The role of oil in macromolecular crystallization.

The different facets of the utilization of oil demonstrate that an individual oil and/or combinations of different oils can influence the outcome of crystallization experiments. The oil can play a part in the control of nucleation, affect the rate of equilibration and consequently determine the size of the forming crystals. Whether used for microbatch, vapour diffusion or for control of nucleation, the presence of oil is a parameter that can contribute to the accuracy, cleanliness and to the increase in the reproducibility of the experiments. Furthermore, the oil has a role in the protection of the trials during the course of their duration and in maintaining the stability of the resulting crystals.

Cloning of a novel prolidase gene from Aureobacterium esteraromaticum.

The prolidase gene from Aureobacterium esteraromaticum was cloned and expressed in Escherichia coli. The cloned enzyme had the same enzymatic properties as the wild-type enzyme. Kinetic analysis of the enzyme indicated that the best substrate was Pro-Hyp, which was not hydrolyzed by other prolidases. Interestingly, there was no homology between the deduced amino acid sequence of A. esteraromaticum prolidase and those of the other sources such as human E. coli and Lactobacillus. However, homology was seen with the yeast hypothetical protein YJL213w, the function of which is unknown. These findings indicate that the A. esteraromaticum prolidase is a novel enzyme different from other prolidases reported to date.

Hydrostatic pressure induces conformational and catalytic changes on two alcohol dehydrogenases but no oligomeric dissociation.

A comparison between the pressure effects on the catalysis of Thermoanaerobium brockii alcohol dehydrogenase (TBADH: a thermostable tetrameric enzyme) and yeast alcohol dehydrogenase (YADH: a mesostable tetrameric enzyme) revealed a different behaviour. YADH activity is continuously inhibited by an increase of pressure, whereas YADH affinity seems less sensitive to pressure. TBADH activity is enhanced by pressure up to 100 MPa. TBADH affinity for alcoholic substrates increases if pressure increases, was TBADH affinity for NADP decreases when pressure increases. Hypothesis has been raised concerning the dissociation of oligomeric enzymes under high hydrostatic pressure ( < 200 MPa) [1]. But in the case of these two enzymes, unless the oligomers reassociate very quickly (< 1 min), the activity inhibition of YADH at all pressures and TBADH for pressures above 100 MPa is not correlated to subunit dissociation. Hence we suggest that enzymes under pressure encounter a molecular rearrangement which can either have a positive or a negative effect on activity. Finally, we have observed that the catalytic behaviour of alcohol dehydrogenases under pressure is connected to their thermostability.

Cloning, sequencing and expression of the gene encoding NADH oxidase from the extreme anaerobic thermophile Thermoanaerobium brockii.

The gene encoding the enzyme NADH oxidase from the extreme thermophile Thermoanaerobium brockii has been isolated from a recombinant library of genomic DNA and sequenced. An open reading frame corresponds to the 651 amino acids of the enzyme's subunit, which include characteristic FAD- and NADH-binding sequences, as well as cysteines which are involved in the FeS cluster present in the enzyme. The enzyme is expressed either from its own promoter or from vector promoters in Escherichia coli. After heat-treating the recombinant extracts at 70 degrees C, most of the host proteins are denatured, leaving the NADH oxidase 5- to 10-fold enriched.

Crystallization and preliminary X-ray diffraction studies of xylose isomerase from Thermoanaerobacterium thermosulfurigenes strain 4B.

Xylose isomerase from the thermophile Thermoanaerobacterium thermosulfurigenes strain 4B has been crystallized by vapour diffusion from Jeffamine ED 4000 as precipitant. The crystal symmetry is P2(1)2(1)2(1), with unit cell dimensions a = 85.6 A, b = 153.5 A and c = 158.5 A. The protein molecular mass and volume of the unit cell is consistent with the presence of a tetramer of the enzyme in the asymmetric unit. The crystals diffract X-rays from a synchrotron source to 1.7 A resolution.

The cellulose-binding domains from Cellulomonas fimi beta-1, 4-glucanase CenC bind nitroxide spin-labeled cellooligosaccharides in multiple orientations.

The N-terminal cellulose-binding domains CBDN1 and CBDN2 from Cellulomonas fimi cellulase CenC each adopt a jelly-roll beta-sandwich structure with a cleft into which amorphous cellulose and soluble cellooligosaccharides bind. To determine the orientation of the sugar chain within these binding clefts, the association of TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl-4-yl) spin-labeled derivatives of cellotriose and cellotetraose with isolated CBDN1 and CBDN2 was studied using heteronuclear 1H-15N NMR spectroscopy. Quantitative binding measurements indicate that the TEMPO moiety does not significantly perturb the affinity of the cellooligo-saccharide derivatives for the CBDs. The paramagnetic enhancements of the amide 1HN longitudinal (DeltaR1) and transverse (DeltaR2) relaxation rates were measured by comparing the effects of TEMPO-cellotetraose in its nitroxide (oxidized) and hydroxylamine (reduced) forms on the two CBDs. The bound spin-label affects most significantly the relaxation rates of amides located at both ends of the sugar-binding cleft of each CBD. Similar results are observed with TEMPO-cellotriose bound to CBDN1. This demonstrates that the TEMPO-labeled cellooligosaccharides, and by inference strands of amorphous cellulose, can associate with CBDN1 and CBDN2 in either orientation across their beta-sheet binding clefts. The ratio of the association constants for binding in each of these two orientations is estimated to be within a factor of five to tenfold. This finding is consistent with the approximate symmetry of the hydrogen-bonding groups on both the cellooligosaccharides and the residues forming the binding clefts of the CenC CBDs.

Cloning and characterization of the MamI restriction-modification system from Microbacterium ammoniaphilum in Escherichia coli.

The genes encoding a class-IIN restriction-modification (R-M) system (MamI, sequence specificity [symbol: see text] from Microbacterium ammoniaphilum have been cloned in Escherichia coli. The vector used for cloning was plasmid pUC18 modified by the inclusion of three MamI recognition sites. Recombinant clones containing the mamIM gene in its genomic context became fully methylated in vivo and remained completely resistant against digestion with the R.MamI restriction endonuclease (ENase). Determination of the nucleotide (nt) sequence revealed three open reading frames with lengths of 1089 bp (ORF1), 276 bp (ORFc) and 927 bp (ORF2). On the basis of expression and deletion experiments, the 1089-bp ORF1 was assigned to mamIM encoding the M.MamI DNA methyltransferase (MTase). By amino acid sequencing of the N terminus of R.MamI and comparison of the deduced nt sequence with ORF2, the 927-bp ORF2 was identified as the mamIR gene encoding R.MamI. The 276-bp ORFc, located between mamIR and mamIM, is part of the DNA sequence downstream from mamIM shown to be necessary for controlled mamIM expression.

The molecular structure of the Na(+)-translocating F1F0-ATPase of Acetobacterium woodii, as revealed by electron microscopy, resembles that of H(+)-translocating ATPases.

The Na(+)-translocating F1F0-ATPase of Acetobacterium woodii was examined by electron microscopy. After reconstitution into proteoliposomes, knobs typical for the F1 domain were visible on the outside of the membrane. The F1-part of the isolated enzyme showed a hexagonal symmetry suggesting an alpha 3 beta 3 structure, and the F1F0 complex had molecular dimensions very similar to those of H(+)-translocating ATPases of E. coli, chloroplasts, and mitochondria.

Sequence of subunit a of the Na(+)-translocating F1F0-ATPase of Acetobacterium woodii: proposal for residues involved in Na+ binding.

Na+ transport through the F0 domain of Na(+)-F1F0-ATPases involves the combined action of subunits c and a but the residues involved in Na+ liganding in subunit a are unknown. As a first step towards the identification of these residues, we have cloned and sequenced the gene encoding subunit a of the Na(+)-F1F0-ATPase of Acetobacterium woodii. This is the second sequence available now for this subunit from Na(+)-F1F0-ATPases. A comparison of subunit a from Na(+)-F1F0-ATPases with those from H(+)-translocating enzymes unraveled structural similarity in a C-terminal segment including the ultimate and penultimate transmembrane helix. Seven residues are conserved in this region and, therefore, likely to be involved in Na+ liganding.

Molecular cloning of CWP1: a gene encoding a Saccharomyces cerevisiae cell wall protein solubilized with Rarobacter faecitabidus protease I.

A yeast cell wall glycoprotein with a molecular weight of 40,000, named gp40, was solubilized from SDS-extracted cell wall of Saccharomyces cerevisiae by incubation with Rarobacter faecitabidus protease I, which is a yeast-lytic enzyme. Based on its amino acid sequence, we cloned and sequenced the gene encoding the precursor of gp40, named CWP1; cell wall protein gene. The DNA sequence of the CWP1 gene was identical to YKL443, an open reading frame identified in a genome sequencing program for yeast chromosome XI. This gene encoded a serine-rich protein of 239 amino acids with a molecular weight of 24,267. The presence of hydrophobic sequences in the N- and C-termini of the CWP1 protein suggests that it is secreted as a glycosylphosphatidylinositol-anchored protein and is subsequently integrated into the cell wall. Since a gene disruption experiment showed no growth defect, the CWP1 gene is not essential for growth. Mutant CWP1 protein deficient in the C-terminal hydrophobic sequence was secreted into the culture medium, not anchored to the cell wall, thereby indicating that this hydrophobic sequence plays a crucial role in anchoring to the cell wall. Homology between the CWP1 protein and TIP1 family of cold shock proteins suggests that they belong to a new family of cell wall proteins.

Multiple xylanases of Cellulomonas fimi are encoded by distinct genes.

Cellulomonas fimi genomic DNA encoding xylanase activity has been cloned and expressed in Escherichia coli. As judged by DNA hybridization and restriction analysis, twelve xylanase-positive clones carried a minimum of four different xylanase (xyn) genes. The encoded enzymes were devoid of cellulase activity but three of the four bound to Avicel.

Anaerostipes caccae gen. nov., sp. nov., a new saccharolytic, acetate-utilising, butyrate-producing bacterium from human faeces.

Two strains of a previously undescribed Eubacterium-like bacterium were isolated from human faeces. The strains are Gram-variable, obligately anaerobic, catalase negative, asporogenous rod-shaped cells which produced acetate, butyrate and lactate as the end products of glucose metabolism. The two isolates displayed 99.9% 16S rRNA gene sequence similarity to each other and treeing analysis demonstrated the faecal isolates are far removed from Eubacterium sensu stricto and that they represent a new subline within the Clostridium coccoides group of organisms. Based on phenotypic and phylogenetic criteria, it is proposed that the two strains from faeces be classified as a new genus and species, Anaerostipes caccae. The type strain of Anaerostipes caccae is NCIMB 13811T (= DSM 14662T).

Cloning of endoglucanase genes from Cellulomonas biazotea into E. coli and S. cerevisiae using shuttle vector YEp24.

We constructed a SmaI genomic library of Cellulomonas biazotea DNA in E. coli and in the S. cerevisiae shuttle vector, YEP 24. Three clone were identified that conferred the ability for E. coli or S. cerevisiae transformants to produce carboxymethylcellulase (CMCase). Cells transformed with these clones were compared with one another and with nontransformed cells for hyper-production of CMCase. In vivo and in vitro studies indicated that the CMCase genes were fully expressed and the enzyme activity was located extracellularly. The optimum pH and temperature for the CMCase thus cloned were pH 7 and 50 degrees C, respectively, as was the case for the donor.

Cloning and expression of beta-glucosidase genes in Escherichia coli and Saccharomyces cerevisiae using shuttle vector pYES 2.0.

Genes for beta-glucosidase (Bgl) isolated from a genomic library of the cellulolytic bacterium, Cellulomonas biazotea, were cloned in pUC18 in its SacI cloning site and transformed to E. coli. Ten putative recombinants showed blackening zones on esculin plates, yellow zones on pNPG plates, in liquid culture and on native polyacrylamide gel electrophoresis activity gels. They fell into three distinct groups. Three representative E. coli clones carried recombinant plasmids designated pRM54, pRM1 and pRM17. The genes were located on 5.6-, 3.7- and 1.84-kb fragments, respectively. Their location was obtained by deletion analysis which revealed that 5.5, 3.2, and 1.8 kb fragments were essential to code for BglA, BglB, and BglC, respectively, and conferred intracellular production of beta-glucosidase on E. coli. Expression of the bgl genes resulted in overproduction of beta-glucosidase in the three clones. Secretion occurred into the periplasmic fractions. Three inserts carrying bgl genes from the representative recombinant E. coli were isolated with SacI, ligated in the shuttle vector pYES 2.0 in its SacI site and transformed to E. coli and S. cerevisiae. The recombinant plasmids were redesignated pRPG1, pRPG2 and pRPG3 coding for BglA1, BglB1 and BglC1. The cloned genes conferred extracellular production of beta-glucosidase on S. cerevisiae and enabled it to grow on cellobiose and salicin. The gall promoter of shuttle vector pYES 2.0 enabled the organisms to produce twice more beta-glucosidase than that supported by the lacZ-promoter of pUC18 plasmid in E. coli. The cloned gene can be used as a selection marker for introducing recombinant plasmids in wild strains of S. cerevisiae. The enzyme produced by bgl+ yeast and E. coli recombinants resembles that of the donor with respect to temperature and pH requirement for maximum activity. Other enzyme properties of the beta-glucosidases from S. cerevisiae were substantially the same as those from C. biazotea.

Crystal structure of the catalytic domain of the beta-1,4-glycanase cex from Cellulomonas fimi.

beta-1,4-Glycanases, principally cellulases and xylanases, are responsible for the hydrolysis of plant biomass. The bifunctional beta-1,4-xylanase/glucanase Cex from the bacterium Cellulomonas fimi, one of a large family of cellulases/xylanases, depolymerizes oligosaccharides and releases a disaccharide unit from the substrate nonreducing end. Hydrolysis occurs with net retention of the anomeric configuration of the sugar through a double-displacement mechanism involving a covalent glycosyl-enzyme intermediate. The active site nucleophile, Glu233, has been unambiguously identified by trapping of such an intermediate [Tull et al. (1991) J. Biol. Chem. 266, 15621-15625] and the acid/base catalyst, Glu127, by detailed kinetic analysis of mutants [MacLeod et al. (1994) Biochemistry 33, 6371-6376]. However, little is known about the enzyme's overall folding and its active site architecture. We report here the high-resolution crystal structure of the catalytic domain of Cex. The atomic structure refinement results in a model that includes 2400 protein atoms and 45 water molecules, with an R-factor of 0.217 for data extending to 1.8-A resolution. The protein forms an eight-parallel-stranded alpha/beta-barrel, which is a novel folding pattern for a microbial beta-glycanase. The active site, inferred from the location of Glu233, Glu127, and other conserved residues, is an open cleft on the carboxy-terminal end of the alpha/beta-barrel. An extensive hydrogen-bonding network stabilizes the ionization states of the key residues; in particular, the Asp235-His205-Glu233 hydrogen-bonding network may play a role in modulating the ionization state of Glu233 and in controlling local charge balance during the reaction.

Purification and characterization of a prolidase from Aureobacterium esteraromaticum.

An EDTA-insensitive prolidase (proline dipeptidase, EC 3.4.13.9) was isolated from a cell-free extract of Aureobacterium esteraromaticum IFO 3752. The enzyme was purified almost to homogeneity using acetone precipitation, hydrophobic chromatography, ion-exchange chromatography, and gel-permeation chromatography. The enzyme has a molecular weight of about 440,000 by gel permeation chromatography, and about 40,000 by SDS polyacrylamide gel electrophoresis. The isoelectric point was 4.6. The enzyme hydrolyzed aminoacylprolines such as Ser-Pro. Thr-Pro, Gly-Pro, Ala-Pro, Ile-Pro, Leu-Pro, and Pro-Pro. It also hydrolyzed Gly-Hyp and Pro-Hyp. The rate of hydrolysis for Pro-Hyp was the highest among the substrates tested. Optimum pH for hydrolyzing Pro-Hyp was 9.0 and the enzyme was stable in the pH range from 5 to 10. The optimum temperature was estimated to be 45 degrees C using 10 min of reaction. At least 90% of the initial activity remained after 30 min of incubation at 60 degrees C. p-Chloromercuribenzoic acid and o-phenanthrolin inhibited the enzyme's activity while EDTA did not. Addition of Mn2+ ion did not stimulate activity. These results suggest either that the metal ion in the enzyme may be tightly bound to the polypeptide chain, or that the enzyme is not a metallo-enzyme but a thiol-enzyme.

Immunological versatility and carbon regulation of Cellulomonas fimi endo-1,4-beta-glucanases.

More than 10 protein molecules with endo-1,4-beta-glucanase activity were identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and zymogram in Cellulomonas fimi culture supernatants, grown in CMC as carbon source. These molecules are shown to belong to at least four immunologically different groups, against three of which polyclonal antibodies were raised. The protein species used as antigens showed significant differences in cross reactivity, carbon regulation, and affinity to crystalline cellulose. Three intracellular precursors of the first group were detected, two of which were under carbon catabolite control with the third apparently being synthesized constitutively. In the extracellular environment this group showed the largest versatility in protein molecules. The second group appeared to originate from two intracellular precursors both synthesized constitutively and subject to minor extracellular modifications as compared to the first group. The main extracellular protein of this group showed high affinity toward crystalline cellulose. One intracellular precursor was identified for the third group, which was subject to carbon catabolite control. Only one extracellular molecule without binding ability to crystalline cellulose corresponded to this precursor, indicating that the latter was resistant to proteolytic modifications after excretion. It appears that the C. fimi cellulases are more complex than expected and reconstitution of the whole system will be difficult.

Changes in the molecular-size distribution of insoluble celluloses by the action of recombinant Cellulomonas fimi cellulases.

Specific patterns of attacks of cotton, bacterial cellulose and bacterial microcrystalline cellulose (BMCC) by recombinant cellulases of Cellulomonas fimi were investigated. Molecular-size distributions of the celluloses were determined by high-performance size-exclusion chromatography. Chromatography of cotton and bacterial celluloses revealed single major peaks centered over progressively lower molecular-mass positions during attack by endoglucanase CenA. In advanced stages, a second peak appeared at very low average size (approx. 11 glucosyl units); ultimate weight losses were approximately 30%. The isolated catalytic domain of CenA, p30, gave results very similar to those with complete CenA. CenA did not effectively depolymerize or solubilize BMCC significantly. Molecular-size distributions of cotton and bacterial cellulose incubated with endoglucanases CenB or CenD exhibited one major peak regardless of incubation time; low-molecular-mass fragments did not accumulate. Weight losses were 40 and 35% respectively. The single peak shifted to lower-molecular-mass positions as incubation continued, but high-molecular-mass material persisted. CenB and CenD readily attacked and solubilized BMCC (approx. 70%). We conclude that CenA attacks cellulose by preferentially cleaving completely through the cellulose microfibrils at the amorphous sites, and much more slowly by degrading the crystalline surfaces. Conversely, CenB and CenD cleave the amorphous regions much less efficiently while vigorously degrading the surfaces of the crystalline regions of the microfibrils.

Crystallographic observation of a covalent catalytic intermediate in a beta-glycosidase.

The three-dimensional structure of a catalytically competent glycosyl-enzyme intermediate of a retaining beta-1,4-glycanase has been determined at a resolution of 1.8 A by X-ray diffraction. A fluorinated slow substrate forms an alpha-D-glycopyranosyl linkage to one of the two invariant carboxylates, Glu 233, as supported in solution by 19F-NMR studies. The resulting ester linkage is coplanar with the cyclic oxygen of the proximal saccharide and is inferred to form a strong hydrogen bond with the 2-hydroxyl of that saccharide unit in natural substrates. The active-site architecture of this covalent intermediate gives insights into both the classical double-displacement catalytic mechanism and the basis for the enzyme's specificity.