Naturally occurring modification of the canonical A, G, C, and T bases can be found in the DNA of cellular organisms and viruses from all domains of life. Bacterial viruses (bacteriophages) are a particularly rich but still underexploited source of such modified variant nucleotides. The modifications conserve the coding and base-pairing functions of DNA, but add regulatory and protective functions. In prokaryotes, modified bases appear primarily to be part of an arms race between bacteriophages (and other genomic parasites) and their hosts, although, as in eukaryotes, some modifications have been adapted to convey epigenetic information. The first half of this review catalogs the identification and diversity of DNA modifications found in bacteria and bacteriophages. What is known about the biogenesis, context, and function of these modifications are also described. The second part of the review places these DNA modifications in the context of the arms race between bacteria and bacteriophages. It focuses p...

http://pubs.acs.org/doi/pdf/10.1021/acs.chemrev.6b00114

Biosynthesis and Function of Modified Bases in Bacteria and Their Viruses

Hydrolytic enzymes are versatile biocatalysts and find increasing applications in organic synthesis and a considerable number of industrial processes using these enzymes have been commercialized. Within this class, lipases (E.C. 3.1.1.3) and carboxyl esterases (E.C. 3.1.1.1) are frequently used as they accept a broad range of non-natural substrates, are usually very stable in organic solvents and exhibit good to excellent stereoselectivity.

Activity of lipases and esterases towards tertiary alcohols: insights into structure-function relationships.

Esterases form a diverse class of enzymes of largely unknown physiological role. Because many drugs and pesticides carry ester functions, the hydrolysis of such compounds forms at least one potential biological function. Carboxylesterases catalyze the hydrolysis of short chain aliphatic and aromatic carboxylic ester compounds. Esterases, D-alanyl-D-alanine-peptidases (DD-peptidases) and beta-lactamases can be grouped into two distinct classes of hydrolases with different folds and topologically unrelated catalytic residues, the one class comprising of esterases, the other one of beta-lactamases and DD-peptidases. The chemical reactivities of esters and beta-lactams towards hydrolysis are quite similar, which raises the question of which factors prevent esterases from displaying beta-lactamase activity and vice versa. Here we describe the crystal structure of EstB, an esterase isolated from Burkholderia gladioli. It shows the protein to belong to a novel class of esterases with homology to Penicillin binding proteins, notably DD-peptidase and class C beta-lactamases. Site-directed mutagenesis and the crystal structure of the complex with diisopropyl-fluorophosphate suggest Ser75 within the "beta-lactamase" Ser-x-x-Lys motif to act as catalytic nucleophile. Despite its structural homology to beta-lactamases, EstB shows no beta-lactamase activity. Although the nature and arrangement of active-site residues is very similar between EstB and homologous beta-lactamases, there are considerable differences in the shape of the active site tunnel. Modeling studies suggest steric factors to account for the enzyme's selectivity for ester hydrolysis versus beta-lactam cleavage.

EstB from Burkholderia gladioli: a novel esterase with a beta-lactamase fold reveals steric factors to discriminate between esterolytic and beta-lactam cleaving activity

Carboxylesterases containing the sequence motif GGGX catalyze hydrolysis of esters of chiral tertiary alcohols, albeit at only low to moderate enantioselectivity towards three model substrates (linalyl acetate, methyl-1-pentin-1-yl acetate, 2-phenyl-3-butin-2-yl acetate). In order to understand the molecular mechanism of enantiorecognition and to improve enantioselectivity towards this interesting substrate class, the interaction of both enantiomers with the substrate binding sites of acetylcholinesterases and p-nitrobenzyl esterase from Bacillus subtilis was modeled and correlated to experimental enantioselectivity. For all substrate-enzyme pairs, enantiopreference and ranking by enantioselectivity could be predicted by the model. In p-nitrobenzyl esterase, one of the key residues in determining enantioselectivity was G105: exchange of this residue by alanine led to a six-fold increase of enantioselectivity (E=19) towards 2-phenyl-3-butin-2-yl acetate. However, the effect of this mutation is personalized: towards the substrate linalyl acetate, the same mutant had a reversed enantiopreference. Thus, depending on the substrate structure, the same mutant had either increased enantioselectivity or opposite enantiopreference compared to wild type enzyme.

/pdf/a-molecular-mechanism-of-enantiorecognition-of-tertiary-3s3do85ol9.pdf

A Molecular Mechanism of Enantiorecognition of Tertiary Alcohols by Carboxylesterases

The discovery of ∼20-kb gene clusters containing a family of paralogs of tRNA guanosine transglycosylase genes, called tgtA5, alongside 7-cyano-7-deazaguanine (preQ0) synthesis and DNA metabolism genes, led to the hypothesis that 7-deazaguanine derivatives are inserted in DNA. This was established by detecting 2’-deoxy-preQ0 and 2’-deoxy-7-amido-7-deazaguanosine in enzymatic hydrolysates of DNA extracted from the pathogenic, Gram-negative bacteria Salmonella enterica serovar Montevideo. These modifications were absent in the closely related S. enterica serovar Typhimurium LT2 and from a mutant of S. Montevideo, each lacking the gene cluster. This led us to rename the genes of the S. Montevideo cluster as dpdA-K for 7-deazapurine in DNA. Similar gene clusters were analyzed in ∼150 phylogenetically diverse bacteria, and the modifications were detected in DNA from other organisms containing these clusters, including Kineococcus radiotolerans, Comamonas testosteroni, and Sphingopyxis alaskensis. Comparative genomic analysis shows that, in Enterobacteriaceae, the cluster is a genomic island integrated at the leuX locus, and the phylogenetic analysis of the TgtA5 family is consistent with widespread horizontal gene transfer. Comparison of transformation efficiencies of modified or unmodified plasmids into isogenic S. Montevideo strains containing or lacking the cluster strongly suggests a restriction–modification role for the cluster in Enterobacteriaceae. Another preQ0 derivative, 2’-deoxy-7-formamidino-7-deazaguanosine, was found in the Escherichia coli bacteriophage 9g, as predicted from the presence of homologs of genes involved in the synthesis of the archaeosine tRNA modification. These results illustrate a deep and unexpected evolutionary connection between DNA and tRNA metabolism.

/pdf/novel-genomic-island-modifies-dna-with-7-deazaguanine-52w41e5v5j.pdf

Novel genomic island modifies DNA with 7-deazaguanine derivatives

Detection of a new enzyme for stereoselective hydrolysis of linalyl acetate using simple plate assays for the characterization of cloned esterases from Burkholderia gladioli.

The parCBA operon, which together with the parDE operon constitutes an efficient stabilization system of the broad-host-range plasmid RP4, encodes a 20 kDa polypeptide (ParB), which exhibits sequence homology to nucleases. The ParB protein was overexpressed by means of an inducible tac-promoter system. Plate assays with herring sperm DNA as substrate provided evidence for nuclease activity. The ParB nuclease shows specificity for circular DNA substrates and linearizes them regardless of the presence in cis of parts of the RP4 partitioning region. The nuclease activity in vitro is stimulated by the presence of Ca2+ ions. EDTA (5 mM) completely inhibits nuclease activity. By restriction analysis of the ParB-linearized products, cleavage of circular DNA substrates taking place preferentially at specific sites was demonstrated. Run-off sequencing and primer extension analysis of ParB-linearized plasmid DNA revealed a specific target for ParB action adjacent to an AT-rich region containing palindromic sequence elements on a pBR322-derived plasmid.

/pdf/the-parb-protein-encoded-by-the-rp4-par-region-is-a-ca2-hh3qikltv3.pdf

The ParB protein encoded by the RP4 par region is a Ca2+-dependent nuclease linearizing circular DNA substrates

Applying an agar plate assay, a novel gene encoding an esterase from Bacillus subtilis Est4B was cloned and highly overexpressed in E. coli . The protein sequence revealed high homology to the srf D gene, which encodes the fourth open reading frame, a putative thioesterase gene from the surfactin synthetase gene cluster. It was almost identical to an unpublished sequence from a putative surfactin synthetase cluster from B. subtilis A13. The enzyme (Est4B1) was produced in E. coli , purified to homogeneity and crystallized. Computational prediction of the protein fold showed high structural similarity of Est4B1 to haloperoxidases and dehalogenases and much lower similarity to lipases and esterases. However, by primary sequence analysis we found a typical esterase/thioesterase/lipase motif. Biocatalytic activity on several model esterase substrates was detected and quantified. This is the first time enzymatic activity could be shown for this type of independent putative thioesterases and this enzyme may serve as a model protein to solve the structure of this type of hydrolytic enzymes, which is commonly found in gene clusters of non-ribosomal peptide synthetases.

Cloning, expression and characterization of a new 2-Cl-propionic acid ester hydrolase from B. subtilis

The two new esterases, EP6 and EP10, from Pseudomonas marginata were studied with respect to their substrate specificities and selectivities. Esters of aliphatic and cyclic secondary alcohols were shown to be substrates for EP10, which could be accepted stereoselectively in hydrolysis and transesterification reactions. EP6 showed no selectivity in hydrolysis of the studied substrates.

New Pseudomonas esterases by genetic engineering

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Papers

Detection of a new enzyme for stereoselective hydrolysis of linalyl acetate using simple plate assays for the characterization of cloned esterases from Burkholderia gladioli.

The ParB protein encoded by the RP4 par region is a Ca2+-dependent nuclease linearizing circular DNA substrates

Cloning, expression and characterization of a new 2-Cl-propionic acid ester hydrolase from B. subtilis

New Pseudomonas esterases by genetic engineering