Ionic network at the C-terminus of the beta-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus: Functional role in the quaternary structure thermal stabilization

Biochemical, crystallographic, and computational data support the hypothesis that electrostatic interactions are among the dominant forces in stabilizing hyperthermophilic proteins. The thermostable beta-glycosidase from the hyperthermophile Sulfolobus solfataricus (Ssbeta-gly) is an interesting model system for the study of protein adaptation to high temperatures. The largest ion-pair network of Ssbeta-gly is located at the tetrameric interface of the molecule; in this paper, key residues in this region were modified by site-directed mutagenesis and the stability of the mutants was analyzed by kinetics of thermal denaturation. All mutations produced faster enzyme inactivation, suggesting that the C-terminal ionic network prevents the dissociation into monomers, which is the limiting step in the mechanism of Ssbeta-gly inactivation. Moreover, the calculated reaction order showed that the mechanism of inactivation depends on the mutation introduced, suggesting that intermediates maintaining enzymatic activity are produced during the inactivation transition of some, but not all, mutants. Molecular models of each mutant allow us to rationalize the experimental evidence and give support to the current theories on the mechanism of ion pair stabilization in proteins from hyperthermophiles.     

Specific granules of human eosinophils have lysosomal characteristics: presence of lysosome-associated membrane proteins and acidification upon cellular activation

Eosinophils possess characteristic specific granules. Their content may be important during host defense but it can also cause damage after release at sites of inflammation. We investigated possible lysosomal characteristics of these granules. Lysosome-associated membrane protein (LAMP)-1 and 2, were detected by Western blot, subcellular fractionation, and immunoelectron microscopy (IEM) and were localized to the membrane of specific granules and in vesicles of the cytoplasm, separate from secretory vesicles. No binding of mannose 6-phosphate receptor to proteins of specific granules could be detected, indicating that they are dephosphorylated and mature. Cellular activation by interleukin-5 caused acidification of specific granules, as detected by pH-dependent probes. The acidification was inhibited by concanamycin A (inhibitor of vacuolar H(+)-ATPase). Activation of eosinophils by serum-treated zymosan (STZ) caused degranulation into STZ-containing phagosomes and incorporation of LAMPs to their membranes. In conclusion, specific granules of eosinophils can be regarded as specialized primary lysosomes, a feature that may be important for their function and integrity.     

The role of pilin glycan in neisserial pathogenesis

The pilus of pathogenic Neisseria is a polymer composed mainly of the glycoprotein, pilin. Recent investigations significantly enhanced characterization of pilin glycan (Pg) from N. gonorrhoeae (gonococcus, GC) and N. meningitidis (meningococcus, MC). Several pilin glycosylation genes were discovered recently from these bacteria and some of these genes transfer sugars previously unknown to be present in neisserial pili. Due to these findings, glycans of GC and MC pilin are now considered more complex. Furthermore, various Pg can be expressed by different strains and variants of GC, as well as MC. Intra-species variation of Pg between different groups of GC or MC can partly be due to polymorphisms of glycosylation genes. In pilus of pathogenic Neisseria, alternative glycoforms are also produced due to phase-variation (Pv) of pilin glycosylation genes. Most remarkably, the pgtA (pilin glycosyl transferase A) gene of GC can either posses or lack the ability of Pv. Many GC strains carry the phase-variable (Pv+) pgtA, whereas others carry the allele lacking Pv (Pv–). Mostly, the GC isolates from disseminated gonococcal infection (DGI) carry Pv+ pgtA but organisms from uncomplicated gonorrhea (UG) contain the Pv– allele. This data suggests that Pv of pgtA facilitates DGI, whereas constitutive expression of the Pv– pgtA may promote UG. Additional implications of Pg in various physiological and pathogenic mechanisms of Neisseria can also be envisaged based on various recent data.     

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.     

Environmental cDNA analysis of the genes involved in lignocellulose digestion in the symbiotic protist community of Reticulitermes speratus

To clarify the lignocellulolytic process of the lower termite symbiotic protistan system, we constructed a cDNA library from an as yet uncultivated symbiotic protist community of the lower termite Reticulitermes speratus. The library was constructed by the biotinylated CAP trapper method and analyzed by one-pass sequencing. Phylogenetic analysis of actin orthologs confirmed that the resulting library reflected the intact organismal and mRNA composition of the symbiotic system. The contents of the library included abundant numbers of lignocellulolytic genes of the glycosyl hydrolase family orthologs (families 3, 5, 7, 8, 10, 11, 26, 43, 45 and 62). Our results clearly indicated that a multiple family of glycosyl hydrolase enzymes was involved in the protistan cellulose degradation system. The data also suggested that the most extensively expressed enzyme was glycosyl hydrolase family 7, a cellobiohydrolase ortholog. This family of enzymes enables the degradation of crystalline cellulose, the principal component of wood biomass.     

Systematic comparison of catalytic mechanisms of hydrolysis and transfer reactions classified in the EzCatDB database

Catalytic mechanisms of 270 enzymes from 131 superfamilies, mainly hydrolases and transferases, were analyzed based on their enzyme structures. A method of systematic comparison and classification of the catalytic reactions was developed. Hydrolysis and transfer reactions closely resemble one another, displaying common mechanisms, single displacement, and double displacement. These displacement mechanisms might be further subclassified according to the type of catalytic factors and nucleophilic substitution involved. Several types of catalytic factors exist: nucleophile, acid, base, stabilizer, modulator, cofactors. Nucleophilic substitution might be categorized as S(N)1/S(N)2 (or dissociative/associative) reactions. The classification indicates that some mechanisms favor particular types of catalytic factors. In hydrolyses of amide bonds and phosphoric ester bonds, mechanisms with single displacement tend to use inorganic cofactors such as zinc and magnesium ions as important catalysts, whereas those with double displacement frequently do not use such cofactors. In contrast, hydrolyses of O-glycoside bond rarely use such cofactors, with one exception. The trypsin-like hydrolytic reaction, which is catalyzed by the classic catalytic triad comprising serine/histidine/aspartate, can be considered as a "super-reaction" because it is observed in at least three nonhomologous enzymes, whereas most reactions are singlets without any nonhomologous enzymes. By dividing complex reactions into several reactions, correlations between active site structures and catalytic functions can be suggested. This classification method is applicable to other reactions such as elimination and isomerization. Furthermore, it will facilitate annotation of enzyme functions from 3D patterns of enzyme active sites. The classification is available at http://mbs.cbrc.jp/EzCatDB/RLCP/index.html.     

米曲霉耐热木聚糖酶纯化及酶学特性研究

对米曲霉原始发酵液中耐热木聚糖酶进行纯化和酶学特性研究,利用甘蔗渣为碳源培养米曲霉,通过超滤和阴离子交换柱两步纯化得到木聚糖酶XynH1,分子量35．402kDa,利用飞行时间质谱和SDS—PAGE分析,推断XynH1为XylanaseXynF1,分子量为35．402kDa。XynH1属于糖苷水解酶家族10,酶活为442．2IU／nag,最适pH和温度分别为pH6．0和65℃,80℃以下及pH4．0～10．5范围内较稳定。     

GENPLAT: an Automated Platform for Biomass Enzyme Discovery and Cocktail Optimization

The high cost of enzymes for biomass deconstruction is a major impediment to the economic conversion of lignocellulosic feedstocks to liquid transportation fuels such as ethanol. We have developed an integrated high throughput platform, called GENPLAT, for the discovery and development of novel enzymes and enzyme cocktails for the release of sugars from diverse pretreatment/biomass combinations. GENPLAT comprises four elements: individual pure enzymes, statistical design of experiments, robotic pipeting of biomass slurries and enzymes, and automated colorimeteric determination of released Glc and Xyl. Individual enzymes are produced by expression in Pichia pastoris or Trichoderma reesei, or by chromatographic purification from commercial cocktails or from extracts of novel microorganisms. Simplex lattice (fractional factorial) mixture models are designed using commercial Design of Experiment statistical software. Enzyme mixtures of high complexity are constructed using robotic pipeting into a 96-well format. The measurement of released Glc and Xyl is automated using enzyme-linked colorimetric assays. Optimized enzyme mixtures containing as many as 16 components have been tested on a variety of feedstock and pretreatment combinations.GENPLAT is adaptable to mixtures of pure enzymes, mixtures of commercial products (e.g., Accellerase 1000 and Novozyme 188), extracts of novel microbes, or combinations thereof. To make and test mixtures of ˜10 pure enzymes requires less than 100 μg of each protein and fewer than 100 total reactions, when operated at a final total loading of 15 mg protein/g glucan. We use enzymes from several sources. Enzymes can be purified from natural sources such as fungal cultures (e.g., Aspergillus niger, Cochliobolus carbonum, and Galerina marginata), or they can be made by expression of the encoding genes (obtained from the increasing number of microbial genome sequences) in hosts such as E. coli, Pichia pastoris, or a filamentous fungus such as T. reesei. Proteins can also be purified from commercial enzyme cocktails (e.g., Multifect Xylanase, Novozyme 188). An increasing number of pure enzymes, including glycosyl hydrolases, cell wall-active esterases, proteases, and lyases, are available from commercial sources, e.g., Megazyme, Inc. (www.megazyme.com), NZYTech (www.nzytech.com), and PROZOMIX (www.prozomix.com).Design-Expert software (Stat-Ease, Inc.) is used to create simplex-lattice designs and to analyze responses (in this case, Glc and Xyl release). Mixtures contain 4-20 components, which can vary in proportion between 0 and 100%. Assay points typically include the extreme vertices with a sufficient number of intervening points to generate a valid model. In the terminology of experimental design, most of our studies are "mixture" experiments, meaning that the sum of all components adds to a total fixed protein loading (expressed as mg/g glucan). The number of mixtures in the simplex-lattice depends on both the number of components in the mixture and the degree of polynomial (quadratic or cubic). For example, a 6-component experiment will entail 63 separate reactions with an augmented special cubic model, which can detect three-way interactions, whereas only 23 individual reactions are necessary with an augmented quadratic model. For mixtures containing more than eight components, a quadratic experimental design is more practical, and in our experience such models are usually statistically valid.All enzyme loadings are expressed as a percentage of the final total loading (which for our experiments is typically 15 mg protein/g glucan). For "core" enzymes, the lower percentage limit is set to 5%. This limit was derived from our experience in which yields of Glc and/or Xyl were very low if any core enzyme was present at 0%. Poor models result from too many samples showing very low Glc or Xyl yields. Setting a lower limit in turn determines an upper limit. That is, for a six-component experiment, if the lower limit for each single component is set to 5%, then the upper limit of each single component will be 75%. The lower limits of all other enzymes considered as "accessory" are set to 0%. "Core" and "accessory" are somewhat arbitrary designations and will differ depending on the substrate, but in our studies the core enzymes for release of Glc from corn stover comprise the following enzymes from T. reesei: CBH1 (also known as Cel7A), CBH2 (Cel6A), EG1(Cel7B), BG (β-glucosidase), EX3 (endo-β1,4-xylanase, GH10), and BX (β-xylosidase).     

High-throughput Saccharification Assay for Lignocellulosic Materials

Polysaccharides that make up plant lignocellulosic biomass can be broken down to produce a range of sugars that subsequently can be used in establishing a biorefinery. These raw materials would constitute a new industrial platform, which is both sustainable and carbon neutral, to replace the current dependency on fossil fuel. The recalcitrance to deconstruction observed in lignocellulosic materials is produced by several intrinsic properties of plant cell walls. Crystalline cellulose is embedded in matrix polysaccharides such as xylans and arabinoxylans, and the whole structure is encased by the phenolic polymer lignin, that is also difficult to digest ¹. In order to improve the digestibility of plant materials we need to discover the main bottlenecks for the saccharification of cell walls and also screen mutant and breeding populations to evaluate the variability in saccharification ². These tasks require a high throughput approach and here we present an analytical platform that can perform saccharification analysis in a 96-well plate format. This platform has been developed to allow the screening of lignocellulose digestibility of large populations from varied plant species. We have scaled down the reaction volumes for gentle pretreatment, partial enzymatic hydrolysis and sugar determination, to allow large numbers to be assessed rapidly in an automated system.This automated platform works with milligram amounts of biomass, performing ball milling under controlled conditions to reduce the plant materials to a standardised particle size in a reproducible manner. Once the samples are ground, the automated formatting robot dispenses specified and recorded amounts of material into the corresponding wells of 96 deep well plate (Figure 1). Normally, we dispense the same material into 4 wells to have 4 replicates for analysis. Once the plates are filled with the plant material in the desired layout, they are manually moved to a liquid handling station (Figure 2). In this station the samples are subjected to a mild pretreatment with either dilute acid or alkaline and incubated at temperatures of up to 90°C. The pretreatment solution is subsequently removed and the samples are rinsed with buffer to return them to a suitable pH for hydrolysis. The samples are then incubated with an enzyme mixture for a variable length of time at 50°C. An aliquot is taken from the hydrolyzate and the reducing sugars are automatically determined by the MBTH colorimetric method.     

Impact and efficiency of GH10 and GH11 thermostable endoxylanases on wheat bran and alkali-extractable arabinoxylans

The results of a comparative study of two thermostable (1-->4)-beta-xylan endoxylanases using a multi-technical approach indicate that a GH11 xylanase is more useful than a GH10 xylanase for the upgrading of wheat bran into soluble oligosaccharides. Both enzymes liberated complex mixtures of xylooligosaccharides. 13C NMR analysis provided evidence that xylanases cause the co-solubilisation of beta-glucan, which is a result of cell-wall disassembly. The simultaneous use of both xylanases did not result in a synergistic action on wheat bran arabinoxylans, but instead led to the production of a product mixture whose profile resembled that produced by the action of the GH10 xylanase alone. Upon treatment with either xylanase, the diferulic acid levels in residual bran were unaltered, whereas content in ferulic and p-coumaric acids were unequally decreased. With regard to the major differences between the enzymes, the products resulting from the action of the GH10 xylanase were smaller in size than those produced by the GH11 xylanase, indicating a higher proportion of cleavage sites for the GH10 xylanase. The comparison of the kinetic parameters of each xylanase using various alkali-extractable arabinoxylans indicated that the GH10 xylanase was most active on soluble arabinoxylans. In contrast, probably because GH11 xylanase can better penetrate the cell-wall network, this enzyme was more efficient than the GH10 xylanase in the hydrolysis of wheat bran. Indeed the former enzyme displayed a nearly 2-fold higher affinity and a 6.8-fold higher turnover rate in the presence of this important by-product of the milling industry.