Tapani Reinikainen

Extreme Halophiles Synthesize Betaine from Glycine by Methylation

Glycine betaine is a compatible solute, which is able to restore and maintain osmotic balance of living cells. It is synthesized and accumulated in response to abiotic stress. Betaine acts also as a methyl group donor and has a number of important applications including its use as a feed additive. The known biosynthetic pathways of betaine are universal and very well characterized. A number of enzymes catalyzing the two-step oxidation of choline to betaine have been isolated. In this work we have studied a novel betaine biosynthetic pathway in two phylogenically distant extreme halophiles,Actinopolyspora halophila and Ectothiorhodospira halochloris. We have identified a three-step series of methylation reactions from glycine to betaine, which is catalyzed by two methyltransferases, glycine sarcosine methyltransferase and sarcosine dimethylglycine methyltransferase, with partially overlapping substrate specificity. The methyltransferases from the two organisms show high sequence homology. E. halochlorismethyltransferase genes were successfully expressed inEscherichia coli, and betaine accumulation and improved salt tolerance were demonstrated.

The difference in affinity between two fungal cellulose-binding domains is dominated by a single amino acid substitution

Cellulose‐binding domains (CBDs) form distinct functional units of most cellulolytic enzymes. We have compared the cellulose‐binding affinities of the CBDs of cellobiohydrolase I (CBHI) and endoglucanase I (EGI) from the fungus Trichoderma reesei. The CBD of EGI had significantly higher affinity than that of CBHI. Four variants of the CBHI CBD were made in order to identify the residues responsible for the increased affinity in EGI. Most of the difference could be ascribed to a replacement of a tyrosine by a tryptophan on the flat cellulose‐binding face.

Domain function in Trichoderma reesei cellobiohydrolases

Abstract The filamentous fungus, Trichoderma reesei produces all of the enzymatic activities required for efficient hydrolysis of highly ordered crystalline cellulose. All the principal enzymes involved in cellulose hydrolysis possess two functional domains, one directly involved in catalysis and the other in substrate recognition and binding. The availability of high-resolution three dimensional structures of the two domains allows genetic engineering to be used for detailed structure-function studies of these important enzymes.

Trichoderma reesei cellobiohydrolase I with an endoglucanase cellulose-binding domain: action on bacterial microcrystalline cellulose

Cellulolytic enzymes consist of distinct catalytic and cellulose-binding domains (CBDs). The presence of a CBD improves the binding and activity of cellulases on insoluble substrates but has no influence on their activities on soluble substrates. Structural and biochemical studies of a fungal CBD from Trichoderma reesei cellobiohydrolase I have revealed a wedge shaped structure with a flat cellulose binding surface containing three essential tyrosine residues. The face of the wedge is strictly conserved in all fungal CBDs while many differences occur on the other face of the wedge. Here we have studied the importance of these differences on the function of the T. reesei CBHI by replacing its CBD by a homologous CBD from the endoglucanase, EGI. Our data shows that, apart from slightly improved affinity of the hybrid enzyme, the domain exchange does not significantly influence the function of CBHI.

Characterization of Glycine Sarcosine N-Methyltransferase and Sarcosine Dimethylglycine N-Methyltransferase

ABSTRACT Glycine betaine is accumulated in cells living in high salt concentrations to balance the osmotic pressure. Glycine sarcosineN-methyltransferase (GSMT) and sarcosine dimethylglycineN-methyltransferase (SDMT) of Ectothiorhodospira halochloris catalyze the threefold methylation of glycine to betaine, with S-adenosylmethionine acting as the methyl group donor. These methyltransferases were expressed inEscherichia coli and purified, and some of their enzymatic properties were characterized. Both enzymes had high substrate specificities and pH optima near the physiological pH. No evidence of cofactors was found. The enzymes showed Michaelis-Menten kinetics for their substrates. The apparent Km andVmax values were determined for all substrates when the other substrate was present in saturating concentrations. Both enzymes were strongly inhibited by the reaction productS-adenosylhomocysteine. Betaine inhibited the methylation reactions only at high concentrations.

Hydrolysis of barley (1→3), (1→4)-β-d-glucan by a cellobiohydrolase II preparation from Trichoderma reesei

Abstract The molecular weight of commercial barley β-glucan was 250,000 as determined by dual angle laser light scattering. 1H NMR analysis showed the polymer to contain 29% (1→3)-linkages and 71% (1→4)-linkages. The barley β-glucan was readily hydrolysed by a highly purified cellobiohydrolase II (CBHII) preparation from Trichoderma reesei. NMR data demonstrated that the cellulase preparation degraded only (1→4)-linkages in the β-glucan chain. Neither internal G(1→3)G(1→4)G nor reducing end G(1→3)G(1→4)G(1→4)G sequences were hydrolysed. The main hydrolysis products were: cellobiose, β- d -Glcp-(1→3)-β- d -Glcp-(l→4)- d -Glcp, β- d -Glcp-(1→3)-β- d -Glcp-(1→4)-β- d -Glcp-(1→4)- d -Glcp and β- d -Glcp-(1→4)-β- d -Glc d -(1→3)-β- d -Glcp-(1→4)-β- d -Glcp. Statistical models of the glucan linkage sequence were fitted to the relative fragment concentrations after CBHII and lichenase degradations. The hydrolysate compositions are well reproduced by a second order Markov chain. All degradation data are consistent with the assumed degradation mechanisms of the two enzymes, including the hypothesis that hydrolysis by CBHII depends on the glycosidic bond orientation.

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

Site directed mutagenesis has been performed to test hypotheses concerning the putative active sites of Trichoderma reesci cellobiohydrolase I and endoglucanase I. It is shown that mutagenesis of the residue 1:126, previously proposed to be the proton donor in CBHI, did not totally inactive the enzyme while mutagenesis of the residue 1:127 in the homologous enzyme EG1 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.

Low-level endoglucanase contamination in a Trichoderma reesei cellobiohydrolase II preparation affects its enzymatic activity on β-glucan

A routinely employed and specific affinity chromatography purification method was used to isolate cellobiohydrolase II (CBHII) from a Trichoderma reesei culture supernatant. Two different identically purified batches of CBHII were found to have distinctly different properties in barley β-glucan hydrolysis. The abilities of the two preparations to produce small oligosaccharides and reduce the viscosity of β-glucan were substantially different, but no contamination or heterogeneity was detected in sodium dodecyl sulfate-electrophoresis used to assess the protein purity. Furthermore, the specific activities of the preparations on cellotetraose substrate were similar. Careful analysis with specific chromogenic oligosaccharide substrates showed that the considerable variation in β-glucan hydrolysis was caused by a minor endoglucanase contamination consisting of only 0.4% of the total protein. The contamination is possibly caused by nonspecific interactions between the proteins and chromatographic column material or by unfavorable protein-protein interactions during the chromatographic separation. The data demonstrate clearly the caution needed in the interpretation of hydrolysis studies on polymeric substrates with cellulolytic enzyme preparations.

Comparison of the adsorption properties of a single-chain antibody fragment fused to a fungal or bacterial cellulose-binding domain

Trichoderma reesei cellobiohydrolase I (CBHI) and Cellulomonas fimi cellulase-xylanase (Cex) both have distinct C-terminal cellulose-binding domains which belong to different CBD sequence families. Two fusion proteins comprising a single-chain antibody fragment (OxscFv) against 2-phenyloxazolone fused to the two CBDs (CBDCBHI or CBDCex) were constructed. The binding properties of the fusion proteins were studied on different cellulosic substrates. It was shown that the CBDCex binds the fusion protein to cellulose more effectively than the CBDCBHI; however, once immobilized, both fusion proteins could be eluted from cellulose only with denaturing agents or very low or high pH. Both fusion proteins retained equally well their ability to bind the hapten recognized by their antibody part.

Modes of action of two Trichoderma reesei cellobiohydrolases

Abstract Trichoderma reesei degrades native cellulose utilizing a set of cellulolytic enzymes dominated by two cellobiohydrolases, CBHI and CBHII. These enzymes exhibit the typical two domain architecture of fungal cellulases, and both act primarily as exoglucanases liberating cellobiose from the ends of the polymeric cellulose chains. The three dimensional structures of the catalytic domains of CBHI and CBHII revealed that their active sites are situated in tunnels formed by long loops on the enzymes surface. The active sites of homologous endoglucanases lack these loops and have more open active sites permitting catalytic activity in the internal positions of cellulose chains. Site-directed mutagenesis and structural studies have identified the key catalytic residues of both CBHI and CBHII. Similarly, the primary interaction surface of the cellulose-binding domain has been defined and residues responsible for its tight binding to cellulose identified.