RESUMO
Relaxases act as DNA selection sieves in conjugative plasmid transfer. Most plasmid relaxases belong to the HUH endonuclease family. TrwC, the relaxase of plasmid R388, is the prototype of the HUH relaxase family, which also includes TraI of plasmid F. In this article we demonstrate that TrwC processes its target nic-site by means of a highly secure double lock and key mechanism. It is controlled both by TrwC-DNA intermolecular interactions and by intramolecular DNA interactions between several nic nucleotides. The sequence specificity map of the interaction between TrwC and DNA was determined by systematic mutagenesis using degenerate oligonucleotide libraries. The specificity map reveals the minimal nic sequence requirements for R388-based conjugation. Some nic-site sequence variants were still able to form the U-turn shape at the nic-site necessary for TrwC processing, as observed by X-ray crystallography. Moreover, purified TrwC relaxase effectively cleaved ssDNA as well as dsDNA substrates containing these mutant sequences. Since TrwC is able to catalyze DNA integration in a nic-site-containing DNA molecule, characterization of nic-site functionally active sequence variants should improve the search quality of potential target sequences for relaxase-mediated integration in any target genome.
Assuntos
Conjugação Genética , DNA Nucleotidiltransferases/química , DNA Nucleotidiltransferases/metabolismo , DNA Bacteriano/química , DNA Bacteriano/metabolismo , Plasmídeos/genética , Domínio Catalítico , DNA Nucleotidiltransferases/genética , DNA de Cadeia Simples/química , Biblioteca Gênica , MutaçãoRESUMO
Directed evolution of Pseudomonas aeruginosa lipase by the use of combinatorial active site saturation test (CAST) criteria provided a highly enantioselective mutant (Leu162Phe) for kinetic resolution of an axially chiral allene, p-nitrophenyl 4-cyclohexyl-2-methylbuta-2,3-dienoate (E=111); the high enantioselectivity of the Leu162Phe mutant was rationalized by pi-pi stacking.
Assuntos
Alcadienos/química , Lipase/química , Pseudomonas aeruginosa/enzimologia , Evolução Molecular Direcionada , Cinética , Modelos Biológicos , Conformação Molecular , Estrutura Molecular , Mutação , Pseudomonas aeruginosa/genética , EstereoisomerismoRESUMO
TrwC is a relaxase protein, which starts and finishes DNA processing during bacterial conjugation in plasmid R388. TrwC recognizes a specific sequence of DNA (25 nucleotides) in the donor cell: the nic-site. As a model example, a single transversion C24G in nic avoids DNA processing by TrwC. Using this simple model, our objective was to obtain a proof of principle that TrwC specificity can be changed. Several structures of DNA-TrwC complexes were used as reference to design a focused saturation mutagenesis library (NNK) randomizing amino acid Lys262, since its side chain seems to sterically hinder the recognition of the C24G nic mutation by wild-type TrwC. Using bacterial conjugation as an in vivo selection system, several TrwC variants were found that show changes in substrate specificity. These variants were also tested in a competitive assay to evaluate their conjugation efficiency.
Assuntos
Conjugação Genética , DNA Nucleotidiltransferases/química , DNA Nucleotidiltransferases/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Engenharia de Proteínas/métodos , Alanina/metabolismo , Substituição de Aminoácidos , Sequência de Bases , Sítios de Ligação/genética , Cisteína/metabolismo , DNA Nucleotidiltransferases/genética , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Modelos Genéticos , Modelos Moleculares , Dados de Sequência Molecular , Estrutura Molecular , Mutagênese Sítio-Dirigida , Plasmídeos , Ligação Proteica/genética , Especificidade por Substrato/genética , Valina/metabolismoRESUMO
Iterative saturation mutagenesis (ISM) is a new and efficient method for the directed evolution of functional enzymes. It reduces the necessary molecular biological work and the screening effort drastically. It is based on a Cartesian view of the protein structure, performing iterative cycles of saturation mutagenesis at rationally chosen sites in an enzyme, a given site being composed of one, two or three amino acid positions. The basis for choosing these sites depends on the nature of the catalytic property to be improved, e.g., enantioselectivity, substrate acceptance or thermostability. In the case of thermostability, sites showing highest B-factors (available from X-ray data) are chosen. The pronounced increase in thermostability of the lipase from Bacillus subtilis (Lip A) as a result of applying ISM is illustrated here.
Assuntos
Evolução Molecular Direcionada/métodos , Enzimas/química , Enzimas/genética , Mutagênese , Bacillus subtilis/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Códon/química , Estabilidade Enzimática , Lipase/química , Lipase/genética , Modelos Moleculares , Mutagênese Sítio-Dirigida/métodos , Estrutura Terciária de ProteínaRESUMO
An earlier experimental study, which involved the directed evolution of enantioselective lipase variants from Pseudomonas aeruginosa as catalysts in the hydrolytic kinetic resolution of 2-methyl-decanoic acid p-nitrophenyl ester, provided a mutant with six mutations. Consequently, the selectivity factor was found to increase from E = 1.1 for the wild-type to E = 51 for the best mutant. Only one of the amino acid exchanges in this mutant was found to occur next to the binding pocket, the other mutations being remote. Our previous theoretical analysis with molecular-dynamics simulations helped to unveil the source of enhanced enantioselectivity: a relay mechanism that involves two of the six mutations was shown to induce strong cooperativity. In this investigation, single, double, and triple mutants were constructed and tested as enantioselective catalysts. This study supports our original postulate regarding the relay mechanism, offers further mechanistic insight into the role of individual mutations, and provides mutants that display even higher enantioselectivity (E of up to 64).
Assuntos
Evolução Molecular Direcionada , Catálise , Cristalografia por Raios X , Primers do DNA/química , Ésteres , Técnicas Genéticas , Cinética , Lipase/química , Modelos Químicos , Modelos Moleculares , Conformação Molecular , Mutação , Pseudomonas aeruginosa/metabolismo , Software , EstereoisomerismoRESUMO
In a previous paper, the combinatorial active-site saturation test (CAST) was introduced as an effective strategy for the directed evolution of enzymes toward broader substrate acceptance. CASTing comprises the systematic design and screening of focused libraries around the complete binding pocket, but it is only the first step of an evolutionary process because only the initial libraries of mutants are considered. In the present study, a simple method is presented for further optimization of initial hits by combining the mutational changes obtained from two different libraries. Combined lipase mutants were screened for hydrolytic activity against six notoriously difficult substrates (bulky carboxylic acid esters) and improved mutants showing significantly higher activity were identified. The enantioselectivity of the mutants in the hydrolytic kinetic resolution of two substrates was also studied, with the best mutant-substrate combination resulting in a selectivity factor of E=49. Finally, the catalytic profile of the evolved mutants in the hydrolysis of simple nonbranched carboxylic acid esters, ranging from acetate to palmitate, was studied for theoretical reasons.