-glucanases when grown on laminarin, a β-1,3/1,6-glucan, as the sole carbon source. To characterize one of the major unknown β-1, 3-glucanases with a molecular mass of 83 kDa, identification, cloning, and heterologous over-expression were carried out using the total genomic information of P. chrysosporium. The cDNA encoding this enzyme included an ORF of 2337 bp and the deduced amino acid sequence contains a predicted signal peptide of 26 amino acids and the mature protein of 752 amino acids. The amino acid sequence showed a significant similarity with glycoside hydrolase family 55 enzymes from filamentous fungi and was named Lam55A. Since the recombinant Lam55A expressed in the methylotrophic yeast Pichia pastoris degraded branched β-1,3/1,6-glucan as well as linear β-1,3-glucan, the kinetic features of the enzyme were compared with those of other β-1,3-glucanases.     

Cloning and Characterization of the Glycoside Hydrolases That Remove Xylosyl Groups from 7-β-xylosyl-10-deacetyltaxol and Its Analogues

Paclitaxel, a natural antitumor compound, is produced by yew trees at very low concentrations, causing a worldwide shortage of this important anticancer medicine. These plants also produce significant amounts of 7-β-xylosyl-10-deacetyltaxol, which can be bio-converted into 10-deacetyltaxol for the semi-synthesis of paclitaxel. Some microorganisms can convert 7-β-xylosyl-10-deacetyltaxol into 10-deacetyltaxol, but the bioconversion yield needs to be drastically improved for industrial applications. In addition, the related β-xylosidases of these organisms have not yet been defined. We set out to discover an efficient enzyme for 10-deacetyltaxol production. By combining the de novo sequencing of β-xylosidase isolated from Lentinula edodes with RT-PCR and the rapid amplification of cDNA ends, we cloned two cDNA variants, Lxyl-p1–1 and Lxyl-p1–2, which were previously unknown at the gene and protein levels. Both variants encode a specific bifunctional β-d-xylosidase/β-d-glucosidase with an identical ORF length of 2412 bp (97% identity). The enzymes were characterized, and their 3.6-kb genomic DNAs (G-Lxyl-p1–1, G-Lxyl-p1–2), each harboring 18 introns, were also obtained. Putative substrate binding motifs, the catalytic nucleophile, the catalytic acid/base, and potential N-glycosylation sites of the enzymes were predicted. Kinetic analysis of both enzymes showed kcat/K_m of up to 1.07 s⁻¹mm⁻¹ against 7-β-xylosyl-10-deacetyltaxol. Importantly, at substrate concentrations of up to 10 mg/ml (oversaturated), the engineered yeast could still robustly convert 7-β-xylosyl-10-deacetyltaxol into 10-deacetyltaxol with a conversion rate of over 85% and a highest yield of 8.42 mg/ml within 24 h, which is much higher than those reported previously. Therefore, our discovery might lead to significant progress in the development of new 7-β-xylosyl-10-deacetyltaxol-converting enzymes for more efficient use of 7-β-xylosyltaxanes to semi-synthesize paclitaxel and its analogues. This work also might lead to further studies on how these enzymes act on 7-β-xylosyltaxanes and contribute to the growing database of glycoside hydrolases.The protection and sustainable utilization of natural resources are among the most important and global problems of the 21st century. Paclitaxel (Taxol^®) is mainly isolated from slow-growing yew trees (genus Taxus, family Taxaceae) and is known as a “blockbuster drug ” showing unique active mechanisms (1), with prominent activity against various cancers (including ovarian, breast, lung, head, and neck carcinomas and the AIDS-related Kaposi sarcoma) (2). However, the source of paclitaxel has always been a top concern, because its content in the plant is extremely low, and it is isolated in “large ” amounts (∼0.02%) only from the bark of the tree (3). A 100-year-old tree might yield 3 kg of bark, which provides enough paclitaxel for one 300-mg dose (4). To preserve the Taxus resource and alleviate some of the pressure on the source, several approaches have been employed to prepare paclitaxel or its analog Taxotere, including chemical semi-synthesis from the precursor 10-deacetylbaccatin III (DB),¹ which is readily available from the twigs of yew trees such as Taxus baccata (5, 6); isolation from the twigs of nursery trees including T. chinensis var. mairei and T. media (hybrid); paclitaxel-producing endophytic strain fermentation (7, 8); and Taxus cell and tissue culture (9). The first two approaches might partially relieve this pressure, but they still cannot meet the growing market demand.7-β-xylosyltaxanes are much more abundant and are extracted simultaneously with paclitaxel and DB from various species of yew (10–12), but generally they are dealt with as byproducts. Among these analogues, 7-β-xylosyl-10-deacetyltaxol (XDT) can be obtained with a yield of as much as 0.5% (from dried stem bark) (13). These 7-β-xylosyltaxanes can be hydrolyzed via chemical or biological methods to give the corresponding 7-hydroxyltaxanes, including 10-deacetyltaxol (DT) and DB, for the semi-synthesis of paclitaxel. In contrast to the chemical approach, which utilizes periodate or other oxidizing agents and a substituted hydrazine in the reactions to remove the sugar, the biological approach is an enzymatic process that releases the d-xylose from 7-xylosyltaxanes through the specific β-xylosidase and is therefore considered to be environmentally friendly.β-xylosidases (EC3.2.1.37) belong to glycoside hydrolase (GH) or glycosidase (EC3.2.1.X) families 3, 30, 39, 43, 52, and 54 (14). However, the filamentous fungal β-xylosidases have hitherto been described as belonging only to GH families 3, 43, and 54 (15). Many kinds of β-xylosidases have been purified from different organisms, such as bacteria (16–19), fungi (20–23), and plants (24, 25). Some β-xylosidase genes, such as those from bacteria (18, 19, 26, 27) or from fungi (23, 28–30), have been cloned and characterized. However, none of these enzymes have been reported to be active against 7-β-xylosyltaxanes. In fact, a lot of commercially available xylosidases, xylanases, and other glycosidases do not have any activity specific for removing xylose from 7-β-xylosyltaxanes (31). Some bacterial isolates, such as Moraxella sp. (ATCC 55475) (31, 32), Cellulosimicrobium cellulans XZ-5 (CCTCC No. M207130) (33), and Enterobacter sp. (CGMCC 2487) (34), have been reported to have the ability to convert XDT to DT. But these strains gave low yields of DT (0.23, 0.4, and 0.76 mg/ml, respectively (31–34)), which is probably due to the ubiquitous low enzyme levels in the native organisms. The related β-xylosidases of these organisms have not yet been defined. Our lab discovered that a fungal species, Lentinula edodes, could transform XDT into DT, but a similarly low yield was also observed (supplemental Fig. S1). Thus, cloning and characterization of the specific enzyme from the fungus might lead to a new biocatalytic route of preparation for 7-hylosyltaxanes for the semi-synthesis of paclitaxel or its analogues. Here, we present a strategy in which we combine protein de novo sequencing with RT-PCR and the rapid amplification of cDNA ends (RACE) to mine the targeted β-xylosidase gene from this fungus. Moreover, yeast engineered with such a heterologous gene can robustly convert 7-β-xylosyltaxanes into 7-hydroxyltaxanes.     

Characterization of Enzymes in the Oxidation of 1,2-Propanediol to <Emphasis Type="SmallCaps">d</Emphasis>-(−)-Lactic Acid by <Emphasis Type="Italic">Gluconobacter oxydans</Emphasis> DSM 2003

Although Gluconobacter oxydans can convert 1,2-propanediol to d-(−)-lactic acid, the enzyme(s) responsible for the conversion has remain unknown. In this study, the membrane-bound alcohol dehydrogenase (ADH) of Gluconobacter oxydans DSM 2003 was purified and confirmed to be essential for the process of d-(−)-lactic acid production by gene knockout and complementation studies. A 25 percent decrease in d-(−)-lactic acid production was found for the aldehyde dehydrogenase (ALDH) deficient strain of G. oxydans DSM 2003, indicating that this enzyme is involved in the reaction but not necessary. It is the first report that reveals the function of ADH and ALDH in the biooxidation of 1,2-propanediol to d-(−)-lactic acid by G. oxydans DSM 2003.