The three-dimensional crystal structure of the catalytic core of cellobiohydrolase I from Trichoderma reesei.

Cellulose is the major polysaccharide of plants where it plays a predominantly structural role. A variety of highly specialized microorganisms have evolved to produce enzymes that either synergistically or in complexes can carry out the complete hydrolysis of cellulose. The structure of the major cellobiohydrolase, CBHI, of the potent cellulolytic fungus Trichoderma reesei has been determined and refined to 1.8 angstrom resolution. The molecule contains a 40 angstrom long active site tunnel that may account for many of the previously poorly understood macroscopic properties of the enzyme and its interaction with solid cellulose. The active site residues were identified by solving the structure of the enzyme complexed with an oligosaccharide, o-iodobenzyl-1-thio-beta-cellobioside. The three-dimensional structure is very similar to a family of bacterial beta-glucanases with the main-chain topology of the plant legume lectins.

Characterization of a birch (Betula pendula Roth.) embryogenic gene, BP8.

We have isolated the birch homologue (BP8) for the carrot embryogenic gene DC8 by heterologous hybridization. The birch BP8 gene encodes a putative protein of 53 kDa, showing 52% sequence identity with the DC8 gene at the amino acid level. The putative BP8 protein contains 20 repeats of 11 amino acids and thus belongs to the group of LEA proteins isolated from such plants as carrot, cotton and wheat. Northern hybridization of mRNA isolated from birch cells representing different stages of somatic embryogenesis and non-embryogenetic material with a PB8 probe gave no signals, suggesting a low expression level of the BP8 gene.

Efficient secretion of murine Fab fragments by Escherichia coli is determined by the first constant domain of the heavy chain.

Fab fragments of IgG1 and IgG3 subclass antibodies which bind to 2-phenyloxazolone (Ox) were produced in Escherichia coli. The signal sequences of the Fd and L chains were correctly processed, the fragments were secreted into the periplasmic space and released into the culture medium upon prolonged cultivations. The yields of active Ox IgG1 and Ox IgG3 Fab fragments after one-step purification from the culture medium by affinity chromatography were 2 micrograms/ml and 0.5 micrograms/ml, respectively. The majority of the purified Ox IgG1 Fab was properly assembled, but in the case of Ox IgG3, the preparation was found to consist of a complete L chain and C-terminally degraded fragments of the Fd chain. A deletion up to the interchain disulfide bond in the first constant domain (CH1) of the Ox IgG3 Fd chain led to proper assembly of the truncated Fab fragment. The production level of the truncated fragment was comparable to that of the Ox IgG1 Fab and its hapten-binding activity similar to that of the idiotype monoclonal antibody. The temperature stability of the Ox IgG1 Fab was similar to that of the intact antibody. However, both of the Ox IgG3 Fab fragments showed reduced stability, suggesting that the CH1 domain contributes significantly to the thermal stability of the Fab fragment.

Efficient production of antibody fragments by the filamentous fungus Trichoderma reesei.

We have engineered the filamentous fungus Trichoderma reesei to assemble and secrete immunologically authentic engineered Fab antibody fragments into the culture medium. A major improvement in yield was achieved by fusing the heavy Fd chain to the T. reesei cellulase, CBHI. The yields of secreted, immunologically active Fab and CBHI-Fab fusion were 1 mg/l and 150 mg/l, respectively. The Fab fragment can be released from the fusion protein CBHI-Fab by an extracellular T. reesei protease. There was no detectable difference in affinity for the antigen between the engineered Fab and the idiotypic antibody.

A Ca(2+)-independent protein kinase C from fission yeast.

A protein kinase C homologue of Schizosaccharomyces pombe, pkc1+, was isolated from a genomic library by screening with the Saccharomyces cerevisiae PKC1 probe. From its primary sequence and biochemical properties, we conclude that S. pombe pkc1+ encodes a phospholipid-activated Ca(2+)-independent protein kinase, homologous to the delta/epsilon classes of mammalian protein kinase C. Gene disruption experiments show that pkc1+ is not essential for cell viability; however, overexpression of the protein leads to an abnormal cell morphology and a block in cell separation following mitosis suggestive of a role in cell division. In vitro phosphorylation experiments reveal several potential pkc1+ substrates.

Prevention of transfusion-associated HIV transmission with the use of a transfusion protocol for under 5s.

Recently there has been an increasing realization that for the prevention of transfusion-associated HIV infection, HIV screening alone is insufficient. In 1988, a treatment protocol for children under 5 years with severe anaemia, was introduced in Ekwendeni Hospital. Initial results showed that it was possible to reduce the number of blood transfusions without increasing the mortality rate. By January 1992 the protocol was being widely disregarded by ward staff, and transfusion rates had increased. Strict enforcement of the transfusion protocol from the 16 January produced a sharp drop in the percentage transfused from 44% to 11%. Mortality rates remained similar throughout the period of the study. Thus, it would appear to be possible, with the use of the transfusion protocol, to reduce the number of blood transfusions carried out, without affecting mortality rates, and hence to reduce the risk of transfusion-associated HIV.

Characterization of the genes of the 2,3-butanediol operons from Klebsiella terrigena and Enterobacter aerogenes.

The genes involved in the 2,3-butanediol pathway coding for alpha-acetolactate decarboxylase, alpha-acetolactate synthase (alpha-ALS), and acetoin (diacetyl) reductase were isolated from Klebsiella terrigena and shown to be located in one operon. This operon was also shown to exist in Enterobacter aerogenes. The budA gene, coding for alpha-acetolactate decarboxylase, gives in both organisms a protein of 259 amino acids. The amino acid similarity between these proteins is 87%. The K. terrigena genes budB and budC, coding for alpha-ALS and acetoin reductase, respectively, were sequenced. The 559-amino-acid-long alpha-ALS enzyme shows similarities to the large subunits of the Escherichia coli anabolic alpha-ALS enzymes encoded by the genes ilvB, ilvG, and ilvI. The K. terrigena alpha-ALS is also shown to complement an anabolic alpha-ALS-deficient E. coli strain for valine synthesis. The 243-amino-acid-long acetoin reductase has the consensus amino acid sequence for the insect-type alcohol dehydrogenase/ribitol dehydrogenase family and has extensive similarities with the N-terminal and internal regions of three known dehydrogenases and one oxidoreductase.

Modelling of the lignin peroxidase LIII of Phlebia radiata: use of a sequence template generated from a 3-D structure.

A model of the lignin peroxidase LIII of Phlebia radiata was constructed on the basis of the structure of cytochrome c peroxidase (CCP). Because of the low percentage of amino acid identity between the CCP and the lignin peroxidase LIII of Phlebia radiata, alignment of the sequences was based on the generation of a template from a knowledge of the 3-D structure of CCP and consensus sequences of lignin peroxidases. This approach gave an alignment in which all the insertions in the lignin peroxidase were placed at loop regions of CCP, with a 21.1% identity for these two proteins. The model was constructed using this alignment and the computer program COMPOSER, which assembles the model as a series of rigid fragments derived from CCP and other proteins. Manual intervention was required for some of the longer loop regions. The alpha-helices forming the structural framework, and especially the haem environment of CCP, are conserved in the LIII model and the core is close packed without holes. A possible site of the substrate oxidation at the haem edge of LIII is discussed.

Investigation of the function of mutated cellulose-binding domains of Trichoderma reesei cellobiohydrolase I.

The function of the cellulose-binding domain (CBD) of the cellobiohydrolase I of Trichoderma reesei was studied by site-directed mutagenesis of two amino acid residues identified by analyzing the 3D structure of this domain. The mutant enzymes were produced in yeast and tested for binding and activity on crystalline cellulose. Mutagenesis of the tyrosine residue (Y492) located at the tip of the wedge-shaped domain to alanine or aspartate reduced the binding and activity on crystalline cellulose to the level of the core protein lacking the CBD. However, there was no effect on the activity toward small oligosaccharide (4-methylumbelliferyl beta-D-lactoside). The mutation tyrosine to histidine (Y492H) lowered but did not destroy the cellulose binding, suggesting that the interaction of the pyranose ring of the substrate with an aromatic side chain is important. However, the catalytic activity of this mutant on crystalline cellulose was identical to the other two mutants. The mutation P477R on the edge of the other face of the domain reduces both binding and activity of CBHI. These results support the hypothesis that both surfaces of the CBD are involved in the interaction of the binding domain with crystalline cellulose.

Saccharomyces cerevisiae cells secreting an Aspergillus niger beta-galactosidase grow on whey permeate.

We describe the construction of a lactose-utilizing Saccharomyces cerevisiae that expresses the cDNA for a secreted, thermostable beta-galactosidase (lacA) from Aspergillus niger. Yeast cells expressing the lacA gene from the yeast ADH1 promotor on a multicopy plasmid secrete up to 40% of the total beta-galactosidase activity into the growth medium. The secreted product is extensively N-glycosylated, and cells expressing the lacA gene grow on whey permeate (4% w/v lactose) with a doubling time of 1.6 hours. Such strains may offer a solution to the increasing problem of waste whey disposal.

Production of functional IgM Fab fragments by Saccharomyces cerevisiae.

The aim of this study was to express and secrete functional mouse IgM fragments in yeast. The heavy chain cDNA was truncated at two different sites, yielding genes coding for the complete VH domain. In one of the truncated genes, the CH1 domain is complete, while in the other gene 18 bp are missing from the 3' terminus of the CH1 region. Both shortened genes were coexpressed in Saccharomyces cerevisiae with a cDNA gene encoding a full length mouse Ig light chain. We show that only the longer form of the truncated heavy chain together with the light chain produced and secreted functional IgM Fab fragments.

An active single-chain antibody containing a cellulase linker domain is secreted by Escherichia coli.

Single-chain antibodies consist of the variable, antigen-binding domains of antibodies joined to a continuous polypeptide by genetically engineered peptide linkers. We have used the flexible interdomain linker region of a fungal cellulase to link together the variable domains of an anti-2-phenyloxazolone IgG1 and show here that the resulting single-chain antibody is efficiently secreted and released to the culture medium of Escherichia coli. The yield of affinity-purified single-chain antibody is 1-2 mg/l of culture medium and its affinity and stability are comparable to those of the corresponding native IgG.

Promoter structure and expression of the 3-phosphoglycerate kinase-encoding gene (pgk1) of Trichoderma reesei.

Transcription of the 3-phosphoglycerate kinase (PGK)-encoding gene (pgk1) of Trichoderma reesei results in two transcripts due to two main transcription start points (tsp) which are differentially regulated during the growth cycle. The nucleotide sequence of the promoter reveals a number of putative regulatory elements present also in the PGK promoter of Saccharomyces cerevisiae: a 20-nt long sequence similar to the CTTCC-repeat region of the upstream activating sequence UAS, the eukaryotic heat-shock consensus sequence, HSE, and a putative eukaryotic cAMP regulatory sequence. The functionality of the putative HSE sequence was examined, but no clear effect could be seen on the total amount of pgk1 mRNA at elevated temperatures nor on transcription initiation from the upstream tsp, preceded by the HSE sequence.

Isolation and structural analysis of the laccase gene from the lignin-degrading fungus Phlebia radiata.

We have isolated and characterized a gene coding for the laccase of the lignin-degrading fungus Phlebia radiata. The gene has nine introns and recognizable fungal promoter elements. Sequences homologous to the consensus eukaryotic heat-shock regulatory element can be found in the promoter. RNA hybridization results indicate that this gene is regulated at the transcriptional level. The derived laccase amino acid sequence shows homology to plant ascorbate oxidases, suggesting that the basic structure of the laccase is similar to the three-fold repeated beta-barrel of the ascorbate oxidases. Potential copper ligands and a residue carrying the prosthetic group pyrroloquinoline quinone (PQQ) in the laccase protein can be identified by homology. The intron/exon structure of the laccase gene suggests that this protein could have evolved by exon shuffling.

Enzyme production by recombinant Trichoderma reesei strains.

The production of both homologous and heterologous proteins with the cellulolytic filamentous fungus Trichoderma reesei is described. Biotechnically important improvements in the production of cellulolytic enzymes have been obtained by genetic engineering methodology to construct strains secreting novel mixtures of cellulases. These improvements have been achieved by gene inactivation and promoter changes. The strong and highly inducible promoter of the gene encoding the major cellulase, cellobiohydrolase I (CBHI) has also been used for the production of eukaryotic heterologous proteins in Trichoderma. The expression and secretion of active calf chymosin is described in detail.

Site-directed mutagenesis of the putative catalytic residues of Trichoderma reesei cellobiohydrolase I and endoglucanase I.

Site directed mutagenesis has been performed to test hypotheses concerning the putative active sites of Trichoderma reesei cellobiohydrolase I and endoglucanase I. It is shown that mutagenesis of the residue E126, previously proposed to be the proton donor in CBHI, did not totally inactivate the enzyme while mutagenesis of the residue E127 in the homologous enzyme EGI resulted in complete loss of activity. These results are compared with those obtained in similar studies of other glucanases and the effects on enzymatic activity of hyperglycosylation of the yeast produced cellulases are discussed.

Three-dimensional structure of cellobiohydrolase II from Trichoderma reesei.

The enzymatic degradation of cellulose is an important process, both ecologically and commercially. The three-dimensional structure of a cellulase, the enzymatic core of CBHII from the fungus Trichoderma reesei reveals an alpha-beta protein with a fold similar to but different from the widely occurring barrel topology first observed in triose phosphate isomerase. The active site of CBHII is located at the carboxyl-terminal end of a parallel beta barrel, in an enclosed tunnel through which the cellulose threads. Two aspartic acid residues, located in the center of the tunnel are the probable catalytic residues.

Molecular modelling study of antigen binding to oxazolone-specific antibodies: the Ox1 idiotypic IgG and its mature variant with increased affinity to 2-phenyloxazolone.

Structural models of the variable domains of the murine anti-2-phenyloxazolone IgG (Ox1 idiotype) and its somatic variant, which has higher affinity to the hapten 2-phenyloxazolone, were constructed by computer-aided model building using known structures of highly homologous immunoglobulins as templates. Molecular dynamics simulations were used to dock the hapten between the VL and VH domains. The hapten is predicted to bind to slightly different sites in the two models. Hypotheses concerning the role of a number of preferred mutations in anti-oxazolone variants are presented. These can be tested by mutagenesis and crystallography. In particular, the higher binding affinities of the different antibody variants are shown to correlate with better complementarity of electrostatics. The molecular dynamic simulations also suggest that two mobile tryptophans at the mouth of the pocket may play an important role in the binding of hapten.