Improvement Of Polyhydroxyalkanoate Synthase From Wautersia Eutropha By In Vitro Evolution [QH460. N842 2005 f rb].

Polyhydroxyalkanoates (PHA) are polyesters synthesized by various bacteria as intracelullar carbon and energy storage material under excess carbon and limiting nutrient conditions. In the recent decades, PHAs have attracted considerable attention due to similarity in their physical properties to petrochemical-based polyesters

Chitinase: diversity, limitations, and trends in engineering for suitable applications

Chitinases catalyze the degradation of chitin, a ubiquitous polymer generated from the cell walls of fungi, shells of crustaceans, and cuticles of insects. They are gaining increasing attention in medicine, agriculture, food and drug industries, and environmental management. Their roles in the degradation of chitin for the production of industrially useful products and in the control of fungal pathogens and insect pests render them attractive for such purposes. However, chitinases have diverse sources, characteristics, and mechanisms of action that seem to restrain optimization procedures and render standardization techniques for enhanced practical applications complex. Hence, results of laboratory trials are not usually consistent with real-life applications. With the growing field of protein engineering, these complexities can be overcome by modifying or redesigning chitinases to enhance specific features required for specific applications. In this review, the variations in features and mechanisms of chitinases that limit their exploitation in biotechnological applications are compiled. Recent attempts to engineer chitinases for improved efficiency are also highlighted

In silico design of potentially functional artificial metallo-haloalkane dehalogenase containing catalytic zinc

Artificial metalloenzymes are unique as they combine the good features of homogeneous and enzymatic catalysts, and they can potentially improve some difficult catalytic assays. This study reports a method that can be used to create an artificial metal-binding site prior to proving it to be functional in a wet lab. Haloalkane dehalogenase was grafted into a metal-binding site to form an artificial metallo-haloalkane dehalogenase and was studied for its potential functionalities in silico. Computational protocols regarding dynamic metal docking were studied using native metalloenzymes and functional artificial metalloenzymes. Using YASARA Structure, a simulation box covering template structure was created to be filled with water molecules followed by one mutated water molecule closest to the metal-binding site to metal ion. A simple energy minimization step was subsequently run using an AMBER force field to allow the metal ion to interact with the metal-binding residues. Long molecular dynamic simulation using YASARA Structure was performed to analyze the stability of the metal-binding site and the distance between metal-binding residues. Metal ions fluctuating around 2.0 Å across a 20 ns simulation indicated a stable metal-binding site. Metal-binding energies were predicted using FoldX, with a native metalloenzyme (carbonic anhydrase) scoring 18.0 kcal/mol and the best mutant model (C1a) scoring 16.4 kcal/mol. Analysis of the metal-binding site geometry was performed using CheckMyMetal, and all scores for the metalloenzymes and mutant models were in an acceptable range. Like native metalloenzymes, the metal-binding site of C1a was supported by residues in the second coordination shell to maintain a more coordinated metal-binding site. Short-chain multihalogenated alkanes (1,2-dibromoethane and 1,2,3-trichloropropane) were able to dock in the active site of C1a. The halides of the substrate were in contact with both the metal and halide-stabilizing residues, thus indicating a better stabilization of the substrate. The simple catalytic mechanism proposed is that the metal ion interacted with halogen and polarized the carbon–halogen bond, thus making the alpha carbon susceptible to attack by nucleophilic hydroxide. The interaction between halogen in the metal ion and halide-stabilizing residues may help to improve the stabilization of the substrate–enzyme complex and reduce the activation energy. This study reports a modified dynamic metal-docking protocol and validation tests to verify the metal-binding site. These approaches can be applied to design different kinds of artificial metalloenzymes or metal-binding sites

Engineering T1 lipase for degradation of poly-(R)-3-hydroxybutyrate

Enzymes with broad substrate specificities that can act on a wide range of substrates would be valuable for industrial applications. T1 lipase is known to have broad substrate specificity in its native form, with active site residues that are similar to polyhydroxylalkanoate (PHA) depolymerase (PhaZ). PhaZ6 from Pseudomonas lemoignei (PhaZ6Pl) is one of PhaZs that can degrade semicrystalline poly-(R)-3-hydroxybutyrate [P(3HB)]. The objective of this study is to enable T1 lipase to degrade semicrystalline P(3HB) similar to PhaZ6Pl while maintaining its native function. Structural analyses on PhaZ6Pl built structure revealed that it does not contain a lid, as opposed to T1 lipase. Therefore, T1 lipase were designed by removing its lid region. This was performed by using Bacillus subtilis lipase A (BSLA) as the reference for T1 lipase modification as the latter does not have a lid region and that its structure fits almost perfectly with T1 lipase based on their superimposed structures. A total of three variants of T1 lipase without lid were successfully designed, namely D1 (without α6–loop–α7), D2 (without α6) and D3 (α6 and loop) in the lid region. All the variants showed PHA depolymerase activity towards P(3HB), with D2 variant exhibiting the highest activity amongst other variants. Further analysis on D2 showed that it was able to maintain its native hydrolytic activity towards olive oil, albeit with decrement in its catalytic efficiency. Results obtained in this study highlighted the fact that native T1 lipase is a versatile hydrolase enzyme which does not only perform triglyceride degradation but also P(3HB) degradation by simply removing the helix 6 which was specifically proven to affect catalytic activity and substrate specificity of the enzyme

Asymmetric Michael reaction catalyzed by mimicked peptides

Peptides mimicked from active site of promiscuous aldo-ketoreductase were synthesized and tested as asymmetry catalysts in the Michael adduct reaction of aldehydes or ketones with nitroolefins to furnish the corresponding γ-nitroaldehydes, γ-nitroketones with up to 93 % yield, 99:1 dr and 71 % ee at room temperature and on eco-friendly solvents. Aspartic acid residue as second amino acid produced greater enantioselectivity

Enantioselectivity investigation of short polar peptides with different positions in the Michael reaction

This work reports the effectiveness of short polar peptides as asymmetric catalysts in Michael reactions to attain good yields of enantio- and diastereoselective isomers. In a comparison, glutamic acid and histidine produced greater ee and yields when they were applied as the second amino acid in short trimeric peptides. These short polar peptides were found to be efficient organocatalysts for the asymmetric Michael addition reaction in water

Novel octapeptide as an asymmetric catalyst for Michael reaction in aqueous media

In this work, three forms of a novel octapeptide have been evaluated as asymmetric catalysts for the Michael reaction. Low quantity catalyst loading, ecofriendly solvents, and reusability of organocatalyst successfully applied to attain excellent yields and moderate enantioselectivities in the Michael reaction

Rational design of mimetic peptides based on aldo-ketoreductase enzyme as asymmetric organocatalysts in aldol reactions

Peptides as a kind of important chiral scaffold are broadly identified for their obvious advantages, diverse structures and accessibility. Based on promiscuous aldo-keto-reductase enzymes, several mimetic peptides were designed which were synthesized and tested as multifunctional organocatalysts in direct asymmetric aldol reactions. The corresponding aldol products were produced with high yields (up to 97%) and excellent diastereoselectivities (up to 99/1) and enantioselectivities (>98%) under mild reaction selectivity and enantioselectivity. The secondary structures of peptide catalysts provide an understanding of their mechanism

CsoR metalloregulatory protein: function, mechanism and relevance

Transition metals are required constituent in bacterial metabolism to assist in some enzymatic reactions. However, intracellular accumulations of these metal ions are harmful to the bacteria as it can trigger unnecessary redox reactions. To overcome this condition, metalloregulatory proteins assist organisms to adapt to sudden elevated and deprived metal ion concentration in the environment via metal homeostasis. CsoR protein is a copper(I) [Cu(I)] sensing operon repressor that is found to be present in all major classes of eubacteria. This metalloregulatory protein binds to the operator region in its apo state under Cu(I) limiting condition and detaches off from the regulatory region when it binds to the excess cytosolic Cu(I) ion, thus derepressing the expression of genes involved in Cu(I) homeostasis. CsoR proteins exist in dimeric and tetrameric states and form certain coordination geometries upon attachment with Cu(I). Certain CsoR proteins have also been found to possess the ability to bind to other types of metals with various binding affinities in some Gram positive bacteria. The role of this metalloregulatory protein in host pathogen interaction and its relation to bacterial virulence are also discussed

Computational design of artificial metallo-haloalkane dehalogenase

Haloalkane dehalogenases (HLDs) can catalyze conversion of some toxic haloalkanes to corresponding harmless alcohols. The limiting factors of native HLDs are their slow product releasing step, low activity against non-natural substrates and synthetic substrates. By creating an artificial metallo-HLD might provide solutions for these problems as metalloenzymes can provide certain advantages like high turnover rate, better stabilization of substrate-enzyme docking and broader substrate specificity. Metallo-HLDs are expected to carry out hydrolysis of haloalkane in 1 step catalysis and with higher KM. Nowadays, computational studies have been improved and commonly used by researchers to validate some structural designs before engineer the proteins in the lab. Computational studies using molecular dynamic simulation software and online molecular tools had largely increase the rate of success in protein engineering. In this work, through the computational design starting from template and metal binding site selection, in silico mutation, in silico metal docking, several validation of metal binding site and in silico docking of substrates had successful created 2 model of metallo-HLDs. These computational approaches had been validated using native metalloenzyme and functional artificial metalloenzyme as positive controls