X-ray structure of acarbose bound to amylomaltase from Thermus aquaticus. Implications for the synthesis of large cyclic glucans.

As a member of the alpha-amylase superfamily of enzymes, amylomaltase catalyzes either the transglycosylation from one alpha-1,4 glucan to another or an intramolecular cyclization. The latter reaction is typical for cyclodextrin glucanotransferases. In contrast to these enzymes, amylomaltase catalyzes the formation of cyclic glucans with a degree of polymerization larger than 22. To characterize the factors that determine the size of the synthesized cycloamyloses, we have analyzed the X-ray structure of amylomaltase from Thermus aquaticus in complex with the inhibitor acarbose, a maltotetraose derivative, at 1.9 A resolution. Two acarbose molecules are bound to the enzyme, one in the active site groove at subsite -3 to +1 and a second one approximately 14 A away from the nonreducing end of the acarbose bound to the catalytic site. The inhibitor bound to the catalytic site occupies subsites -3 to +1. Unlike the situation in other enzymes of the alpha-amylase family, the inhibitor is not processed and the inhibitory cyclitol ring of acarbose, which mimicks the half chair conformation of the transition state, does not bind to catalytic subsite -1. The minimum ring size of cycloamyloses produced by this enzyme is proposed to be determined by the distance of the specific substrate binding sites at the active site and near Tyr54 and by the size of the 460s loop. The 250s loop might be involved in binding of the substrate at the reducing end of the scissile bond.     

Crystal structure of a UDP-glucose-specific glycosyltransferase from a Mycobacterium species

Glycosyltransferases (GTs) are a large and ubiquitous family of enzymes that specifically transfer sugar moieties to a range of substrates. Mycobacterium tuberculosis contains a large number of GTs, many of which are implicated in cell wall synthesis, yet the majority of these GTs remain poorly characterized. Here, we report the high resolution crystal structures of an essential GT (MAP2569c) from Mycobacterium avium subsp. paratuberculosis (a close homologue of Rv1208 from M. tuberculosis) in its apo- and ligand-bound forms. The structure adopted the GT-A fold and possessed the characteristic DXD motif that coordinated an Mn(2+) ion. Atypical of most GTs characterized to date, MAP2569c exhibited specificity toward the donor substrate, UDP-glucose. The structure of this ligated complex revealed an induced fit binding mechanism and provided a basis for this unique specificity. Collectively, the structural features suggested that MAP2569c may adopt a "retaining" enzymatic mechanism, which has implications for the classification of other GTs in this large superfamily.     

Characterization of crystals of the thermostable DNA polymerase I from thermus aquaticus

Thermus aquaticus DNA polymerase I is an enzyme that is of both physiological and technological interest. It carries out template-directed polymerization of DNA at elevated temperatures and is widely used in polymerase chain reaction (PCR). We have obtained crystals of the enzyme that diffracts X-rays to at least 3.0 Å resolution in a cubic space group. Determination of the three-dimensional structure of the native enzyme along with those of relevant complexes will greatly enhance our knowledge of molecular events involved in DNA replication, will permit improvements in PCR, and will add to our knowledge of the structural bases of thermo stability in proteins. © 1995 Wiley-Liss, Inc.     

Thermus aquaticus ATCC 33923 amylomaltase gene cloning and expression and enzyme characterization: production of cycloamylose

The amylomaltase gene of the thermophilic bacterium Thermus aquaticus ATCC 33923 was cloned and sequenced. The open reading frame of this gene consisted of 1,503 nucleotides and encoded a polypeptide that was 500 amino acids long and had a calculated molecular mass of 57,221 Da. The deduced amino acid sequence of the amylomaltase exhibited a high level of homology with the amino acid sequence of potato disproportionating enzyme (D-enzyme) (41%) but a low level of homology with the amino acid sequence of the Escherichia coli amylomaltase (19%). The amylomaltase gene was overexpressed in E. coli, and the enzyme was purified. This enzyme exhibited maximum activity at 75 degrees C in a 10-min reaction with maltotriose and was stable at temperatures up to 85 degrees C. When the enzyme acted on amylose, it catalyzed an intramolecular transglycosylation (cyclization) reaction which produced cyclic alpha-1,4-glucan (cycloamylose), like potato D-enzyme. The yield of cycloamylose produced from synthetic amylose with an average molecular mass of 110 kDa was 84%. However, the minimum degree of polymerization (DP) of the cycloamylose produced by T. aquaticus enzyme was 22, whereas the minimum DP of the cycloamylose produced by potato D-enzyme was 17. The T. aquaticus enzyme also catalyzed intermolecular transglycosylation of maltooligosaccharides. A detailed analysis of the activity of T. aquaticus ATCC 33923 amylomaltase with maltooligosaccharides indicated that the catalytic properties of this enzyme differ from those of E. coli amylomaltase and the plant D-enzyme.     

Use of random and saturation mutageneses to improve the properties of Thermus aquaticus amylomaltase for efficient production of cycloamyloses

Amylomaltase from Thermus aquaticus catalyzes intramolecular transglycosylation of alpha-1,4 glucans to produce cyclic alpha-1,4 glucans (cycloamyloses) with degrees of polymerization of 22 and higher. Although the amylomaltase mainly catalyzes the transglycosylation reaction, it also has weak hydrolytic activity, which results in a reduction in the yield of the cycloamyloses. In order to obtain amylomaltase with less hydrolytic activity, random mutagenesis was perfromed for the enzyme gene. Tyr54 (Y54) was identified as the amino acid involved in the hydrolytic activity of the enzyme. When Y54 was replaced with all other amino acids by site-directed mutagenesis, the hydrolytic activities of the mutated enzymes were drastically altered. The hydrolytic activities of the Y54G, Y54P, Y54T, and Y54W mutated enzymes were remarkably reduced compared with that of the wild-type enzyme, while those of the Y54F and Y54K mutated enzymes were similar to that of the wild-type enzyme. Introducing an amino acid replacement at Y54 also significantly affected the cyclization activity of the amylomaltase. The Y54A, Y54L, Y54R, and Y54S mutated enzymes exhibited cyclization activity that was approximately twofold higher than that of the wild-type enzyme. When the Y54G mutated enzyme was employed for cycloamylose production, the yield of cycloamyloses was more than 90%, and there was no decrease until the end of the reaction.     

Molecular cloning of the gene,expression in E. coli and purification of the thermus aquaticus DNA polymerase I

The Thermus aquaticus DNA Polymerase I gene was isolated by using standard recombinant DNA techniques and cloned into an E. coli vector in front of the lac promoter. E. coli cultures produced thermostable polymerase when induced with the analog IPTG. A purification procedure is shown which renders an enzymatic preparation suitable for PCR reactions.     

Crystal structures of a multifunctional triterpene/flavonoid glycosyltransferase from Medicago truncatula

Glycosylation is a ubiquitous reaction controlling the bioactivity and storage of plant natural products. Glycosylation of small molecules is catalyzed by a superfamily of glycosyltransferases (GTs) in most plant species studied to date. We present crystal structures of the UDP flavonoid/triterpene GT UGT71G1 from Medicago truncatula bound to UDP or UDP-glucose. The structures reveal the key residues involved in the recognition of donor substrate and, by comparison with other GT structures, suggest His-22 as the catalytic base and Asp-121 as a key residue that may assist deprotonation of the acceptor by forming an electron transfer chain with the catalytic base. Mutagenesis confirmed the roles of these key residues in donor substrate binding and enzyme activity. Our results provide an initial structural basis for understanding the complex substrate specificity and regiospecificity underlying the glycosylation of plant natural products and other small molecules. This information will direct future attempts to engineer bioactive compounds in crop plants to improve plant, animal, and human health and to facilitate the rational design of GTs to improve the storage and stability of novel engineered bioactive compounds.     

Crystal structure of SsfS6, the putative C‐glycosyltransferase involved in SF2575 biosynthesis

The molecule known as SF2575 from Streptomyces sp. is a tetracycline polyketide natural product that displays antitumor activity against murine leukemia P388 in vivo. In the SF2575 biosynthetic pathway, SsfS6 has been implicated as the crucial C‐glycosyltransferase (C‐GT) that forms the C‐C glycosidic bond between the sugar and the SF2575 tetracycline‐like scaffold. Here, we report the crystal structure of SsfS6 in the free form and in complex with TDP, both at 2.4 Å resolution. The structures reveal SsfS6 to adopt a GT‐B fold wherein the TDP and docked putative aglycon are consistent with the overall C‐glycosylation reaction. As one of only a few existing structures for C‐glycosyltransferases, the structures described herein may serve as a guide to better understand and engineer C‐glycosylation. Proteins 2013; 81:1277–1282. © 2013 Wiley Periodicals, Inc.     

Crystal structure of cyclic Lys48-linked tetraubiquitin

Lys48-linked polyubiquitin chains serve as a signal for protein degradation by 26S proteasomes through its Ile44 hydrophobic patches interactions. The individual ubiquitin units of each chain are conjugated through an isopeptide bond between Lys48 and the C-terminal Gly76 of the preceding units. The conformation of Lys48-linked tetraubiquitin has been shown to change dynamically depending on solution pH. Here we enzymatically synthesized a wild-type Lys48-linked tetraubiquitin for structural study. In the synthesis, cyclic and non-cyclic species were obtained as major and minor fractions, respectively. This enabled us to solve the crystal structure of tetraubiquitin exclusively with native Lys48-linkages at 1.85 Å resolution in low pH 4.6. The crystallographic data clearly showed that the C-terminus of the first ubiquitin is conjugated to the Lys48 residue of the fourth ubiquitin. The overall structure is quite similar to the closed form of engineered tetraubiquitin at near-neutral pH 6.7, previously reported, in which the Ile44 hydrophobic patches face each other. The structure of the second and the third ubiquitin units [Ub(2)-Ub(3)] connected through a native isopeptide bond is significantly different from the conformations of the corresponding linkage of the engineered tetraubiquitins, whereas the structures of Ub(1)-Ub(2) and Ub(3)-Ub(4) isopeptide bonds are almost identical to those of the previously reported structures. From these observations, we suggest that the flexible nature of the isopeptide linkage thus observed contributes to the structural arrangements of ubiquitin chains exemplified by the pH-dependent closed-to-open conformational transition of tetraubiquitin.     

Direct cloning of gene encoding a novel amylomaltase from soil bacterial DNA for large-ring cyclodextrin production

The aim of this study was to isolate a novel amylomaltase gene from community DNA of soil samples collected from Ban Nong Khrok hot spring in Thailand without bacterial cultivation. Using PCR, a 1.5 kb full-length gene was amplified and ligated with pGEM®-T easy vector to transform into Escherichia coli DH5 α for sequencing. The obtained gene encoding an amylomaltase consisted of 1,503 bp that translated into 500 amino acids. Amino acid sequence deduced from this gene was highly homologous with that of amylomaltase from Thermus thermophillus ATCC 33923. In order to express the enzyme, the cloned gene was subcloned into plasmid pET-17b and introduced into E. coli BL21(DE3). The maximum expression was observed when the cloned cells were cultured at 37°C for 6 h with 0.5 mM IPTG induction. By 10% SDS-PAGE, the relative molecular mass of the purified amylomaltase was approximately 58 kDa. This enzyme was optimally active at 70°C and pH 9.0. In addition, the enzyme could hydrolyze pea starch to yield the largering cyclodextrins with degrees of polymerization of 23 and higher. It is noted that CD29 was the product in the largest quantity under all tested conditions.     

The production of glucans via glucansucrases from Lactobacillus satsumensis isolated from a fermented beverage starter culture

Several starter cultures used in the production of fermented beverages were screened for lactic acid bacteria that produced water-insoluble polysaccharides from sucrose. The strain producing the greatest amount was identified as Lactobacillus satsumensis by its 16S RNA sequence and was deposited in the ARS culture collection as NRRL B-59839. This strain produced at least two α-d-glucans from sucrose. One was a water-soluble dextran, consisting of predominantly α-(1?→?6)-linked d-glucose units, and the other was a water-insoluble glucan containing both α-(1?→?6)-linked and α-(1?→?3)-linked d-glucose units. The culture fluid was found to contain glucansucrases responsible for the two glucans, and no significant level of fructansucrase was detected. Glucansucrase activity was not present in the culture fluid when the bacteria were grown on glucose, fructose, or raffinose as the carbon source. Although the water-soluble glucans produced by cell-free enzyme and by cell suspensions were essentially identical, the same was not true for the water-insoluble glucans. The water-insoluble glucan produced by cell-free culture fluid contained a higher proportion of α-(1?→?3)-linked d-glucose units than the water-insoluble glucan produced by cell suspensions.     

Scaling-up the production of thermostable lipolytic enzymes from Thermus aquaticus YT1

The lipolytic enzymes synthesized by Thermusaquaticus YT1 present extremely interesting properties of thermostability (more than 70% of activity after 12 days at 80°C and a half-life time of 1 h at 95°C), which point out the interest of proposing efficient strategies to successfully tackle the scale-up of the production process. In this study,viable scaling-up of the production process was implemented,and relevant aspects affecting the enzyme synthesis, such as the mineral composition of the culture medium, the aeration and the agitation have been evaluated.A strategy combining the modification of the culture medium and the aeration degree was also approached by adding perfluorocarbons, compounds which improve the availability of oxygen in the culture medium. An opposite response of biomass and lipolytic activity to the aeration conditions was found between scales (about 600 U L(-1) at high aeration levels in flask vs. 150 U L(-1) at high aeration rates in reactor), which further demonstrates the important role of the hydrodynamic conditions on the suitable development of the biological process. In all cases, the cultures were kinetically characterized and the Luedeking and Piret model turned out to be a valuable tool to conclude that the produced lipolytic enzyme is a growth-associated metabolite, no matter the medium and the scale.     

Crystal structure of a cyclic AMP-independent mutant of catabolite gene activator protein

Escherichia coli NCR91 synthesizes a mutant form of catabolite gene activator protein (CAP) in which alanine 144 is replaced by threonine. This mutant, which also lacks adenylate cyclase activity, has a CAP phenotype; in the absence of cAMP it is able to express genes that normally require cAMP. CAP91 has been purified and crystallized with cAMP under the same conditions as used to crystallize the wild type CAP X cAMP complex. X-ray diffraction data were measured to 2.4-A resolution and the CAP91 structure was determined using initial model phases from the wild type structure. A difference Fourier map calculated between CAP91 and wild type showed the 2 alanine to threonine sequence changes in the dimer and also a change in orientation of cysteine 178 in one of the subunits. The CAP91 coordinates were refined by restrained least squares to an R factor of 0.186. Differences in the atomic positions of the wild type and mutant protein structures were analyzed by a vector averaging technique. There were small changes that included concerted motions in the small domains, in the hinge between the two domains and in an adjacent loop between beta-strands 4 and 5. The mutation at residue 144 apparently causes changes in the position of some protein atoms that are distal to the mutation site.     

Crystal structure of Mip, a prolylisomerase from Legionella pneumophila

The human pathogen Legionella pneumophila, the etiological agent of the severe and often fatal Legionnaires' disease, produces a major virulence factor, termed 'macrophage infectivity potentiator protein' (Mip), that is necessary for optimal multiplication of the bacteria within human alveolar macrophages. Mip exhibits a peptidyl prolyl cis-trans isomerase (PPIase) activity, which appears to be important for infection. Here we report the 2.4 A crystal structure of the Mip protein from L. pneumophila Philadelphia 1 and the 3.2 A crystal structure of its complex with the drug FK506. Each monomer of the homodimeric protein consists of an N-terminal dimerization module, a long (65 A) connecting alpha-helix and a C-terminal PPIase domain exhibiting similarity to human FK506-binding protein. In view of the recent significant increase in the number of reported cases of Legionnaires' disease and other intracellular infections, these structural results are of prime interest for the design of new drugs directed against Mip proteins of intracellular pathogens.     

Crystal structure of the platelet activator convulxin, a disulfide-linked alpha4beta4 cyclic tetramer from the venom of Crotalus durissus terrificus

Convulxin (CVX), a C-type lectin, isolated from the venom of the South American rattlesnake Crotalus durissus terrificus, causes cardiovascular and respiratory disturbances and is a potent platelet activator which binds to platelet glycoprotein GPVI. The structure of CVX has been solved at 2.4A resolution to a crystallographic residual of 18.6% (R(free)=26.4%). CVX is a disulfide linked heterodimer consisting of homologous alpha and beta chains. The heterodimers are additionally linked by disulfide bridges to form cyclic alpha(4)beta(4)heterotetramers. These domains exhibit significant homology to the carbohydrate-binding domains of C-type lectins, to the factor IX-binding protein (IX-bp), and to flavocetin-A (Fl-A) but sequence and structural differences are observed in both the domains in the putative Ca(2+)and carbohydrate binding regions.