RESUMEN
Evolution has led to the development of a gigantic repertoire of microbial genes that can be exploited for industrial purposes. Due to microevolutionary processes, this gene pool is constantly varied and adapted to the prevalent environmental and physiological conditions. It though remains unclear to what extent gene variants coexist in natural habitats and to what extent they vanish due to competition. Here, we tapped the pool of gene variants of the serine protease Subtilisin Carlsberg present in soil habitats, demonstrating a high degree of (micro) diversity on a genetic level, as well as on a functional level. A set of 51 mature enzyme variants each carrying two to eight amino acid changes were recovered. While some mutations were only present in single variants, other changes appear to be rather conserved even across different habitats. The observed spectrum of biochemical properties makes persistent gene variants a potent source for biotechnologically relevant enzymes, expanding the toolbox of metagenomic approaches.
Asunto(s)
Variación Genética , Metagenómica , Microbiología del Suelo , Subtilisinas/genética , Secuencia de Aminoácidos , Secuencia de Bases , Datos de Secuencia Molecular , MutaciónRESUMEN
We constructed stabilized variants of beta-lactamase (BLA) from Enterobacter cloacae by combinatorial recruitment of consensus mutations. By aligning the sequences of 38 BLA homologs, we identified 29 positions where the E.cloacae gene differs from the consensus sequence of lactamases and constructed combinatorial libraries using mixtures of mutagenic oligonucleotides encompassing all 29 positions. Screening of 90 random isolates from these libraries identified 15 variants with significantly increased thermostability. The stability of these isolates suggest that all tested mutations make additive contributions to protein stability. A statistical analysis of sequence and stability data identified 11 mutations that made stabilizing contributions and eight mutations that destabilized the protein. A second-generation library recombining these 11 stabilizing mutations led to the identification of BLA variants that showed further stabilization. The most stable variant had a mid-point of thermal denaturation (Tm) that was 9.1 degrees C higher than the starting molecule and contained eight consensus mutations. Incubation of three stabilized BLA variants with several proteases showed that all tested isolates have significantly increased resistance to proteolysis. Our data demonstrate that combinatorial consensus mutagenesis (CCM) allows the rapid generation of protein variants with improved thermal and proteolytic stability.
Asunto(s)
Técnicas Químicas Combinatorias/métodos , Mutagénesis , Ingeniería de Proteínas/métodos , Proteínas/genética , Secuencia de Aminoácidos , Enterobacter cloacae/enzimología , Enterobacter cloacae/genética , Enterobacter cloacae/metabolismo , Estabilidad de Enzimas , Biblioteca de Genes , Calor , Datos de Secuencia Molecular , Mutación , Proteínas/química , Proteínas/metabolismo , beta-Lactamasas/química , beta-Lactamasas/genética , beta-Lactamasas/metabolismoRESUMEN
We describe the modelling of the structure of the highly alkaline subtilisin protease OPTICLEAN from Bacillus alcalophilus. The model was developed through modelling by homology. We used the structure of subtilisin Carlsberg from the Brookhaven protein databank (entry 1CSE) as start structure. Amino acid changes and deletions were performed with the graphic protein design program BRAGI. Force field calculations and molecular dynamic simulations were made with AMBER 3.0 on a Multiflow TRACE 14/300. The comparison of the model and the later solved X-ray structure of OPTICLEAN shows a high similarity between the two structures, but there were also remarkable deviations between the two structures in some loop regions. The comparison shows that the deviations are due to difficulties in the prediction of correct main chain torsion angles of the prolines and the selection of correct loops in deletion or insertion regions. Strategies to avoid these mistakes are discussed.
Asunto(s)
Modelos Moleculares , Conformación Proteica , Subtilisinas/química , Difracción de Rayos X/métodos , Algoritmos , Secuencia de Aminoácidos , Bacillus/enzimología , Gráficos por Computador , Bases de Datos Factuales , Enlace de Hidrógeno , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteínas Recombinantes/química , Eliminación de SecuenciaRESUMEN
Using a significantly simplified modification procedure, four charged analogues of the coenzyme NAD, N(1)- and N6-(2-hydroxy-3-trimethylammoniumpropyl)-NAD, N(1)- and N6-(3-sulfopropyl)-NAD were prepared. The kinetic parameters of these derivatives and N(1)-(2-aminoethyl)-NAD, N6-(2-aminoethyl)-NAD and tricyclic 1,N6-ethanoadenine-NAD, all with alterations to the adenine moiety, were determined for porcine heart lactate dehydrogenase isoenzyme H4. The coenzyme activity depends on both position and charge of the introduced groups. Modification of the N6-position leads to a 25-250-fold increase of the kcat/Km value compared to the related N(1) derivative. The kcat/Km value for 1,N6-ethanoadenine-NAD is in the range between that of N(1)-(2-aminoethyl)-NAD and N6-(2-aminoethyl)-NAD. In the case of both N(1) and N6 functionalization, the Km values increase from (3-sulfopropyl)-NAD, with a negatively charged substituent at the adenine, over (2-amino-ethyl)-NAD to (2-hydroxy-3-trimethylammoniumpropyl)-NAD with an uncharged and positively charged substituent, respectively, at the adenine. All N6 derivatives are analogues like NAD with respect to Km and/or Vmax and kcat/Km. The conformation of NAD and its derivatives was calculated and their interaction in the active site of lactate dehydrogenase was simulated using the molecular mechanics program AMBER. The significant differences in activity in correlation to porcine heart lactate dehydrogenase isoenzyme H4 could be rationalized by modelling the three-dimensional structure of the NAD site.
Asunto(s)
Adenina/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Miocardio/enzimología , NAD/metabolismo , Adenina/química , Animales , Sitios de Unión , Catálisis , Isoenzimas , Cinética , Conformación Molecular , NAD/análogos & derivados , NAD/química , Relación Estructura-Actividad , PorcinosRESUMEN
The successful attempt is presented to engineer an enzyme with respect to its technical application by the use of computer-aided protein design techniques. Based on a modeled 3-D structure a number of mutants of a subtilisin-like protease was designed with the aim to increase its washing performance. The model of the highly alkaline subtilisin protease OPTICLEAN from Bacillus alcalophilus was developed by the process of 'modeling by homology' starting with the structure of subtilisin Carlsberg 1CSE.BRK from the Brookhaven protein databank. Amino acid changes and deletions were performed with the graphic protein design program BRAGI. Force field calculations and molecular dynamic simulations were made with AMBER 3.0. The comparison of the model and the later solved X-ray structure of OPTICLEAN shows a high similarity between the two structures. On the other hand, interesting deviations between the two structures were observed in some external loop regions. The comparison shows that the deviations are due to difficulties in the prediction of correct main chain torsion angles of additional prolines and the selection of correct loops in deletion or insertion regions.
Asunto(s)
Bacillus/enzimología , Serina Endopeptidasas/química , Secuencia de Aminoácidos , Simulación por Computador , Diseño Asistido por Computadora , Indicadores y Reactivos , Microbiología Industrial , Modelos Moleculares , Datos de Secuencia Molecular , Ingeniería de Proteínas , Homología de Secuencia de Aminoácido , Serina Endopeptidasas/metabolismo , Difracción de Rayos XRESUMEN
The X-ray structure determination, refinement and comparison of two crystal forms of a variant (Asn115Arg) of the alkaline protease from Bacillus alcalophilus is described. Under identical conditions crystals were obtained in the orthorhombic space group P2(1)2(1)2(1) (form I) and the rhombohedral space group R32 (form II). For both space groups the structures of the protease were solved by molecular replacement and refined at 1.85 A resolution. The final R-factors are 17.9% and 17.1% for form I and form II, respectively. The root-mean-square deviation between the two forms is 0.48 A and 0.86 A for main-chain and side-chain atoms, respectively. Due to differences in crystal lattice contacts and packing, the structures of the two crystal forms differ in intermolecular interaction affecting the local conformation of three flexible polypeptide sequences (Ser50-Glu55, Ser99-Gly102, Gly258-Ser259) at the surface of the protein. While the two overall structures are very similar, the differences are significantly larger than the errors inherent in the structure determination. As expected, the differences in the temperature factors in form I and II are correlated with the solvent accessibility of the corresponding amino acid residues. In form II, two symmetry-related substrate binding sites face each other, forming a tight intermolecular interaction. Some residues contributing to this intermolecular interaction are also found to be involved in the formation of the complex between subtilisin Carlsberg and the proteinaceous inhibitor eglin C. This demonstrates that the two symmetry-related molecules interact with each other at the same molecular surface area that is used for binding of substrates and inhibitors.
Asunto(s)
Bacillus/enzimología , Serina Endopeptidasas/química , Cristalización , Enlace de Hidrógeno , Modelos Moleculares , Polimorfismo Genético , Serina Endopeptidasas/metabolismo , Especificidad por Sustrato , Temperatura , Difracción de Rayos XRESUMEN
The crystal structure of an alkaline protease from Bacillus alcalophilus has been determined by X-ray diffraction at 2.4 A resolution. The enzyme crystallizes in space group P2(1)2(1)2(1) with lattice constants a = 53.7, b = 61.6, c = 75.9 A. The structure was solved by molecular replacement using the structure of subtilisin Carlsberg as search model. Refinement using molecular dynamics and restrained least squares methods results in a crystallographic R-factor of 0.185. The tertiary structure is very similar to that of subtilisin Carlsberg. The greatest structural differences occur in loops at the surface of the protein.