The interactions of RNase A with cytidine 3'-monophosphate (3"CMP) and deoxycytidyl-3',5'-deoxyadenosine (d(CpA)) were analyzed by X-ray crystallography. The 3'-CMP complex and the native structure were determined from trigonal crystals, and the d(CpA) complex from monoclinic crystals. The differences between the overall structures are concentrated in loop regions and are relatively small. The protein-inhibitor contacts are interpreted in terms of the catalytic mechanism. The general base His 12 interacts with the 2' oxygen, as does the electrostatic catalyst Lys 41. The general acid His 119 has 2 conformations (A and B) in the native structure and is found in, respectively, the A and the B conformation in the d(CpA) and the 3'-CMP complex. From the present structures and from a comparison with RNase T1, we propose that His 119 is active in the A conformation. The structure of the d(CpA) complex permits a detailed analysis of the downstream binding site, which includes His 119 and Asn 71. The comparison of the present RNase A structures with an inhibitor complex of RNase T1 shows that there are important similarities in the active sites of these 2 enzymes, despite the absence of any sequence homology. The water molecules were analyzed in order to identify conserved water sites. Seventeen water sites were found to be conserved in RNase A structures from 5 different space groups. It is proposed that 7 of those water molecules play a role in the binding of the N-terminal helix to the rest of the protein and in the stabilization of the active site.

Kinetic analyses of bacterial growth, carbohydrate consumption, and metabolite production of 18 Bifidobacterium strains grown on fructose, oligofructose, or inulin were performed. A principal component analysis of the data sets, expanded with the results of a genetic screen concerning the presence of a ␤-fructofuranosidase gene previously encountered in Bifidobacterium animalis subsp. lactis DSM 10140 T , revealed the existence of four clusters among the bifidobacteria tested. Strains belonging to a first cluster could not degrade oligofructose or inulin. Strains in a second cluster could degrade oligofructose, displaying a preferential breakdown mechanism, but did not grow on inulin. Fructose consumption was faster than oligofructose degradation. A third cluster was composed of strains that degraded all oligofructose fractions simultaneously and could partially break down inulin. Oligofructose degradation was substantially faster than fructose consumption. A fourth, smaller cluster consisted of strains that shared high fructose consumption and oligofructose degradation rates and were able to perform partial breakdown of inulin. For all strains, a metabolic shift toward more acetate, formate, and ethanol production, at the expense of lactate production, was observed during growth on less readily fermentable energy sources. No correlation between breakdown patterns and the presence of the ␤-fructofuranosidase gene could be detected. These variations indicate niche-specific adaptation of bifidobacteria and could have in vivo implications on the strain specificity of the stimulatory effect of inulin-type fructans on bifidobacteria.

It is widely accepted that many phase transitions do not follow nucleation pathways as envisaged by the classical nucleation theory. Many substances can traverse intermediate states before arriving at the stable phase. The apparent ubiquity of multi-step nucleation has made the inverse question relevant: does multistep nucleation always dominate single-step pathways? Here we provide an explicit example of the classical nucleation mechanism for a system known to exhibit the characteristics of multi-step nucleation. Molecular resolution atomic force microscopy imaging of the two-dimensional nucleation of the protein glucose isomerase demonstrates that the interior of subcritical clusters is in the same state as the crystalline bulk phase. Our data show that despite having all the characteristics typically associated with rich phase behaviour, glucose isomerase 2D crystals are formed classically. These observations illustrate the resurfacing importance of the classical nucleation theory by re-validating some of the key assumptions that have been recently questioned.

The purification and characterization of triose-phosphate isomerase from the psychrophilic bacterium Vibrio marinus (vTIM) is described. Crystal structures of the vTIM-sulfate complex and the vTIM-2-phosphoglycolate complex (at a 2.7-Å resolution) are also presented. The optimal growth temperature of Vibrio marinus is 15°C. Stability studies show that vTIM is an unstable protein with a half-life of only 10 min at 25°C. The vTIM sequence is most closely related to the sequence of Escherichia coli TIM (eTIM) (66% identity), and several unique structural features described for eTIM are also seen in vTIM, but eTIM is considerably more stable. The T d values of vTIM and eTIM, determined by calorimetric studies, are 41 and 54°C, respectively. Amino acid sequence comparison reveals that vTIM has an alanine in loop 8 (at position 238), whereas all other TIM sequences known to date have a serine. The vTIM mutant A238S was produced and characterized. Compared with wild type, the catalytic efficiency of the A238S mutant is somewhat reduced, and its stability is considerably increased.Triose-phosphate isomerase (TIM 1 ; EC 5.3.1.1) is a dimeric glycolytic enzyme formed by two identical subunits consisting of about 250 residues each. It catalyzes the interconversion of dihydroxyacetone phosphate and D-glyceraldehyde-3-phosphate (see Fig. 1). TIM has been the subject of extensive biophysical, enzymological, and computational studies. Its catalytic properties and mechanism have been studied in detail (1, 2). It has been established that TIM is a very efficient catalyst, since the reaction rates are diffusion-controlled. Neither cofactors nor metal ions are required in this reaction, and there is no evidence of allostery or cooperativity among the subunits.Structurally TIMs do form a well characterized family. To date, x-ray structures of TIM from seven different sources have been solved (wild type or/and in complex with substrate analogues): chicken (3), yeast (4), Trypanosoma brucei (5), Escherichia coli (6), human (7), Bacillus stearothermophilus (8), and Plasmodium falciparum (9). In addition, the structures have been determined of Leishmania mexicana TIM 2 and of Thermotoga maritima TIM. 3 Also, 45 amino acid TIM sequences from a wide variety of organisms have been determined and are available in the data bases.Each TIM monomer has a globular shape with two protruding loops. The globular part is formed by an 8-fold repeat of a ␤-strand, loop, ␣-helix, loop motif. The ␤␣ units fold up in a regular way, such that the ␤-strands (numbered ␤1 to ␤8) form an eight-stranded ␤-barrel surrounded by the eight ␣-helices (numbered ␣1 to ␣8) on the outside. This folding motif is also referred to as the TIM barrel motif. In TIM, the two protruding loops are after ␤-strand three and after ␤-strand six.The TIM barrel structural motif is found for a large number of other enzymes with widely different functions (10) that have little or no sequence homology. As such this stable framework with tolerant sequence variations has already been use...