Accounting for the instantaneous disorder in the enzyme-substrate Michaelis complex to calculate the Gibbs free energy barrier of an enzyme reaction.

Many enzyme reactions present instantaneous disorder. These dynamic fluctuations in the enzyme-substrate Michaelis complexes generate a wide range of energy barriers that cannot be experimentally observed, but that determine the measured kinetics of the reaction. These individual energy barriers can be calculated using QM/MM methods, but then the problem is how to deal with this dispersion of energy barriers to provide kinetic information. So far, the most usual procedure has implied the so-called exponential average of the energy barriers. In this paper, we discuss the foundations of this method, and we use the free energy perturbation theory to derive an alternative equation to get the Gibbs free energy barrier of the enzyme reaction. In addition, we propose a practical way to implement it. We have chosen four enzyme reactions as examples. In particular, we have studied the hydrolysis of a glycosidic bond catalyzed by the enzyme Thermus thermophilus ß-glycosidase, and the mutant Y284P Ttb-gly, and the hydrogen abstraction reactions from C13 and C7 of arachidonic acid catalyzed by the enzyme rabbit 15-lipoxygenase-1.

Comparing Hydrolysis and Transglycosylation Reactions Catalyzed by Thermus thermophilus ß-Glycosidase. A Combined MD and QM/MM Study.

The synthesis of oligosaccharides and other carbohydrate derivatives is of relevance for the advancement of glycosciences both at the fundamental and applied level. For many years, glycosyl hydrolases (GHs) have been explored to catalyze the synthesis of glycosidic bonds. In particular, retaining GHs can catalyze a transglycosylation (T) reaction that competes with hydrolysis (H). This has been done either employing controlled conditions in wild type GHs or by engineering new mutants. The goal, which is to increase the T/H ratio, has been achieved with moderate success in several cases despite the fact that the molecular basis for T/H modulation are unclear. Here we have used QM(DFT)/MM calculations to compare the glycosylation, hydrolysis and transglycosylation steps catalyzed by wild type Thermus thermophilus ß-glycosidase (family GH1), a retaining glycosyl hydrolase for which a transglycosylation yield of 36% has been determined experimentally. The three transition states have a strong oxocarbenium character and ring conformations between 4H3 and 4E. The atomic charges at the transition states for hydrolysis and transglycosylation are very similar, except for the more negative charge of the oxygen atom of water when compared to that of the acceptor Glc. The glycosylation transition state has a stronger S N 2 character than the deglycosylation ones and the proton transfer is less advanced. At the QM(PBE0/TZVP)/MM level, the TS for transglycosylation has shorter O4GLC-C1FUC (forming bond) distance and longer OE2GLU338-C1FUC (breaking) distance than the hydrolysis one, although the HACC proton is closer to the Glu164 base in the hydrolysis TS. The QM(SCC-DFTB)/MM free energy maxima show the inverted situation, although the hydrolysis TS presents significant structural fluctuations. The 3-OHGLC group of the acceptor Glc (transglycosylation) and WAT432 (neighbor water in hydrolysis) are identified to stabilize the oxocarbenium transition states through interaction with O5FUC and O4FUC. The analysis of interaction suggests that perturbing the Glu392-Fuc interaction could increase the T/H ratio, either by direct mutation of this residue or indirectly as reported experimentally in the Asn390I and Phe401S cases. The molecular understanding of similarities and differences between hydrolysis and transglycosylation steps may be of help in the design of new biocatalysts for glycan synthesis.

Polymorphism of genes encoding PmrAB in colistin-resistant strains of Escherichia coli and Salmonella enterica isolated from poultry and swine.

OBJECTIVES: To detect the occurrence of low susceptibility to colistin (polymyxin E), a last-resort antimicrobial, among enterobacteria isolated from samples of animal origin (poultry and swine) and to find out the molecular basis of colistin resistance. METHODS: Salmonella enterica and Escherichia coli were isolated from eggs and swine samples. Bacterial strains were screened for colistin resistance by using MIC determinations interpreted according to EUCAST recommendations. pmrAB genes were amplified by PCR from bacterial isolates and their sequences were characterized. RESULTS: Nine colistin-resistant strains were detected in a collection of 739 enterobacteria (S. enterica and E. coli) isolated from animal samples taken in different environments. Sequences encoding the PmrAB two-component sensor-regulator from two colistin-resistant E. coli strains isolated from swine faeces presented three non-synonymous polymorphisms, producing the variants 39S → I and 81R → S of PmrA and 161V → G of PmrB, among which the involvement of mutations in PmrA-81 and PmrB-161 in resistance to the antimicrobial had been previously shown. No variation at the protein level was detected after analysis of PmrAB sequences from seven colistin-resistant S. enterica strains. CONCLUSIONS: E. coli strains carrying mutations in PmrAB that confer resistance to polymyxins, which might have evolved in vivo and have been rarely detected, are described for the first time in enterobacteria isolated from animals.

Septicemic salmonellosis caused by Salmonella Hessarek in wintering and migrating Song Thrushes (Turdus philomelos) in Spain.

We investigated two mortality events in wintering and migrating Song Thrushes (Turdus philomelos) in Catalonia, northeastern Spain in 2009 and 2010. Both episodes occurred in late February to mid-March during the spring migration. Salmonellosis produced by the serotype Salmonella enterica subsp. enterica serotype Hessarek (S. Hessarek) was identified as the cause of death in both episodes. Poor body condition, marked splenomegaly, and microscopic disseminated intravascular coagulation with numerous intravascular and tissular bacteria were the most consistent findings. Macro-restriction profiling by pulsed-field gel electrophoresis (PFGE) using XbaI was performed for epidemiologic typing of the S. Hessarek isolates. Two clusters were discernible, that are possibly related, with a similarity of 82.8%. Analysis comparing pectoral muscle and subcutaneous fat scores from the Song Thrushes that died from S. Hessarek with those from healthy Song Thrushes from nearby areas during 2009 and 2010 suggest that poor body condition was associated with the S. Hessarek infection.

Unexpected inefficiency of the European pharmacopoeia sterility test for detecting contamination in clostridial vaccines.

Three outbreaks of clostridial disease in ruminants were reported in Spain. Out of 202,525 animals in affected herds, 41,767 were infected and 22,189 died. Epidemiological investigation linked these outbreaks with vaccination with three different commercial anti-clostridial vaccines contaminated with Clostridium sordellii. Vaccines were produced by the same manufacturer. Microbiological and molecular genetic analyses confirmed this association, and isolates of C. sordellii with identical biochemical and pulsed-field gel electrophoresis (PFGE) macrorestriction patterns were isolated from both diseased animals and the epidemiologically related vaccines. Contamination of the commercial vaccines was not detected by the sterility test proposed by the European (EU) Pharmacopoeia. However, growth was obtained when the commercial vaccines were inoculated into specific culture media for Clostridium and incubated for up to 60 days.

Salmonella diversity associated with wild reptiles and amphibians in Spain.

During the spring and summer of 2001, faeces from 166 wild reptiles (94 individuals) and amphibians (72 individuals) from 21 different species found in central Spain were examined for the presence of Salmonella. Thirty-nine reptiles (41.5%) yielded 48 Salmonella isolates, whereas all the amphibians examined were negative. Subspecies Salmonella enterica enterica (I) accounted for up to 50% of isolates. Fourteen isolates (29.2%) belonged to subspecies diarizonae (IIIb), six isolates (12.5%) to subspecies salamae (II), and four isolates (8.3%) to subspecies arizonae (IIIa). Twenty-seven different serotypes were identified. Serotypes Anatum (12.5%), Herzliya (8.3%), Abony, 18:l,v:z, 9,12:z29:1,5 and 38:z10:z53 (6.2%/each) were the most frequently isolated. A high percentage (39.6%) of isolates belonged to serotypes previously associated with environmental sources. Also, 37.5% of isolates belonged to serotypes which had been related to human cases of salmonellosis. From these data, it is concluded that wild reptiles, but apparently not amphibians, may represent an important reservoir of Salmonella in nature and have potential implications for public health.