Biomedical subjects

Lorenzo Segovia

Publications and source records attributed to Lorenzo Segovia.

10 recordsLinked to original sources

PubMed2006

Identification and functional characterization of levS, a gene encoding for a levansucrase from Leuconostoc mesenteroides NRRL B-512 F.

A Leuconostoc mesenteroides NRRL B-512 F levansucrase gene, (levS), was isolated, sequenced and cloned in Escherichia coli. The recombinant enzyme was shown to be a fructosyltransferase producing a polymer identified by (13)C-NMR as levan. Based on sequence analysis, we found that this levansucrase is a mosaic protein, bearing structural features of glucosyltransferases in the amino and carboxy terminal regions similarly to inulosucrase from Leuconostoc citreum. The phylogenetic analysis of the C-terminal region domain of levansucrases from L. mesenteroides demonstrates that they group together into a novel putative sub-family of genes and evolved long before all other glucosyltransferases, while their catalytic domain structure is species related.

Bacterial Proteins↗

PubMed2005

Mimicking natural evolution in metallo-beta-lactamases through second-shell ligand mutations.

Metallo-beta-lactamases (MBLs) represent the latest generation of beta-lactamases. The structural diversity and broad substrate profile of MBLs allow them to confer resistance to most beta-lactam antibiotics. To explore the evolutionary potential of these enzymes, we have subjected the Bacillus cereus MBL (BcII) to a directed evolution scheme, which resulted in an increased hydrolytic efficiency toward cephalexin. A systematic study of the hydrolytic profile, substrate binding, and active-site features of the evolved lactamase reveal that directed evolution has shaped the active site by means of remote mutations to better hydrolyze cephalosporins with small, uncharged C-3 substituents. One of these mutations is found in related enzymes from pathogenic bacteria and is responsible for the increase in that enzyme's hydrolytic profile. The mutations lowered the activation energy of the rate-limiting step rather than improved the affinity of the enzyme toward these substrates. The following conclusions can be made: (i) MBLs are able to expand their substrate spectrum without sacrificing their inherent hydrolytic capabilities; (ii) directed evolution is able to mimic mutations that occur in nature; (iii) the metal-ligand strength is tuned by second-shell mutations, thereby influencing the catalytic efficiency; and (iv) changes in the position of the second Zn(II) ion in MBLs affect the substrate positioning in the active site. Overall, these results show that the evolution of enzymatic catalysis can take place by remote mutations controlling reactivity.

Amino Acid Substitution↗

PubMed2005

Cry11Aa toxin from Bacillus thuringiensis binds its receptor in Aedes aegypti mosquito larvae through loop alpha-8 of domain II.

Bacillus thuringiensis subs israelensis produces Cry toxins active against mosquitoes. Receptor binding is a key determinant for specificity of Cry toxins composed of three domains. We found that exposed loop alpha-8 of Cry11Aa toxin, located in domain II, is an important epitope involved in receptor interaction. Synthetic peptides corresponding to exposed regions in domain II (loop alpha-8, beta-4 and loop 3) competed binding of Cry11Aa to membrane vesicles from Aedes aegypti midgut microvilli. The role of loop alpha-8 of Cry11A in receptor interaction was demonstrated by phage display and site-directed mutagenesis. We isolated a peptide-displaying phage (P5.tox), that recognizes loop alpha-8 in Cry11Aa, interferes interaction with the midgut receptor and attenuates toxicity in bioassay. Loop alpha-8 mutants affected in toxicity and receptor binding were characterized.

Aedes↗

PubMed2005

A truncated isoform of pyroglutamyl aminopeptidase II produced by exon extension has dominant-negative activity.

Thyrotropin-releasing hormone is inactivated in the extracellular space by a membrane-bound peptidase, pyroglutamyl aminopeptidase II (PPII), a member of the M1 family of zinc metallopeptidases. The functional significance of multiple PPII RNA species expression is unknown. We detected, in rat tissues, a RNA species derived from an alternative processing at the exon 14-intron 14 boundary. The alternatively processed RNA encoded a shorter version of PPII (PPII*), lacking part of the C-terminal domain. PPII* was expressed in COS-7 (or C6 glioma) cells but it did not exhibit any PPII activity. Co-transfection of PPII and increasing amounts of PPII* expression vectors resulted in a dose-dependent reduction in PPII activity and the formation of covalent PPII-PPII* heterodimers. PPII* is therefore a powerful dominant-negative isoform of PPII, and heterodimerization may be its mechanism of action. Natural expression of shortened versions of M1 aminopeptidases may constitute a new mode of regulation of their activity.

Alternative Splicing↗

PubMed2005

gammaN-crystallin and the evolution of the betagamma-crystallin superfamily in vertebrates.

The beta and gamma crystallins are evolutionarily related families of proteins that make up a large part of the refractive structure of the vertebrate eye lens. Each family has a distinctive gene structure that reflects a history of successive gene duplications. A survey of gamma-crystallins expressed in mammal, reptile, bird and fish species (particularly in the zebrafish, Danio rerio) has led to the discovery of gammaN-crystallin, an evolutionary bridge between the beta and gamma families. In all species examined, gammaN-crystallins have a hybrid gene structure, half beta and half gamma, and thus appear to be the 'missing link' between the beta and gamma crystallin lineages. Overall, there are four major classes of gamma-crystallin: the terrestrial group (including mammalian gammaA-F); the aquatic group (the fish gammaM-crystallins); the gammaS group; and the novel gammaN group. Like the evolutionarily ancient beta-crystallins (but unlike the terrestrial gammaA-F and aquatic gammaM groups), both the gammaS and gammaN crystallins form distinct clades with members in fish, reptiles, birds and mammals. In rodents, gammaN is expressed in nuclear fibers of the lens and, perhaps hinting at an ancestral role for the gamma-crystallins, also in the retina. Although well conserved throughout vertebrate evolution, gammaN in primates has apparently undergone major changes and possible loss of functional expression.

Algorithms↗

PubMed2004

RNA silencing of rotavirus gene expression.

RNA interference (RNAi) is a double-stranded RNA (dsRNA)-triggered mechanism for suppressing gene expression, which is conserved in evolution and has emerged as a powerful tool to study gene function. Rotaviruses, the leading cause of severe diarrhea in young children, are formed by three concentric layers of protein, and a genome composed of 11 segments of dsRNA. Here, we show that the RNAi machinery can be triggered to silence rotavirus gene expression by sequence-specific short interfering RNAs (siRNAs). RNAi is also useful for the study of the virus-cell interactions, through the silencing of cellular genes that are potentially important for the replication of the virus. Interestingly, while the translation of mRNAs is readily stopped by the RNAi machinery, the viral transcripts involved in virus genome replication do not seem to be susceptible to RNAi. Since gene silencing by RNAi is very efficient and specific, this system could become a novel therapeutic approach for rotavirus and other virus infections, once efficient methods for in vivo delivery of siRNAs are developed. Although the use of RNAi as an antiviral therapeutic tool remains to be demonstrated, there is no doubt that this technology will influence drastically the way postgenomic virus research is conducted.

Gene Expression Regulation, Viral↗

PubMed2004

Phylogenetic distribution of DNA-binding transcription factors in bacteria and archaea.

We have addressed the distribution and abundance of 75 transcription factor (TF) families in complete genomes from 90 different bacterial and archaeal species. We found that the proportion of TFs increases with genome size. The deficit of TFs in some genomes might be compensated by the presence of proteins organizing and compacting DNA, such as histone-like proteins. Nine families are represented in all the bacteria and archaea we analyzed, whereas 17 families are specific to bacteria, providing evidence for regulon specialization at an early stage of evolution between the bacterial and archeal lineages. Ten of the 17 families identified in bacteria belong exclusively to the proteobacteria defining a specific signature for this taxonomical group. In bacteria, 10 families are lost mostly in intracellular pathogens and endosymbionts, while 9 families seem to have been horizontally transferred to archaea. The winged helix-turn-helix (HTH) is by far the most abundant structure (motif) in prokaryotes, and might have been the earliest HTH motif to appear as shown by its distribution and abundance in both bacterial and archaeal cellular domains. Horizontal gene transfer and lineage-specific gene losses suggest a progressive elimination of TFs in the course of archaeal and bacterial evolution. This analysis provides a framework for discussing the selective forces directing the evolution of the transcriptional machinery in prokaryotes.

Amino Acid Sequence↗

PubMed2004

Phylogenetic relationships of rhizobia based on citrate synthase gene sequences.

Partial nucleotide sequences of the citrate synthase (gltA) gene from different rhizobia genera were determined. Tree topologies based on this housekeeping gene were similar to that obtained using 16S rRNA sequences. However gltA appeared to be more reliable at determining phylogenetic relationships of closely related taxa. We propose gltA sequences as an additional tool to be used in molecular phylogenetic studies.

Alphaproteobacteria↗

PubMed2003

Evolutionary engineering of a beta-Lactamase activity on a D-Ala D-Ala transpeptidase fold.

The beta-Lactamase hydrolytic activity has arisen several times from DD-transpeptidases. We have been able to replicate the evolutionary process of beta-Lactamase activity emergence on a PBP2X DD-transpeptidase. Some of the most interesting changes, like modifying the catalytic properties of an enzyme, may require several mutations in concert; therefore it is essential to explore efficiently sequence space by generating the right diversity. We designed a biased combinatorial library in which biochemical and structural information were incorporated by site directed mutagenesis on relevant residues and then subjected to random mutagenesis to allow for mutations in unforeseen positions. We isolated mutants from this library conferring 10-fold higher cefotaxime resistance levels than the background wild-type through mutations exclusively in the coding sequence. We demonstrate that only three substitutions in the DD-transpeptidase active site, two produced by the directed and one by the random mutagenesis, are sufficient to acquire this activity. The purified product of one mutant (MutE) had a 10(5)-fold increase in cefotaxime deacylation rate allowing it to hydrolyze beta-Lactams yet it has apparently conserved DD-peptidase activity. This work is the first to show a possible evolutionary intermediate between a beta-Lactamase and a DD-transpeptidase necessary for the development of antibiotic resistance.

Amino Acid Sequence↗

PubMed1988

Effect of Naturally Occurring nif Reiterations on Symbiotic Effectiveness in Rhizobium phaseoli.

Most naturally occurring strains of Rhizobium phaseoli possess reiteration of the nif genes. Three regions contain nitrogenase structural genes in strain CFN42. Two of these regions (a and b) have copies of nifH, nifD, and nifK, whereas the third region (c) contains only nifH. Strains containing mutations in either nif region a or nif region b had significantly diminished symbiotic effectiveness compared with the wild-type strain on the basis of nodule mass, total nitrogenase activity per plant, nitrogenase specific activity, total nitrogen in the shoot, and percentage of nitrogen. A strain containing mutations in both nif region a and nif region b was totally ineffective. These data indicate that both nif region a and nif region b are needed for full symbiotic effectiveness in R. phaseoli.

Journal Article↗