RESUMO
BACKGROUND: Biocatalysis in organic solvents is nowadays a common practice with a large potential in Biotechnology. Several studies report that proteins which are co-crystallized or soaked in organic solvents preserve their fold integrity showing almost identical arrangements when compared to their aqueous forms. However, it is well established that the catalytic activity of proteins in organic solvents is much lower than in water. In order to explain this diminished activity and to further characterize the behaviour of proteins in non-aqueous environments, we performed a large-scale analysis (1737 proteins) of the conformational diversity of proteins crystallized in aqueous and co-crystallized or soaked in non-aqueous media. RESULTS: Using proteins' experimentally determined conformational diversity taken from CoDNaS database, we found that proteins in non-aqueous media display much lower conformational diversity when compared to the corresponding conformers obtained in water. When conformational diversity is compared between conformers obtained in different non-aqueous media, their structural differences are larger and mostly independent of the presence of cognate ligands. We also found that conformers corresponding to non-aqueous media have larger but less flexible cavities, lower number of disordered regions and lower active-site residue mobility. CONCLUSIONS: Our results show that non-aqueous media conformers have specific structural features and that they do not adopt extreme conformations found in aqueous media. This makes them clearly different from their corresponding aqueous conformers.
Assuntos
Biologia Computacional/métodos , Proteínas/química , Solventes/química , Água/química , Biocatálise , Bases de Dados de Proteínas , Humanos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas/metabolismo , Proteínas ras/química , Proteínas ras/metabolismoRESUMO
Promiscuous activities have been related to the capacity to catalyze reactions different from those a protein has evolved to sustain. In this work, we rethought the serum albumin's promiscuous behavior using evolutionary and structural analysis. We found that the cross aldol condensation of acetone and p-formylbenzonitrile is a promiscuous reaction conserved in humans serum albumin and in closely related albumins from other mammals. Evolutionary analysis indicates that the residues involved in this promiscuous reaction are evolving under positive selection, an evolutionary pattern indicating a putative functional adaptation. Also, key residues are located in an evolutionary conserved cavity connected with the protein surface with an also conserved tunnel and mutations involving these residues are described in human diseases. Overall, our results suggest that albumin could have evolved to sustain a still unknown biological function among the many others it maintains. Our results could contribute to better characterize the serum albumin family and raise questions about the evolution of protein promiscuity and function.
Assuntos
Evolução Molecular , Albumina Sérica , Adaptação Fisiológica , Animais , Catálise , Humanos , Mamíferos , Albumina Sérica/genéticaRESUMO
A new procedure was carried out for the synthesis of nucleoside 5'-monophosphates, involving the use of two enzymes. The first step applied phospholipase D from Streptomyces netropsis and phosphatidylcholine as phosphatidyl donor, to give 5'-(3-sn-phosphatidyl) nucleosides (C, U, A, I). These were selectively hydrolysed in the second step by the action of phospholipase C from Bacillus cereus to produce the respective 5'-nucleotides. Application of this methodology on a preparative scale conducted to 5'-adenosine monophosphate in 63% overall yield from adenosine. The regioselectivity of these enzymes avoids protection steps, the overall synthesis is performed under mild reaction conditions and product isolation is easily achieved.
Assuntos
Nucleotídeos/biossíntese , Monofosfato de Adenosina/biossíntese , Monofosfato de Adenosina/isolamento & purificação , Bacillus cereus/metabolismo , Biocatálise , Estabilidade Enzimática , Hidrólise , Nucleosídeos/química , Nucleosídeos/metabolismo , Nucleotídeos/química , Fosfolipase D/metabolismo , Fosforilação , Streptomyces/enzimologia , Especificidade por Substrato , Fosfolipases Tipo C/metabolismoRESUMO
Nucleosides are valuable bioactive molecules, which display antiviral and antitumour activities. Diverse types of prodrugs are designed to enhance their therapeutic efficacy, however this strategy faces the troublesome selectivity issues of nucleoside chemistry. In this context, the aim of this review is to give an overview of the opportunities provided by biocatalytic procedures in the preparation of nucleoside prodrugs. The potential of biocatalysis in this research area will be presented through examples covering the different types of nucleoside prodrugs: nucleoside analogues as prodrugs, nucleoside lipophilic prodrugs and nucleoside hydrophilic prodrugs.
Assuntos
Biotecnologia , Nucleosídeos , Pró-Fármacos , Biocatálise , NucleotídeosRESUMO
N-Monoacetylated derivatives of ribo- (adenosine, guanosine) and 2'-deoxyribonucleosides (2'-deoxyadenosine and 2'-deoxyguanosine), useful as oligonucleotide building blocks, were obtained in 88-100% by enzymatic chemoselective hydrolysis of the corresponding peracetylated nucleosides. Among the tested hydrolases, most satisfactory results were found with acylase I from Aspergillus melleus and Candida antarctica lipase B. For acylase I, the observed chemoselectivity towards ester hydrolysis, without amide reaction, broadens the information about the selectivity of the enzyme and its synthetic applications in the field of nucleosides.
Assuntos
Aspergillus/enzimologia , Nucleosídeos de Purina/metabolismo , Amidoidrolases/metabolismo , Proteínas Fúngicas/metabolismo , Hidrólise , Lipase/metabolismoRESUMO
In this article, we describe the application of lipases in acylation and alcoholysis reactions on steroids and nucleosides. In the field of steroids, a variety of acetyl and fatty acid derivatives of androstanes, pregnanes, and cholestanes have been prepared through lipase-catalyzed acylation and alcoholysis reactions taking advantage of the high regio- and stereoselectivity of these enzymes. The substrates as well as the products show a high degree of biological activity as neurosteroids, hormones, and glucocorticoids. The regioselective preparation of diacylated nucleosides by means of an enzymatic alcoholysis allowed the synthesis of nucleosides prodrugs or modified nucleosides. The quantitative full deacylation and dealkoxycarbonylation of nucleosides and steroids is a mild synthetic method for the deprotection of these labile compounds. Some of the reported steroid and nucleoside products are novel, and it is not possible to obtain them satisfactorily by following traditional synthetic procedures. The advantages presented by this methodology, such as selectivity, mild reaction conditions, and low environmental impact, make the lipases an important tool in the application of the principles of Green Chemistry, offering a convenient way to prepare derivatives of natural compounds with a great potential in the pharmaceutical industry.
Assuntos
Produtos Biológicos/síntese química , Proteínas Fúngicas/química , Lipase/química , Nucleosídeos/química , Acilação , Androstanos/síntese química , Biocatálise , Candida/química , Colestanos/síntese química , Ácidos Graxos/química , Química Verde , Pregnanos/síntese química , Pró-Fármacos/síntese química , EstereoisomerismoRESUMO
Two new compounds, 2',3'-di-O-ethoxycarbonyluridine and 2',3'-di-O-ethoxycarbonylinosine, were obtained through a Candida antarctica lipase B catalysed regioselective ethanolysis of the corresponding trialcoxycarbonylated nucleosides with benzyl alcohol in 1,4-dioxane at 30 degrees C.
Assuntos
Álcoois/química , Candida/enzimologia , Biologia Computacional/métodos , Nucleosídeos/química , Catálise , Dioxanos/química , Enzimas/química , Proteínas Fúngicas , Lipase/química , Modelos Químicos , Estereoisomerismo , Especificidade por Substrato , TemperaturaRESUMO
Enzymatic hydrolysis of acetylated nucleosides using microbial whole cells has been carried out for the first time. Unlike Candida antarctica B lipase-catalysed alcoholysis, none of the tested microorganisms displayed a common deacetylation profile. Depending on the substrate and the biocatalyst used, 5'-selective deprotection or mixtures of mono O-acetylated products were obtained.