A 2.7-kilobase fragment of DNA from Oerskovia xanthineolytica containing the gene for a beta-1,3-glucanase has been isolated and its complete nucleotide sequence determined. The sequence was found to contain two large open reading frames. Purification of the mature native enzyme and subsequent amino-terminal sequencing defined the glucanase gene in one reading frame which potentially encodes a protein of 548 amino acids. We have expressed this glucanase gene in Escherichia coli under control of the lacUV5 promoter and found the product to be secreted into the periplasm as a mature enzyme of about the same molecular weight as that of the native protein. The recombinant enzyme was purified to near homogeneity by a single step of high performance liquid chromatography. The ability of the recombinant enzyme to digest beta-glucan substrates and to lyse viable yeast cells was found to be indistinguishable from that of the native protein. Deletion of the cysteine-rich carboxyl-terminal 117 amino acids of the enzyme, which also contain two duplicated segments, abolished the lytic activity but did not significantly affect the glucanase function of the protein. The possible involvement of this domain in interaction with the yeast cell wall is discussed.

Previous analysis of tet39 suggests it may be present in other bacterial species. Hence, we investigated the host range of tet39 among bacterial from a poultry waste polluted river in Southwestern Nigeria. Thirteen resistant bacterial isolated from the water and sediment of the polluted river was investigated for the presence of tetracycline resistance genes tetA, tetB, tetC, tet39 and the transposon integrase gene of the Tn916/1545 family by PCR. While tetA, tetB, tetC and integrase genes cannot be detected in any of the organisms, tet39 was detected in eight of the tested organisms including three Gram-positive species. Sequence analysis showed the genes have high sequence identities (> or =99%) with tet39 of Acinetobacter sp. LUH5605, the first and only bacterial genus from which the gene has been reported to date. This is a novel observation. This study shows that apart from Acinetobacter, tet39 is present in other bacterial species tested in this study. This study adds to available information on the occurrence and distribution of tet39 among environmental bacteria and suggests that the gene has a broader host range than previously reported.

Molecular characterisation of a lytic thermoactive beta-1,3-glucanase from Oerskovia xanthineolytica LL-G109 has been performed. A molecular mass of 27 195.6 +/- 1.3 Da and an isoelectric point of 4.85 were determined by electrospray mass spectrometry and from its titration curve, respectively. Its thermoactivity profile shows it to be a heat-stable enzyme with a temperature optimum of 65 degrees C. The secondary structure content of the protein was estimated by circular dichroism to be approx. 25% alpha-helix, 7% random coil, and 68% beta-sheet and beta-turn structure. Nuclear magnetic resonance spectra confirm the high content of beta-structure. Furthermore, the presence of a compact hydrophobic core is indicated by the presence of slowly exchanging amide hydrogens and the enzyme's relatively high resistance to proteolysis. The N-terminal sequences of the intact protein and of a tryptic peptide each exhibit significant similarity to family 16 of glycosyl hydrolases whose overall fold is known to contain almost exclusively beta-sheets and surface loops. Moreover, the sequenced tryptic peptide appears to encompass residues of the Oerskovia xanthineolytica glucanase active site, since it contains a portion of the family 16 active-site motif E-[L/I/V]-D-[L/I/V]-E.

The nucleotide sequence of the betaglIIA gene, encoding the extracellular beta-1,3-glucanase IIA (betaglIIA) of the yeast-lytic actinomycete Oerskovia xanthineolytica LL G109, was determined. Sequence comparison shows that the betaglIIA enzyme has over 80% identity to the betaglII isoenzyme, an endo-beta-1,3-glucanase having low yeast-lytic activity secreted by the same bacterium. The betaglIIA enzyme lacks a glucan- or mannan-binding domain, such as those observed in beta-1,3-glucanases and proteases having high yeast/fungus-lytic activity. It can be included in the glycosyl hydrolase family 16. Gene fusion expression in Bacillus subtilis DN1885 followed by preliminary characterization of the recombinant gene product indicates that betaglIIA has a pI of 3.8 to 4.0 and is active on both laminarin and curdlan, having an acid optimum pH activity (ca. 4.0).

Molecular cloning of the beta gIII gene encoding for an endo-beta-1,3-glucanase (beta gl II) from Oerskovia xanthineolytica LLG109, a yeast-lytic gram-positive bacterium, has been conducted in order to elucidate its primary sequence and subsequently express it into B. subtilis. This endo-beta-1,3-glucanase exhibits low yeast-lytic activity toward viable S. cerevisiae cells, and it has shown ability to selectively permeabilize the yeast cell wall and release intracellular proteins produced by yeast. Highly degenerate oligonucleotides have been used to PCR-amplify a region of the beta-1,3-glucanase II encoding gene from O. xanthineolytica LLG109. The amplified fragment has been cloned and sequenced. The deduced amino acid sequence contains regions identical to the amino acid sequences previously determined by direct sequencing of the purified enzyme from O. xanthineolytica LLG109. By using the 180-bp PCR product as a homologous probe, we have been able to isolate four positive clones harboring plasmids pPF1A, pPF1B, pPF8A, and pPF9A, respectively, from a partial genomic library from O. xanthineolytica LLG109. All four plasmids contained a 2.7-kb BamHI insert that hybridized to the PCR probe under high stringency conditions. The 2.7-kb fragment seemed to be identical in all four cases regarding preliminary partial restriction mapping analysis done on the four plasmids. The 1.5-kb BamHI/KpnI restriction fragment from pPF8A and pPF9A hybridizing with the 180-bp PCR probe is presently being sequenced. The cloning of the lytic beta-1,3-glucanase from O. xanthineolytica LLG109 expands the number of yeast lytic beta-glucanases so far cloned. The availability of the nucleotide sequences of such a family of genes will allow further understanding of the role and mode of action of these enzymes in yeast cell wall degradation. In addition, a more extensive study on the structure and functional relationships of these enzymes will allow us to engineer "tailor-made" lytic beta-1,3-glucanases for use in new and improved large-scale selective cell permeabilization (SCP) and selective protein recovery (SPR) from yeast cells, not only from S. cerevisiae but also from alternative yeast expression systems such as Hansenula polymorpha, Pichia pastoris, and others, which are becoming of increasing importance in biotechnology.

The DNA methyltransferase of the AluI restriction-modification system, from Arthrobacter luteus, converts cytosine to 5-methylcytosine in the sequence AGCT. The gene for this methyltransferase, aluIM, was cloned into Escherichia coli and sequenced. A 525-codon open reading frame was found, consistent with deletion evidence, and the deduced amino acid sequence revealed all ten conserved regions common to 5-methylcytosine methyltransferases. The aluIM sequence predicts a protein of M(r) 59.0k, in agreement with the observed M(r), making M.AluI the largest known methyltransferase from a type II restriction-modification system. M.AluI also contains the largest known variable region of any monospecific DNA methyltransferase, larger than that of most multispecific methyltransferases. In other DNA methyltransferases the variable region has been implicated as the sequence-specific target recognition domain. An in-frame deletion that removes a third of this putative target-recognition region leaves the Alu I methyltransferase still fully active.

Endo-1,3-�]-glucanase from Cellulosimicrobium cellulans is composed of a catalytic domain and a carbohydrate-binding module. We have determined the X-ray crystal structure of the catalytic domain at a high resolution of 1.66?. The overall fold is a sandwich-like �]-jelly roll architecture like the enzymes in the glycoside hydrolase family 16. The substrate-binding cleft has a length and a width of ~28 and ~15?, respectively, which is thought to be capable of accommodating at least six glucopyranose units. Laminarihexaose was placed into the substrate-binding cleft, namely at the subsites +2 to -4 from the reducing end, and the complex structure was analyzed using molecular dynamics simulations (MD) and using a rotamer search of the pocket. During the MD simulations, the substrate fluctuated more than the enzyme, where the residues at the subsites toward the non-reducing end fluctuated more than those toward the reducing end. Little conformational change of the protein was observed for the subsites +1 and +2, indicating that the glucose's position could be tightly restricted inside the pocket. Substrate binding experiments using isothermal titration calorimetry showed that the binding affinity of laminaritriose was higher than that of laminaribiose and similar to those of other longer laminarioligosaccharides. Taken together, the substrates mainly bind to the subsites -1 to -3 with the highest affinity, while the part bound to the reducing end would be hydrolyzed.

Lysosomal storage diseases are treated with human lysosomal enzymes produced in mammalian cells. Such enzyme therapeutics contain relatively low levels of mannose-6-phosphate, which is required to target them to the lysosomes of patient cells. Here we describe a method for increasing mannose-6-phosphate modification of lysosomal enzymes produced in yeast. We identified a glycosidase from C. cellulans that 'uncaps' N-glycans modified by yeast-type mannose-Pi-6-mannose to generate mammalian-type N-glycans with a mannose-6-phosphate substitution. Determination of the crystal structure of this glycosidase provided insight into its substrate specificity. We used this uncapping enzyme together with �\-mannosidase to produce in yeast a form of the Pompe disease enzyme �\-glucosidase rich in mannose-6-phosphate. Compared with the currently used therapeutic version, this form of �\-glucosidase was more efficiently taken up by fibroblasts from Pompe disease patients, and it more effectively reduced cardiac muscular glycogen storage in a mouse model of the disease.