Comparison of family 9 cellulases from mesophilic and thermophilic bacteria

Cellulases containing a family 9 catalytic domain and a family 3c cellulose binding module (CBM3c) are important components of bacterial cellulolytic systems. We measured the temperature dependence of the activities of three homologs: Clostridium cellulolyticum Cel9G, Thermobifida fusca Cel9A, and C. thermocellum Cel9I. To directly compare their catalytic activities, we constructed six new versions of the enzymes in which the three GH9-CBM3c domains were fused to a dockerin both with and without a T. fusca fibronectin type 3 homology module (Fn3). We studied the activities of these enzymes on crystalline cellulose alone and in complex with a miniscaffoldin containing a cohesin and a CBM3a. The presence of Fn3 had no measurable effect on thermostability or cellulase activity. The GH9-CBM3c domains of Cel9A and Cel9I, however, were more active than the wild type when fused to a dockerin complexed to scaffoldin. The three cellulases in complex have similar activities on crystalline cellulose up to 60°C, but C. thermocellum Cel9I, the most thermostable of the three, remains highly active up to 80°C, where its activity is 1.9 times higher than at 60°C. We also compared the temperature-dependent activities of different versions of Cel9I (wild type or in complex with a miniscaffoldin) and found that the thermostable CBM is necessary for activity on crystalline cellulose at high temperatures. These results illustrate the significant benefits of working with thermostable enzymes at high temperatures, as well as the importance of retaining the stability of all modules involved in cellulose degradation.     

The thermostability of DNA-binding protein HU from mesophilic, thermophilic, and extreme thermophilic bacteria

Based on primary structure comparison between four highly homologous DNA-binding proteins (HUs) displaying differential thermostability, we have employed in vitro site-directed mutagenesis to decipher their thermostability mechanism at the molecular level. The contribution of the 11 amino acids that differ between the thermophilic HUBst from Bacillus stearothermophilus (Tm = 61.6 degrees C) and the mesophilic HUBsu from Bacillus subtilis (Tm = 39.7 degrees C) was evaluated by replacing these amino acids in HUBst with their mesophilic counterparts. Among 11 amino acids, three residues, Gly-15, Glu-34, and Val-42, which are highly conserved in the thermophilic HUs, have been found to be responsible for the thermostability of HUBst. These amino acids in combination (HUBst-G15E/E34D/V42I) reduce the thermostability of the protein (Tm = 45.1 degrees C) at the level of its mesophilic homologue HUBsu. By replacing these amino acids in HUBsu with their thermophilic counterparts, the HUBsu-E15G/D34E/142V mutant was generated with thermostability (Tm = 57.8 degrees C) at the level of thermophilic HUBst. Employing the same strategy, we generated several mutants in the extremely thermophilic HUTmar from Thermotoga maritima (Tm = 80.5 degrees C), and obtained data consistent with the previous results. The triplet mutant HUTmar-G15E/E34D/V421 (Tm = 35.9 degrees C) converted the extremely thermophilic protein HUTmar to mesophilic. The various forms of HU proteins were overproduced in Escherichia coli, highly purified, and the thermostability of the mutants confirmed by circular dichroism spectroscopy. The results presented here were elucidated on the basis of the X-ray structure of HUBst and HUTmar (our unpublished results), and their mechanism was proposed at the molecular level. The results clearly show that three individual local interactions located at the helix-turn-helix part of the protein are responsible for the stability of HU proteins by acting cooperatively in a common mechanism for thermostability.     

Isocitrate dehydrogenase from thermophilic and mesophilic bacteria. Isolation and some characteristics

1. Simple methods incorporating the principle of selective enzyme elution from a triazinyl dye adsorbent with a mixture of NADP+ and isocitrate are described for isolating NADP+-linked isocitrate dehydrogenase in pure state from several mesophilic and thermophilic bacteria. 2. Several characteristics of the isocitrate dehydrogenases have been examined, viz. molecular size, amino acid composition including the content of sulphydryl groups, thermostability and structural homology by the criterion of immunological cross-section.     

Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria. I) Isolation and characterization of lactate dehydrogenases from thermophilic and mesophilic bacilli.

Lactate dehydrogenases from thermophilic bacilli (Bacillus stearothermophilus, Bacillus caldotenax) and from mesophilic bacilli (Bacillus X1, Bacillus subtilis) have been isolated by a two-step purification procedure. Only one type (LDH-P4) composed of four identical subunits (Mr 34 000 or 36 000) was found in each bacillus. The tetrameric enzymes were characterized with respect to thermostability, pH and temperature dependence of the pyruvate reduction and the L-lactate oxidation, substrate specificity, saturation kinetics (Km values of pyruvate, lactate, NAD, NADH), pyruvate and oxamate inhibition, and activation by fructose bisphosphate. The thermophilic and mesophilic enzymes differ characteristically in these parameters. Preliminary structural data (amino acid composition, comparative N-terminal sequence analysis) show the expected close phylogenetic relationship (high degree of sequence homology), but also typical differences between thermophilic and mesophilic dehydrogenases, a suitable basis for further comparative studies.     

Stability of the 'L12 stalk' in ribosomes from mesophilic and (hyper)thermophilic Archaea and Bacteria

The ribosomal stalk complex, consisting of one molecule of L10 and four or six molecules of L12, is attached to 23S rRNA via protein L10. This complex forms the so-called ‘L12 stalk’ on the 50S ribosomal subunit. Ribosomal protein L11 binds to the same region of 23S rRNA and is located at the base of the ‘L12 stalk’. The ‘L12 stalk’ plays a key role in the interaction of the ribosome with translation factors. In this study stalk complexes from mesophilic and (hyper)thermophilic species of the archaeal genus Methanococcus and from the Archaeon Sulfolobus solfataricus, as well as from the Bacteria Escherichia coli, Geobacillus stearothermophilus and Thermus thermophilus, were overproduced in E.coli and purified under non-denaturing conditions. Using filter-binding assays the affinities of the archaeal and bacterial complexes to their specific 23S rRNA target site were analyzed at different pH, ionic strength and temperature. Affinities of both archaeal and bacterial complexes for 23S rRNA vary by more than two orders of magnitude, correlating very well with the growth temperatures of the organisms. A cooperative effect of binding to 23S rRNA of protein L11 and the L10/L12₄ complex from mesophilic and thermophilic Archaea was shown to be temperature-dependent.     

Simple efficient methods for the isolation of malate dehydrogenase from thermophilic and mesophilic bacteria.

Malate dehydrogenase from a number of bacteria drawn from several genera and representing the mesophilic, moderately thermophilic and extremely thermophilic classes was isolated by procedures which involve only a small number of steps (in most cases only two), of which the key one is affinity chromatography on 5'-AMP--Sepharose and/or on NAD+--hexane--agarose. Electrophoretic analysis of the native enzymes in polyacrylamide gel and of the denaturated enzymes in sodium dodecyl sulphate/polyacrylamide gel revealed no significant protein impurity in the purified preparations. The yields ranged from about 40% to over 80%. The malate dehydrogenases from the extreme thermophiles and from some of the moderate thermophiles are appreciably less efficient catalytically than their mesophilic homologues.     

Functional 70S hybrid ribosomes from blue-green algae and bacteria

Hybrid 70S ribosomes were produced by combining Anacystis nidulans and Escherichia coli 30S and 50S subunits. Both the A. nidulans 30S-E. coli 50S and E. coli 30S- A. nidulans 50S hybrids were functional in synthesizing protein when tested in a standard in vitro amino acid incorporating system. Both 70S hybrids were inhibited by streptomycin but the degree of inhibition was dependent upon the source of the 30S subunit. The ability to form functional 70S ribosomes from subunits of blue-green algae and bacteria is further evidence of the procaryotic nature of blue-greens and of the functional homology of the two protein synthesizing systems.     

Xylan-hydrolysing enzymes from thermophilic and mesophilic fungi

Screening of 40 mesophilic and 13 thermophilic fungi indicated that enzyme activities capable of degrading oat spelt xylan extensively were produced by only a few of the mesophilic species investigated. The relatively low degree of hydrolysis effected by the enzymes from thermophilic organisms could be explained, in part, by their lack of -xylosidase. Several strains of Aspergillus awamori and Aspergillus phoenicis were notable in producing high xylanase and -xylosidase and low protease activities. Of the fungl tested, 13 produced activities capable of removing O-acetyl, arabinosyl, 4-O-methylglucuronyl, feruloyl and coumaroyl substituents from the backbone of xylan polysaccharides as well as endo-1,4--d-xylanase and -1,4-xylosidase. When the growth medium contained oat spelt xylan as carbon source, higher levels of xylanase, -xylosidase and acetyl xylan esterase were found than in cultures containing meadow fescue grass but the latter were richer in ferulic acid and coumaric acid esterases and 4-O-methylglucuronidase. No single organism or carbon source used was capabie of producing high levels of all the debranching enzymes as well as high levels of enzymes capable of cleaving the glycosidic linkages of the xylan backbone. The best ballnce of enzymes was obtained in cultures of A. awamori IMI 142717 and NRRL 2276 and A. phoenicis IMI 214827. Either of these would be suitable for strain improvement studies.The authors are with The Rowett Research Institute. Bucksburn, Aberdeen, AB2 9SB, UK.T.M. Wood is the corresponding author.     

Structural differences between mesophilic, moderately thermophilic and extremely thermophilic protein subunits: results of a comprehensive survey

BACKGROUND: Proteins from thermophilic organisms usually show high intrinsic thermal stability but have structures that are very similar to their mesophilic homologues. From prevous studies it is difficult to draw general conclusions about the structural features underlying the increased thermal stability of thermophilic proteins. RESULTS: In order to reveal the general evolutionary strategy for changing the heat stability of proteins, a non-redundant data set was compiled comprising all high-quality structures of thermophilic proteins and their mesophilic homologues from the Protein Data Bank. The selection (quality) criteria were met by 64 mesophilic and 29 thermophilic protein subunits, representing 25 protein families. From the atomic coordinates, 13 structural parameters were calculated, compared and evaluated using statistical methods. This study is distinguished from earlier ones by the strict quality control of the structures used and the size of the data set. CONCLUSIONS: Different protein families adapt to higher temperatures by different sets of structural devices. Regarding the structural parameters, the only generally observed rule is an increase in the number of ion pairs with increasing growth temperature. Other parameters show just a trend, whereas the number of hydrogen bonds and the polarity of buried surfaces exhibit no clear-cut tendency to change with growth temperature. Proteins from extreme thermophiles are stabilized in different ways to moderately thermophilic ones. The preferences of these two groups are different with regards to the number of ion pairs, the number of cavities, the polarity of exposed surface and the secondary structural composition.     

Structure and function of L-lactate dehydrogenases from thermophilic and mesophilic bacteria, IV. The primary structure of the mesophilic lactate dehydrogenase from Bacillus subtilis

The complete amino-acid sequence of lactate dehydrogenase from the mesophilic Bacillus subtilis (B. X1) was determined. Approximately 70% of the sequence was obtained by sequence analysis of intact protein (N-terminal sequence) and of four CNBr fragments (CNBr3, CNBr4, CNBr5 and CNBr6). Sequences overlapping the CNBr fragments were determined from polypeptide fragments obtained by cleavage using o-iodosobenzoic acid (cleavage at Trp) or clostripain (cleavage at Arg). The C-terminal amino-acid residue (Asn) was detected by carboxypeptidase Y-degradation. Lactate dehydrogenase from B. subtilis shows a 69% sequence homology to that from the thermophilic strain B. stearothermophilus, and a 34% sequence homology to those from higher organism. The homology of these enzymes is particularly high at the active site regions (the coenzyme and substrate binding sites). The relatively high sequence conservation of the lactate dehydrogenases from B. subtilis and B. stearothermophilus (and from other bacilli) allows a structural comparison of this temperature variants.     

Structural and dynamic aspects of beta-glycosidase from mesophilic and thermophilic bacteria by multitryptophanyl emission decay studies

The tryptophanyl emission decay of beta-glycosidase from the extremophilic archaeon Sulfolobus solfataricus (Sbetagly) has been investigated by frequency domain fluorometry. The data were analyzed in terms of sum of discrete lifetimes as well as in terms of quasi- continuous lifetime distributions of different shape. At neutral pH the emission decay is characterized by two components: a long-lived component, centered at 7.4 ns, and a short one at 2.7 ns, irrespective of the decay scheme used for the interpretation of the experimental results. The effects of an irreversible inhibitor, that is, cyclophellitol, and that of a powerful denaturant such as guanidinium hydrochloride on the dynamics of Sbetagly has been investigated by observing the changes induced in the two components of the tryptophanyl emission decay. The addition of cyclophellitol to native Sbetagly reduces the contribution of the short-lived component but does not affect the long-lived one. Increasing concentrations of guanidinium hydrochloride differently affect the contributions of the two emission components. Higher concentrations were required to unfold the molecular regions containing the long-lived indolic fluorophores. These results indicate that the long-lived contribution arises from tryptophanyl residues deeply clustered in the interior of the protein matrix, whereas the short-lived one includes residues located in less rigid and more solvent accessible regions, some of which might be located in functionally important parts of protein. The knowledge of the crystallographic structure of Sbetagly allowed us to evaluate some average parameters for each tryptophanyl microenvironment in the Sbetagly such as hydrophobicity, structural flexibility, and ability of side chains to act as fluorescence quenchers. These results permitted to divide the tryptophanyl fluorescence of Sbetagly in the contribution of two emitting groups: one consisting of eight closely clustered tryptophans, that is, Trp 33, 36, 60, 84, 151 174, 425, and 433, responsible for the long-lived emission component and the other one, composed of nine tryptophans nearer to the subunit surface, that is, Trp 12, 156, 192, 287, 288, 316, 361, 376, 455, associable to the short-lived emission component. Finally, the examination of the tryptophanyl emission decay of the mesophilic beta-galactosidase from Escherichia coli (Cbetagal) and the Arrhenius analysis of its dependence on temperature indicated that the tryptophanyl environments of the mesophilic enzyme are rather homogeneous in consequence of a larger protein dynamics.     

Occurrence and molecular characterization of cultivable mesophilic and thermophilic obligate anaerobic bacteria isolated from paper mills

The aim of this work was to characterize the cultivable obligate anaerobic bacterial population in paper mill environments. A total of 177 anaerobically grown bacterial isolates were screened for aerotolerance, from which 67 obligate anaerobes were characterized by automated ribotyping and 41 were further identified by partial 16S rDNA sequencing. The mesophilic isolates indicated 11 different taxa (species) within the genus Clostridium and the thermophilic isolates four taxa within the genus Thermoanaerobacterium and one within Thermoanaerobacter (both formerly Clostridium). The most widespread mesophilic bacterium was closely related to C. magnum and occurred in three of four mills. One mill was contaminated with a novel mesophilic bacterium most closely related to C. thiosulfatireducens. The most common thermophile was T. thermosaccharolyticum, occurring in all four mills. The genetic relationships of the mill isolates to described species indicated that most of them are potential members of new species. On the basis of identical ribotypes clay could be identified to be the contamination source of thermophilic bacteria. Automated ribotyping can be a useful tool for the identification of clostridia as soon as comprehensive identification libraries are available.