A highly divergent α-amylase from Streptomyces spp.: An evolutionary perspective.

The present study deals with the genetic changes observed in the protein sequence of an α-amylase from Streptomyces spp. and its structural homologs from Pseudoalteromonas haloplanktis, invertebrates and mammals. The structural homologs are renowned for their important features such as chloride binding triad and a serine-protease like catalytic triad (a triad which is reported to be strictly conserved in all chloride-dependent α-amylases). These conserved regions are essential for allosteric activation of enzyme and conformational stability, respectively. An evaluation of these distinctive features in Streptomyces α-amylases revealed the role of mutations in conserved regions and evolution of chloride-independent α-amylases in Streptomyces spp. Besides, the study also discovers a highly divergent α-amylase from Streptomyces spp. which varies greatly even within the homologs of the same genus. Another very important feature is the number of disulfide bridges in which the structural homologs own eight Cys residues to form four disulfide bridges whereas Streptomyces α-amylases possess only seven Cys to form three disulfide bridges. The study also highlights the unique evolution of carbohydrate binding module 20 domain (CBM20 also known as raw starch binding domain or E domain) in Streptomyces α-amylases which is completely absent in α-amylases of other structural homologs.

Biochemical characterization and structural insights into the high substrate affinity of a dimeric and Ca2+ independent Bacillus subtilis α-amylase.

An extracellular amylase (AmyKS) produced by a newly isolated Bacillus subtilis strain US572 was purified and characterized. AmyKS showed maximal activity at pH 6 and 60°C with a half-life of 10 min at 70°C. It is a Ca2+ independent enzyme and able to hydrolyze soluble starch into oligosaccharides consisting mainly of maltose and maltotriose. When compared to the studied α-amylases, AmyKS presents a high affinity toward soluble starch with a Km value of 0.252 mg ml-1 . Coupled with the size-exclusion chromatography data, MALDI-TOF/MS analysis indicated that the purified amylase is a dimer with a molecular mass of 136,938.18 Da. It is an unusual feature of a non-maltogenic α-amylase. A 3D model and a dimeric model of AmyKS were generated showing the presence of an additional domain suspected to be involved in the dimerization process. This dimer arrangement could explain the high substrate affinity and catalytic efficiency of this enzyme.

Identification of Phenolic Compounds from Nettle as New Candidate Inhibitors of Main Enzymes Responsible on Type-II Diabetes.

BACKGROUND: In medicinal chemistry, the discovery of small organic molecules that can be optimized and lead to a future drug capable of effectively modulating the biological activity of a therapeutic target remains a major challenge. Because of the harmful secondary effects of synthesized therapeutic molecules, the development of research has been oriented towards phytomedicines. Phenolic compounds from medicinal plants are constantly explored for new therapeutic use. METHODS: In this paper, we studied interactions between main enzymes responsible for causing type 2 diabetes mellitus (T2DM) and phenolic compounds from nettle (Urtica dioica L.) using molecular Docking with Molecular Operating Environment Software (MOE). RESULTS: Docking results show a common molecule (secoisolariciresinol), which may form stable complexes with depeptidyl peptidase 4 (DPP-4), alpha-amylase and beta-glucosidase with binding energy of -7.04732084 kcal/mol, -3.82946181 kcal/mol and -4.16077089 kcal/mol respectively. Besides secoisolariciresinol, other phenolic compounds give better docking score than the original co-crystallized ligand for alpha-amylase (PDB ID 5U3A) and beta-glucosidase (PDB ID 1OGS). CONCLUSION: The obtained results are promising for the discovery of new alpha-amylase and betaglucosidase inhibitors. This study also confirms the folk use of nettle as antidiabetic agent.

PEGylated silica-enzyme nanoconjugates: a new frontier in large scale separation of α-amylase.

High resolution is nearly lost at the expense of throughput in most conventional bioseparation methods. Nanoparticles, due to their high surface to volume ratio, are attractiveenzyme carriers, which can boost the performance of extraction manifold. Here, wereport design and application ofa method highly capable of improving the partitioning of α-amylase in aqueous two-phase system of polymer and salt. Silica nanoparticle introduced to the system acts as a bridge that connects the enzyme and polymer. Theconjugated nanoparticles form the major part of the upper phase and thus significantly enhance the protein recovery. A thorough investigation was performed on the structure of the nanoconjugatesas well as analyzing the conformational structure of the enzyme after conjugationto explore anypossible denaturation.

Molten globule-like partially folded state of Bacillus licheniformis α-amylase at low pH induced by 1,1,1,3,3,3-hexafluoroisopropanol.

Effect of 1,1,1,3,3,3-hexafluoroisopropanol (HFIP) on acid-denatured Bacillus licheniformis α -amylase (BLA) at pH 2.0 was investigated by far-UV CD, intrinsic fluorescence, and ANS fluorescence measurements. Addition of increasing HFIP concentrations led to an increase in the mean residue ellipticity at 222 nm (MRE 222 nm) up to 1.5 M HFIP concentration beyond which it sloped off. A small increase in the intrinsic fluorescence and a marked increase in the ANS fluorescence were also observed up to 0.4 M HFIP concentration, both of which decreased thereafter. Far- and near-UV CD spectra of the HFIP-induced state observed at 0.4 M HFIP showed significant retention of the secondary structures closer to native BLA but a disordered tertiary structure. Increase in the ANS fluorescence intensity was also observed with the HFIP-induced state, suggesting exposure of the hydrophobic clusters to the solvent. Furthermore, thermal denaturation of HFIP-induced state showed a non-cooperative transition. Taken together, all these results suggested that HFIP-induced state of BLA represented a molten globule-like state at pH 2.0.

Novel magnetic cross-linked enzyme aggregates (magnetic CLEAs) of alpha amylase.

Novel magnetic cross-linked enzyme aggregates of alpha amylase were prepared by chemical cross-linking of enzyme aggregates with amino functionalized magnetite nanoparticles which can be separated from reaction mixture using magnetic field. Of the initially applied alpha amylase activity 100% was recovered in magnetic CLEAs, whereas only 45% was recovered in CLEAs due to the low content of Lys residues in alpha amylase. Scanning electron microscopy analysis showed that CLEAs and magnetic CLEAs were spherical structures. The CLEAs and magnetic CLEAs displayed a shift in optimal pH towards the acidic side, whereas optimal temperature of magnetic CLEAs was improved compared to free enzyme and CLEAs. Although V(max) of enzyme in CLEAs and magnetic CLEAs did not change, substrate affinity of the enzyme increased. The magnetic CLEAs also enhanced the thermal stability and storage stability. Moreover, the magnetic CLEAs retained 100% initial activity even after 6 cycles of reuse.

Salivary oncocytic cystadenoma with intraluminal crystalloids: a case report with review of the literature.

Oncocytic cystadenoma is a rare benign tumor of major salivary glands that in rare occasions may present histologically with intraluminal crystalloids. We report a case of 53-year-old man with a progressively enlarging lump in the left submandibular region. Ultrasound examination revealed a cystic mass with an intraluminal fluid collection. The tumor was surgically removed. Histologic examination yielded a diagnosis of oncocytic cystadenoma with a high concentration of intraluminal crystalloids. The microscopic features of the crystalloids were compatible with nontyrosine (alpha-amylase) crystalloids. When compared with previously published cases in the literature, this is the first report of oncocytic cystadenoma with intraluminal crystalloids arising in the submandibular gland, and the second reported case of the nontyrosine type of crystalloids occurring in association with this tumor. The nontyrosine crystalloids may be highly characteristic to salivary gland tumors with oncocytic differentiation.

Detection of salivary alpha-amylase and lysozyme exposed on the pellicle formed in situ on different materials.

Amylase and lysozyme are components of the salivary pellicle, exposing considerable enzymatic activity in the immobilized state. The purpose of the present study was to elucidate the influence of different solid substrata on the amount and distribution of amylase and lysozyme exposed on the surface of the salivary pellicle formed in situ. Slabs of titanium, feldspar ceramic, and bovine enamel were fixed on the buccal sites of individual splints worn by three subjects for 3 or 30 min, respectively, to allow pellicle formation. Subsequently, slabs were removed from the splints and rinsed with running water. Detection of amylase and lysozyme was performed by FEI-SEM after gold-immunolabeling of the enzymes. Both enzymes were found to be distributed randomly at the pellicle surface. Irrespective of formation time and substratum, significantly more labeled lysozyme molecules (5.23 +/- 4.5 microm(-2)) were detected compared with amylase (3.4 +/- 2.9 microm(-2)). Neither the substratum nor the pellicle formation time had significant impact on the amount of the respective enzyme that could be detected. This study for the first time provides evidence, that amylase and lysozyme are exposed at the surface of the salivary pellicle formed in situ on titanium and ceramics. Both enzymes are distributed randomly on the surface of the pellicle, irrespective of the underlying substratum.

Ca-binding to Bacillus licheniformis alpha-amylase (BLA).

Ca-induced renaturation of Bacillus licheniformis alpha-amylase in the presence of urea has been employed to determine the binding constants of the ion. The native enzyme is folded at 3M urea while the Ca-depleted protein is largely unfolded at this denaturant concentration. Refolding of the protein has been monitored by circular dichroism and the titration curves have been analyzed assuming a model of three independent binding sites. The stoichiometry has been taken from X-ray studies. The refolded protein exhibits a secondary structure that is similar but not identical to that of the native protein. The binding constants have been used to construct a phase diagram that illustrates the contribution of Ca-binding to the resistance against urea unfolding.

Electron microscopic detection of salivary alpha-amylase in the pellicle formed in situ.

Immunological and biochemical analyses have shown that alpha-amylase is an essential component of the acquired pellicle. After adsorption, this enzyme might act as a receptor for bacterial adherence. However, data indicating that amylase is bound to the pellicle surface in vivo and thus available for adhering bacteria are rare. Therefore, the present study focused on alpha-amylase within the pellicle formed in situ, using gold-immunolabeling electron microscopic techniques. Pellicles were formed by intra-oral exposure of enamel specimens for 30 and 120 min in six subjects. The results obtained by transmission electron microscopy indicate that amylase was randomly distributed in the pellicle layer without any preferential localization within the pellicle. Thus, salivary alpha-amylase might be considered as an important structural component that is even involved in the early stages of pellicle formation. The findings of field emission in-lens scanning electron microscopy provided evidence that the enzyme is located on the pellicle surface. It could be concluded that alpha-amylase might act as a receptor for bacterial adherence to the pellicle in vivo.

Liposomes for microcompartmentation of enzymes and their influence on catalytic activity.

Modular systems for protein coupling have been applied for anchoring enzyme molecules on liposome surfaces. Two cytoplasmic model enzymes, alpha-amylase from Escherichia coli (EC. 3.2.1.1) and guanylate kinase from Saccharomyces cerevisiae (EC. 2.7.4.8), were directly coupled by a histidine-tag or indirectly via strep-tag and streptavidin or streptactin linker to a liposome membrane. Though the catalytic properties of the enzymes are generally maintained, stability and specific activity of the enzymes are modified after coupling and are especially influenced by the lipid used for the liposome assembly.

Identification of amylase crystalloids in cystic lesions of the parotid gland.

OBJECTIVE: To identify alpha-amylase crystalloid formations in parotid specimens obtained by fine needle aspiration. STUDY DESIGN: The study concerned three cases of sialadenitis with crystalloid formation observed between 1993 and 1998. In one of these cases, transmission electron microscopy, mass spectrometry and measurement of amylase activity were used to characterize the nature of the crystalloids. RESULTS: Light microscopy revealed the same crystalloid structure in all three cases. In one case, where the material was saved, a biochemical method made it possible to reveal high amylase activity, while protein electrophoresis and mass spectrometry were used to identify salivary alpha-amylase. CONCLUSION: Crystalloids of salivary alpha-amylase can be identified by May-Grünwald-Giemsa and Papanicolaou stain and can be rapidly confirmed through determination of amylase activity.

Crystal structures of the psychrophilic alpha-amylase from Alteromonas haloplanctis in its native form and complexed with an inhibitor.

Alteromonas haloplanctis is a bacterium that flourishes in Antarctic sea-water and it is considered as an extreme psychrophile. We have determined the crystal structures of the alpha-amylase (AHA) secreted by this bacterium, in its native state to 2.0 angstroms resolution as well as in complex with Tris to 1.85 angstroms resolution. The structure of AHA, which is the first experimentally determined three-dimensional structure of a psychrophilic enzyme, resembles those of other known alpha-amylases of various origins with a surprisingly greatest similarity to mammalian alpha-amylases. AHA contains a chloride ion which activates the hydrolytic cleavage of substrate alpha-1,4-glycosidic bonds. The chloride binding site is situated approximately 5 angstroms from the active site which is characterized by a triad of acid residues (Asp 174, Glu 200, Asp 264). These are all involved in firm binding of the Tris moiety. A reaction mechanism for substrate hydrolysis is proposed on the basis of the Tris inhibitor binding and the chloride activation. A trio of residues (Ser 303, His 337, Glu 19) having a striking spatial resemblance with serine-protease like catalytic triads was found approximately 22 angstroms from the active site. We found that this triad is equally present in other chloride dependent alpha-amylases, and suggest that it could be responsible for autoproteolytic events observed in solution for this cold adapted alpha-amylase.

The crystal structure of porcine pancreatic alpha-amylase in complex with the microbial inhibitor Tendamistat.

The crystal structure of the complex formed between the 498 amino acid residue porcine pancreatic alpha-amylase (PPA) and the 74 amino acid residue inhibitor Tendamistat secreted from Streptomyces tendae, has been determined by multiple isomorphous replacement in a crystal of space group P6(5)22 (a = b = 77.7 A, c = 359.5 A). The model has been refined to an R-factor of 0.194 by Powell minimization applying strong energy constraints based on 17,964 independent reflections in the 7 to 2.5 A resolution range, and obeys standard geometry within 0.011 A in bond lengths and 1.78 degrees in bond angles. The final model consists of all 496 amino acid residues of PPA, 71 amino acid residues of Tendamistat (without the three N-terminal residues), one calcium ion, one chloride ion and 167 water molecules. PPA exhibits the same topological fold in the complex as the uncomplexed PPA recently published by others. About 30% of the water-accessible surface of Tendamistat is in contact with PPA. Four segments of the polypeptide chain, with a total of 15 amino acid residues, are involved in the binding. One segment containing the staggered side-chains of the triplet Trp18, Arg19, Tyr20, typical for this class of inhibitors, binds into the catalytic site. The other segments fill out the groove in the PPA molecule, which also binds the carbohydrate inhibitor acarbose and is assumed to be the substrate-binding region. This extended interaction between Tendamistat and alpha-amylase explains the very high inhibition constant of about 9 x 10(-12) M.

Crystallization and preliminary X-ray studies of wild type and catalytic-site mutant alpha-amylase from Bacillus subtilis.

Recombinant alpha-amylase (EC3.2.1.1) from Bacillus subtilis has been crystallized by the hanging drop vapor diffusion method using polyethylene glycol as precipitant. Crystals of wild-type protein diffract to at least 2.2 A resolution, and belong to the space group P2(1)2(1)2(1) with a = 72.2 A, b = 74.9 A, c = 116.1 A with probably one molecule in the asymmetric unit. A catalytic site mutant created by site-directed mutagenesis has also been grown as isomorphous crystals with a = 72.6 A, b = 74.4 A, c = 116.7 A. Structural studies of both wild-type and mutant proteins will provide a basis for understanding the catalytic mechanism of alpha-amylase.