An extracellular exo-beta-(1,3)-glucanase from Pichia pastoris: purification, characterization, molecular cloning, and functional expression

An extracellular exo-beta-(1,3)-glucanase (designated EXG1) was purified to apparent homogeneity from Pichia pastoris X-33 cultures by ammonium sulfate fractionation, ion-exchange chromatography, and gel filtration. The native enzyme is unglycosylated and monomeric with a molecular mass of approximately 47kDa. At its optimal pH of 6.0, the enzyme shows highest activity among physiological substrates toward laminarin (apparent Km, 3.5 mg/ml; Vmax, 192 micromole glucose produced/min/mg protein) but also hydrolyzes amygdalin and esculin, and the chromogenic substrates p-nitrophenyl-beta-D-glucopyranoside and p-nitrophenyl-beta-D-xylopyranoside. The P. pastoris EXG1 gene was cloned by a PCR-based strategy using genomic DNA as template. This intronless gene predicts an ORF that encodes a primary translation product of 414 amino acids. We believe that this preproprotein is processed sequentially by signal peptidase and a Kex2-like endoprotease to yield a mature protein of 392 amino acids (45,376 Da; pI, 4.46) that shares 36-64% amino acid identity with other yeast exo-beta-(1,3)-glucanases belonging to Glycoside Hydrolase Family 5. It also possesses the eight invariant residues and signature pattern [LIV]-[LIVMFYWGA](2)-[DNEQG]-[LIVMGST]-X-N-E-[PV]-[RHDNSTLIVFY] shown by all Family 5 members. Overexpression of the cloned EXG1 gene in Pichia cells, followed by Ni-CAM HC resin chromatography, yielded milligram quantities of homogeneous recombinant EXG1 in active form for further characterization studies.     

The relationship between yeast cell size and cell division in Candida albicans

The mean size and percentage of budded and unbudded cells of Candida albicans grown in batch culture over a wide range of doubling times have been measured. Cell volume decreased with increased doubling time and a nonlinear approach to an asymptotic minimum was observed. When cells were separated by age according to bud scars, each age showed a similar decrease. During each cell division cycle, size increased slowly during both budded and unbudded periods so that each generation was significantly larger than the preceding. There was no difference in size between the parent portion of budded cells and unbudded cells of the same age. Time-lapse photomicroscopy of cells growing on solid medium showed that cells divide asymmetrically with larger parents having a shorter subsequent cycle time than the smaller daughter, although the time utilized for bud formation was similar. When cells were shifted from a medium supporting a low growth rate and small size to a medium supporting a faster growth rate and larger size, both budded and unbudded cells increased significantly in size. As the doubling time increased, both the budded and unbudded portions of parental and daughter cycles increased.     

Structural characterization of (1-->3)-beta-D-glucans isolated from blastospore and hyphal forms of Candida albicans

Glucans are (1-->3)-beta-linked linear and branched polymers containing anhydroglucose repeat units. They comprise a major portion of the cell wall of saprophytic and pathogenic fungi. Glucans activate a wide range of innate immune responses. They are also released from the fungal cell wall as exopolymers into the blood of patients with fungal infections. Extensive studies have been done on glucans isolated from saprophytic fungi, such as Saccharomyces cerevisiae; however, much less is known about the glucans produced by the polymorphic fungal pathogen Candida albicans. We have undertaken an extensive structural characterization and comparison of glucans isolated from C. albicans blastospores and hyphae using high-resolution, solution-state proton nuclear magnetic resonance spectroscopy (NMR). In addition, we developed a simple and straightforward method for the production of Candida hyphae that resulted in gram quantities of hyphal mass. Also, we compared and contrasted the Candida glucans isolated by two different protocols with those isolated from S. cerevisiae. Isolation protocols provide high purity glucans with source-based structural differences. Structural details provided by this NMR analysis included the degree of polymerization, molecular weight, degree and type of branching, and structural composition. We observed that Candida glucans, derived from blastospores or hyphae, are different compared to those isolated from S. cerevisiae with regard to side-chain branching along the backbone and at the reducing terminus. These structural details are an important prerequisite for biomedical studies on the interaction of isolated fungal cell wall glucans with the innate immune system.     

Cloning and functional expression of alkaline alpha-galactosidase from melon fruit: similarity to plant SIP proteins uncovers a novel family of plant glycosyl hydrolases

Raffinose and stachyose are ubiquitous galactosyl-sucrose oligosaccharides in the plant kingdom which play major roles, second only to sucrose, in photoassimilate translocation and seed carbohydrate storage. These sugars are initially metabolised by alpha-galactosidases (alpha-gal). We report the cloning and functional expression of the first genes, CmAGA1 and CmAGA2, encoding for plant alpha-gals with alkaline pH optima from melon fruit (Cucumis melo L.), a raffinose and stachyose translocating species. The alkaline alpha-gal genes show very high sequence homology with a family of undefined 'seed imbibition proteins' (SIPs) which are present in a wide range of plant families. In order to confirm the function of SIP proteins, a representative SIP gene, from tomato, was expressed and shown to have alkaline alpha-gal activity. Phylogenetic analysis based on amino acid sequences shows that the family of alkaline alpha-gals shares little homology with the known prokaryotic and eukaryotic alpha-gals of glycosyl hydrolase families 27 and 36, with the exception of two cross-family conserved sequences containing aspartates which probably function in the catalytic step. This previously uncharacterised, plant-specific alpha-gal family of glycosyl hydrolases, with optimal activity at neutral-alkaline pH likely functions in key processes of galactosyl-oligosaccharide metabolism, such as during seed germination and translocation of RFO photosynthate.     

Properties of a thermoactive beta-1,3-1,4-glucanase (lichenase) from Clostridium thermocellum expressed in Escherichia coli

A Clostridium thermocellum gene (licB) encoding a thermoactive 1,3-1,4-beta-glucanase (lichenase) with a molecular weight of about 35,000 was localized on a 1.5-kb DNA fragment by cloning and expression in E. coli. The enzyme acts on beta-glucans with alternating beta-1,3- and beta-1,4-linkages such as barley beta-glucan and lichenan, but not on beta-glucans containing only 1,3- or 1,4-glucosidic bonds. It is active over a broad pH range (pH 5-12) and has a temperature optimum around 80 degrees C. The C. thermocellum lichenase is unusually resistant against inactivation by heat, ethanol or ionic detergents. These properties make the enzyme highly suitable for industrial application in the mashing process of beer brewing.     

Candida albicans: Frequency and characterization in oral cancer (Stage I) from smokers and drinkers

The frequency and the biotype of Candida albicans, from patients with epidermoid carcinoma of the oral mucosa (stage I) were evaluated. The patients chosen were habitual drinkers and smokers, aged 34 to 81 years who had not submitted previously to any treatment. They exhibited ulcero-vegetative lesions, mainly on the floor of the mouth, palate and tongue and were classified as stage TNM 100 - TNM 200. Samples from the buccal mucosa were collected for mycological study including: identification of yeasts, serotyping, determination of exo-enzymes as proteinase and phospholipase as well as "killer" assay for biotype characterization. Positive cultives for yeasts were observed in 51.5% of the patients(17/33), being 21.2% represented by C. albicans, all serotype A. The "killer" test demonstrated two different biotypes of C. albicans, namely 211(71.4%) and 611(28.6%), with high levels of proteinase (Prz < 0.30), while phospholipase presented intermediary levels (Pz > 0.29 and =/< 0.69). These data suggested a potentiality to virulence of C. albicans, although did not show an association of a particular biotype with the carcinogenic factors present or with the development of oral epidermoid carcinoma in this initial stage.     

Identification and characterization of a xyloglucan-specific family 74 glycosyl hydrolase from Streptomyces coelicolor A3(2)

The sco6545 gene of Streptomyces coelicolor A3(2) was nominated as a putative cellulase with 863 mature-form amino acids (90.58 kDa). We overexpressed and purified Sco6545 and demonstrated that the protein is not a cellulase but a xyloglucan-specific glycosyl hydrolase which cleaves xyloglucan at unbranched glucose residues.     

阴道白色念珠菌致病株与携带株两相性与毒力关系研究

目的探讨阴道白色念珠菌致病株和携带株菌丝相和酵母相分泌型酸性蛋白酶和细胞外磷脂酶活性以及与其毒力的关系。方法分别采用牛奶平板和卵黄培养基法检测白色念珠菌致病株和携带株200株分泌型酸性蛋白酶和细胞外磷脂酶的活力,分别将致病产酶株的菌丝相和孢子相菌悬液（5×10^6CFU／ral）注射小鼠尾静脉,1个月内观察小鼠死亡率及平均存活时间,以平均存活时间评价菌株毒力。结果白色念珠菌致病株和携带株分泌型酸性蛋白酶检出率分别为83．3％和35．7％（P〈0．01）;细胞外磷脂酶阳性率分别为87．5％和39．3％（P〈0．01）。动物实验结果表明,白色念珠菌致病株菌丝相分泌型酸性蛋白酶和细胞外磷脂酶的活力均显著高于孢子相（P〈0．01,P〈0．005）;注射菌丝相白色念珠菌的小鼠死亡率高于注射孢子相的小鼠（P〈0．01）,且平均存活期短于注射孢子相的小鼠（P〈0．01）。结论分泌型酸性蛋白酶和细胞外磷脂酶是白色念珠菌重要毒力因子,致病株毒力高于携带株,菌丝相毒力高于酵母相。     

The structure of Mycobacterium tuberculosis MPT51 (FbpC1) defines a new family of non-catalytic alpha/beta hydrolases

Mycobacterium tuberculosis, the causative agent of tuberculosis, is known to secrete a number of highly immunogenic proteins that are thought to confer pathogenicity, in part, by mediating binding to host tissues. Among these secreted proteins are the trimeric antigen 85 (Ag85) complex and the related MPT51 protein, also known as FbpC1. While the physiological function of Ag85, a mycolyltransferase required for the biosynthesis of the cell wall component alpha,alpha'-trehalose dimycolate (or cord factor), has been identified recently, the function of the closely related MPT51 (approximately 40% identity with the Ag85 components) remains to be established. The crystal structure of M.tuberculosis MPT51, determined to 1.7 A resolution, shows that MPT51, like the Ag85 components Ag85B and Ag85C2, folds as an alpha/beta hydrolase, but it does not contain any of the catalytic elements required for mycolyltransferase activity. Moreover, the absence of a recognizable alpha,alpha'-trehalose monomycolate-binding site and the failure to detect an active site suggest that the function of MPT51 is of a non-enzymatic nature and that MPT51 may in fact represent a new family of non-catalytic alpha/beta hydrolases. Previous experimental evidence and the structural similarity to some integrins and carbohydrate-binding proteins led to the hypothesis that MPT51 might have a role in host tissue attachment, whereby ligands may include the serum protein fibronectin and small sugars.     

Cloning and sequence analysis of a rapamycin-binding protein-encoding gene (RBP1) from Candida albicans.

Rapamycin (Rm) and FK506 are macrolide antifungal agents that exhibit potent immunosuppressive properties in higher eukaryotes which are mediated through interaction with specific receptor proteins (FKBPs or RBPs, for FK506- and Rm-binding proteins, respectively). These proteins possess peptidyl-prolyl cis-trans isomerase (PPIase) activity in vitro which is inhibited by the binding of Rm and FK506. We previously isolated a gene encoding an RBP from Saccharomyces cerevisiae, and demonstrated that null mutations in this gene (called RBP1) result in a recessive Rm-resistant (RmR) phenotype. We now have cloned the Candida albicans RBP1 gene via complementation of the RmR phenotype in S. cerevisiae. The predicted C. albicans RBP exhibits 61%, 52% and 49% amino acid (aa) sequence identity with RBPs (FKBPs) from S. cerevisiae, Neurospora crassa and human cells (FKBP-12), respectively. Furthermore, several of the aa residues identified as being important for drug binding in human FKBP-12 are conserved within the C. albicans RBP.     

Minor groove binding of a bis-quaternary ammonium compound: the crystal structure of SN 7167 bound to d(CGCGAATTCGCG)2.

The X-ray crystal structure of the complex between the synthetic antitumour and antiviral DNA binding ligand SN 7167 and the DNA oligonucleotide d(CGCGAATTCGCG)2 has been determined to an R factor of 18.3% at 2.6 A resolution. The ligand is located within the minor groove and covers almost 6 bp with the 1-methylpyridinium ring extending as far as the C9-G16 base pair and the 1-methylquinolinium ring lying between the G4-C21 and A5-T20 base pairs. The ligand interacts only weakly with the DNA, as evidenced by long range contacts and shallow penetration into the groove. This structure is compared with that of the complex between the parent compound SN 6999 and the alkylated DNA sequence d(CGC[e6G]AATTCGCG)2. There are significant differences between the two structures in the extent of DNA bending, ligand conformation and groove binding.     

The structure of apo and holo forms of xylose reductase,a dimeric aldo-keto reductase from Candida tenuis

Xylose reductase is a homodimeric oxidoreductase dependent on NADPH or NADH and belongs to the largely monomeric aldo-keto reductase superfamily of proteins. It catalyzes the first step in the assimilation of xylose, an aldose found to be a major constituent monosaccharide of renewable plant hemicellulosic material, into yeast metabolic pathways. It does this by reducing open chain xylose to xylitol, which is reoxidized to xylulose by xylitol dehydrogenase and metabolically integrated via the pentose phosphate pathway. No structure has yet been determined for a xylose reductase, a dimeric aldo-keto reductase or a family 2 aldo-keto reductase. The structures of the Candida tenuis xylose reductase apo- and holoenzyme, which crystallize in spacegroup C2 with different unit cells, have been determined to 2.2 A resolution and an R-factor of 17.9 and 20.8%, respectively. Residues responsible for mediating the novel dimeric interface include Asp-178, Arg-181, Lys-202, Phe-206, Trp-313, and Pro-319. Alignments with other superfamily members indicate that these interactions are conserved in other dimeric xylose reductases but not throughout the remainder of the oligomeric aldo-keto reductases, predicting alternate modes of oligomerization for other families. An arrangement of side chains in a catalytic triad shows that Tyr-52 has a conserved function as a general acid. The loop that folds over the NAD(P)H cosubstrate is disordered in the apo form but becomes ordered upon cosubstrate binding. A slow conformational isomerization of this loop probably accounts for the observed rate-limiting step involving release of cosubstrate. Xylose binding (K(m) = 87 mM) is mediated by interactions with a binding pocket that is more polar than a typical aldo-keto reductase. Modeling of xylose into the active site of the holoenzyme using ordered waters as a guide for sugar hydroxyls suggests a convincing mode of substrate binding.     

The relationship between the solvent-accessible surface area of a protein and the number of native contacts in its structure

The degree of protein folding is characterized either by the solvent-accessible surface area (S _ASA ) or the actual number of native contacts (N _cont). Evidently, these values should correlate with each other, as a decrease in S _ASA caused by the change in protein conformation during its folding must be accompanied by the corresponding increase in the number of native contacts. It is shown that this correlation does exist and is very strong (the correlation coefficient exceeds 99%), which can be used for an accurate and rapid estimation of the protein surface area from the number of native contacts. Among the methods commonly used for calculating the native contacts, the atom-atom approach gives the best fit if hydrogen atoms are taken into account and the cutoff value for the distance between the centers of atoms is taken to be 8 Å. The latter means that two layers of surface atoms are required to shield the protein core from the solvent.     

Isolation of a chitin synthase gene (CHS1) from Candida albicans by expression in Saccharomyces cerevisiae

Chitin synthase activity was studied in yeast and hyphal forms of Candida albicans. pH-activity profiles showed that yeast and hyphae contain a protease-dependent activity that has an optimum at pH 6.8. In addition, there is an activity that is not activated by proteolysis in vitro and which shows a peak at pH 8.0. This suggests there are two distinct chitin synthases in C. albicans. A gene for chitin synthase from C. albicans (CHS1) was cloned by heterologous expression in a Saccharomyces cerevisiae chs1 mutant. Proof that the cloned chitin synthase is a C. albicans membrane-bound zymogen capable of chitin biosynthesis in vitro was based on several criteria. (i) the CHS1 gene complemented the S. cerevisiae chs1 mutation and encoded enzymatic activity which was stimulated by partial proteolysis; (ii) the enzyme catalyses incorporation of [14C]-GlcNAc from the substrate, UDP[U-14C]-GlcNAc, into alkali-insoluble chitin; (iii) Southern analysis showed hybridization of a C. albicans CHS1 probe only with C. albicans DNA and not with S. cerevisiae DNA; (iv) pH profiles of the cloned enzyme showed an optimum at pH 6.8. This overlaps with the pH-activity profiles for chitin synthase measured in yeast and hyphal forms of C. albicans. Thus, CHS1 encodes only part of the chitin synthase activity in C. albicans. A gene for a second chitin synthase in C. albicans with a pH optimum at 8.0 is proposed. DNA sequencing revealed an open reading frame of 2328 nucleotides which predicts a polypeptide of Mr 88,281 with 776 amino acids. The alignment of derived amino acid sequences revealed that the CHS1 gene from C. albicans (canCHS1) is homologous (37% amino acid identity) to the CHS1 gene from S. cerevisiae (sacCHS1).