The Role of a Loop in the Non-catalytic Domain B on the Hydrolysis/Transglycosylation Specificity of the 4-α-Glucanotransferase from Thermotoga maritima.

The mechanism by which glycoside hydrolases control the reaction specificity through hydrolysis or transglycosylation is a key element embedded in their chemical structures. The determinants of reaction specificity seem to be complex. We looked for structural differences in domain B between the 4-α-glucanotransferase from Thermotoga maritima (TmGTase) and the α-amylase from Thermotoga petrophila (TpAmylase) and found a longer loop in the former that extends towards the active site carrying a W residue at its tip. Based on these differences we constructed the variants W131G and the partial deletion of the loop at residues 120-124/128-131, which showed a 11.6 and 11.4-fold increased hydrolysis/transglycosylation (H/T) ratio relative to WT protein, respectively. These variants had a reduction in the maximum velocity of the transglycosylation reaction, while their affinity for maltose as the acceptor was not substantially affected. Molecular dynamics simulations allow us to rationalize the increase in H/T ratio in terms of the flexibility near the active site and the conformations of the catalytic acid residues and their associated pKas.

Modulating Glycoside Hydrolase Activity between Hydrolysis and Transfer Reactions Using an Evolutionary Approach.

The proteins within the CAZy glycoside hydrolase family GH13 catalyze the hydrolysis of polysaccharides such as glycogen and starch. Many of these enzymes also perform transglycosylation in various degrees, ranging from secondary to predominant reactions. Identifying structural determinants associated with GH13 family reaction specificity is key to modifying and designing enzymes with increased specificity towards individual reactions for further applications in industrial, chemical, or biomedical fields. This work proposes a computational approach for decoding the determinant structural composition defining the reaction specificity. This method is based on the conservation of coevolving residues in spatial contacts associated with reaction specificity. To evaluate the algorithm, mutants of α-amylase (TmAmyA) and glucanotransferase (TmGTase) from Thermotoga maritima were constructed to modify the reaction specificity. The K98P/D99A/H222Q variant from TmAmyA doubled the transglycosydation/hydrolysis (T/H) ratio while the M279N variant from TmGTase increased the hydrolysis/transglycosidation ratio five-fold. Molecular dynamic simulations of the variants indicated changes in flexibility that can account for the modified T/H ratio. An essential contribution of the presented computational approach is its capacity to identify residues outside of the active center that affect the reaction specificity.

Deep Eutectic Solvents as New Reaction Media to Produce Alkyl-Glycosides Using Alpha-Amylase from Thermotoga maritima.

Deep Eutectic Solvents (DES) were investigated as new reaction media for the synthesis of alkyl glycosides catalyzed by the thermostable α-amylase from Thermotoga maritima Amy A. The enzyme was almost completely deactivated when assayed in a series of pure DES, but as cosolvents, DES containing alcohols, sugars, and amides as hydrogen-bond donors (HBD) performed best. A choline chloride:urea based DES was further characterized for the alcoholysis reaction using methanol as a nucleophile. As a cosolvent, this DES increased the hydrolytic and alcoholytic activity of the enzyme at low methanol concentrations, even when both activities drastically dropped when methanol concentration was increased. To explain this phenomenon, variable-temperature, circular dichroism characterization of the protein was conducted, finding that above 60 °C, Amy A underwent large conformational changes not observed in aqueous medium. Thus, 60 °C was set as the temperature limit to carry out alcoholysis reactions. Higher DES contents at this temperature had a detrimental but differential effect on hydrolysis and alcoholysis reactions, thus increasing the alcoholyisis/hydrolysis ratio. To the best of our knowledge, this is the first report on the effect of DES and temperature on an enzyme in which structural studies made it possible to establish the temperature limit for a thermostable enzyme in DES.

CRISPR-Cas system presents multiple transcriptional units including antisense RNAs that are expressed in minimal medium and upregulated by pH in Salmonella enterica serovar Typhi.

The CRISPR-Cas system is involved in bacterial immunity, virulence, gene regulation, biofilm formation and sporulation. In Salmonella enterica serovar Typhi, this system consists of five transcriptional units including antisense RNAs. It was determined that these genetic elements are expressed in minimal medium and are up-regulated by pH. In addition, a transcriptional characterization of cas3 and ascse2-1 is included herein.

Exopolyphosphatase of Pseudomonas aeruginosa is essential for the production of virulence factors, and its expression is controlled by NtrC and PhoB acting at two interspaced promoters.

The exopolyphosphatase (Ppx) of Pseudomonas aeruginosa is encoded by the PA5241 gene (ppx). Ppx catalyses the hydrolysis of inorganic polyphosphates to orthophosphate (Pi). In the present work, we identified and characterized the promoter region of ppx and its regulation under environmental stress conditions. The role of Ppx in the production of several virulence factors was demonstrated through studies performed on a ppx null mutant. We found that ppx is under the control of two interspaced promoters, dually regulated by nitrogen and phosphate limitation. Under nitrogen-limiting conditions, its expression was controlled from a σ(54)-dependent promoter activated by the response regulator NtrC. However, under Pi limitation, the expression was controlled from a σ(70) promoter, activated by PhoB. Results obtained from the ppx null mutant demonstrated that Ppx is involved in the production of virulence factors associated with both acute infection (e.g. motility-promoting factors, blue/green pigment production, C6-C12 quorum-sensing homoserine lactones) and chronic infection (e.g. rhamnolipids, biofilm formation). Molecular and physiological approaches used in this study indicated that P. aeruginosa maintains consistently proper levels of Ppx regardless of environmental conditions. The precise control of ppx expression appeared to be essential for the survival of P. aeruginosa and the occurrence of either acute or chronic infection in the host.

Choline catabolism, σ54 factor and NtrC are required for the full expression of the Pseudomonas aeruginosa phosphorylcholine phosphatase gene.

Choline favors the pathogenesis of Pseudomonas aeruginosa because hemolytic phospholipase C and phosphorylcholine phosphatase (PchP) are synthesized as a consequence of its catabolism. The experiments performed here resulted in the identification of the factors that regulate both the catabolism of choline and the gene coding for PchP. We have also identified and characterized the promoter of the pchP gene, its transcriptional organization and the factors that affect its expression. Deletion analyses reveal that the region between -188 and -68 contains all controlling elements necessary for pchP expression: a hypothetical -12/-24 promoter element, a consensus sequence for the integration host factor (-141/-133), and a palindromic sequence resembling a binding site for a potential enhancer binding protein (-190/-174). Our data also demonstrate that choline catabolism and NtrC (nitrogen regulatory protein) are necessary for the full expression of pchP and is partially dependent on σ(54) factor.

Systematic discovery of analogous enzymes in thiamin biosynthesis.

In all genome-sequencing projects completed to date, a considerable number of 'gaps' have been found in the biochemical pathways of the respective species. In many instances, missing enzymes are displaced by analogs, functionally equivalent proteins that have evolved independently and lack sequence and structural similarity. Here we fill such gaps by analyzing anticorrelating occurrences of genes across species. Our approach, applied to the thiamin biosynthesis pathway comprising approximately 15 catalytic steps, predicts seven instances in which known enzymes have been displaced by analogous proteins. So far we have verified four predictions by genetic complementation, including three proteins for which there was no previous experimental evidence of a role in the thiamin biosynthesis pathway. For one hypothetical protein, biochemical characterization confirmed the predicted thiamin phosphate synthase (ThiE) activity. The results demonstrate the ability of our computational approach to predict specific functions without taking into account sequence similarity.

A Neurospora crassa mutant altered in the regulation of L-amino acid oxidase.

The isolation and characterization of a Neurospora crassa mutant altered in L-amino oxidase regulation is reported. The previously isolated gln-1bR8 strain, which only synthesizes the glutamine synthetase alpha monomer and lacks the beta monomer, was used as parental strain. A mutant derivative of strain was selected for its ability to grow on minimal medium in the presence of DL-methionine-SR-sulfoximine (MSO), an inhibitor of glutamine synthetase activity. This gln-1bR8;MSOR mutant overcame the inhibitory effect of MSO by increasing the activity of L-amino acid oxidase, an enzyme capable of degrading this compound. In contrast with the wild-type strain, the L-amino acid oxidase of the MSOR mutant was resistant to glutamine repression; in fact, it was induced by this amino acid but repressed by ammonium. This mutant is different from other nitrogen regulatory N. crassa mutants reported and is only altered in the regulation of L-amino acid oxidase. The MSOR mutation is epistatic to nit-2 since the nit2;MSOR double mutant regulated the L-amino acid oxidase in the same way as the MSOR single mutant.