ABSTRACT
Biosynthesis of 1,3-propanediol (1,3-PD) by 1,3-propanediol oxidoreductase (PDOR) is often limited by the stability issues. To address this issue, the goal of the present study was to engineer the Clostridium butyricum PDOR dimeric interface. The interface exists between the chains and plays a role in the synthesis of 1,3-PD, which is hindered by the increased temperature and pH. Herein, we engineered PDOR by HotSpot Wizard 3.0 and molecular dynamics simulations, improving its thermal stability, pH tolerance, and catalytic properties with respect to the wild-type PDOR activity at 37 °C. Compared to the activity of the wild-type PDOR, the N298C mutant showed 0.5-fold greater activity at pH 8.0, while the P299E mutant showed significantly increased activity of over five fold at pH 4.0. Further structural comparisons between the wild-type and P299E mutant revealed that the extraordinary stability of the P299E mutant could be due to the formation of additional hydrogen bonds and salt bridges. The N298C mutant also exhibits thermal stability at a broad range of temperature at pH 8 with respect to wild-type PDOR and other mutants. The molecular dynamics simulations revealed that stability profiles of P299E mutants at pH 4.0 are attributed to identical root mean square deviation values and stable conformations in the motif region present in the dimer interface of the enzyme. These findings suggest that the dimer interface motifs are essential for the compactness and stability of the PDOR enzyme; therefore, engineering the PDOR using a structure-guided approach could aid in improving its activity and stability under various physiological conditions (pH and temperature).
Subject(s)
Clostridium butyricum , Alcohol Oxidoreductases , Clostridium butyricum/genetics , Enzyme Stability , Molecular Dynamics Simulation , TemperatureABSTRACT
Plants produce interesting secondary metabolites that are a valuable source of both medicines for human use, along with significant advantages for the manufacturer species. The active compounds which lead to these instrumental effects are generally secondary metabolites produced during various plant growth phases, which provide the host survival advantages while affecting human health inadvertently. Different chemical classes of secondary metabolites are biosynthesized by the plant type III polyketide synthases (PKSs). They are simple homodimeric proteins with the unique mechanistic potential to produce a broad array of secondary metabolites by utilizing simpler starter and extender units. These PKS derived products are majorly the precursors of some important secondary metabolite pathways leading to products such as flavonoids, stilbenes, benzalacetones, chromones, acridones, xanthones, cannabinoids, aliphatic waxes, alkaloids, anthrones, and pyrones. These secondary metabolites have various pharmaceutical, medicinal and industrial applications which make biosynthesizing type III PKSs an important tool for bioengineering purposes. Because of their structural simplicity and ease of manipulation, these enzymes have garnered interest in recent years due to their application in the generation of unnatural natural polyketides and modified products in the search for newer drugs for a variety of health problems. The following review covers the biosynthesis of a variety of type III PKS-derived secondary metabolites, their biological relevance, the associated enzymes, and recent research.
ABSTRACT
Biofilm is a mode of living employed by many pathogenic and environmental microbes to proliferate as multicellular aggregates on inert inanimate or biological substrates. Several microbial diseases are associated with biofilms that pose challenges in treatment with antibiotics targeting individual cells. Bacteria in biofilms secrete exopolymeric substances that contribute to architectural stability and provide a secure niche to inhabiting cells. Quorum sensing (QS) plays essential roles in biofilm development. Pathogenic bacteria in biofilms utilize QS mechanisms to activate virulence and develop antibiotic resistance. This review is a brief overview of biofilm research and provides updates on recent understandings on biofilm development, antibiotic resistance and transmission, and importance of QS mechanisms. Strategies to combat biofilm associated diseases including anti-biofilm substances, quorum quenching molecules, bio-surfactants and competitive inhibitors are briefly discussed. The review concludes with updates on recent approaches utilized for biofilm inhibition and provides perspectives for further research in the field.
ABSTRACT
Type III polyketide synthases (PKSs) biosynthesize varied classes of metabolites with diverse bio-functionalities. Inherent promiscuous substrate specificity, multiple elongations of reaction intermediates and several modes of ring-closure, confer the proteins with the ability to generate unique scaffolds from limited substrate pools. Structural studies have identified crucial amino acid residues that dictate type III PKS functioning, though cyclization specific residues need further investigation. PKSIIINc, a functionally and structurally characterized type III PKS from the fungus, Neurospora crassa, is known to biosynthesize alkyl-resorcinol, alkyl-triketide- and alkyl-tetraketide-α-pyrone products. In this study, we attempted to identify residue positions governing cyclization specificity in PKSIIINc through comparative structural analysis. Structural comparisons with other type III PKSs revealed a motif with conserved hydroxyl/thiol groups that could dictate PKSIIINc catalysis. Site-directed mutagenesis of Cys120 and Ser186 to Ser and Cys, respectively, altered product profiles of mutant proteins. While both C120S and S186C proteins retained wild-type PKSIIINc product activity, S186C favoured lactonization and yielded higher amounts of the α-pyrone products. Notably, C120S gained new cyclization capability and biosynthesized acyl-phloroglucinol in addition to wild-type PKSIIINc products. Generation of alkyl-resorcinol and acyl-phloroglucinol by a single protein is a unique observation in fungal type III PKS family. Mutation of Cys120 to bulky Phe side-chain abrogated formation of tetraketide products and adversely affected overall protein stability as revealed by molecular dynamics simulation studies. Our investigations identify residue positions governing cyclization programming in PKSIIINc protein and provide insights on how subtle variations in protein cores dictate product profiles in type III PKS family.
ABSTRACT
Mycobacterial pathogenesis is hallmarked by lipidic polyketides that decorate the cell envelope and mediate infection. However, factors mediating persistence remain largely unknown. Dynamic cell wall remodeling could facilitate the different pathogenic phases. Recent studies have implicated type III polyketide synthases (PKSs) in cell wall alterations in several bacteria. Comparative genome analysis revealed several type III pks gene clusters in mycobacteria. In this study, we report the functional characterization of two novel type III PKSs, MMAR_2470 and MMAR_2474, in Mycobacterium marinum. These type III pkss belong to a unique pks genomic cluster conserved exclusively in pathogenic mycobacteria. Cell-free reconstitution assays and high-resolution mass spectrometric analyses revealed methylated polyketide products in independent reactions of both proteins. MMAR_2474 protein exceptionally biosynthesized methylated alkyl-resorcinol and methylated acyl-phloroglucinol products from the same catalytic core. Structure-based homology modeling, product docking, and mutational studies identified residues that could facilitate the distinctive catalysis of these proteins. Functional investigations in heterologous mycobacterial strain implicated MMAR_2474 protein to be vital for mycobacterial survival in stationary biofilms. Our investigations provide new insights into type III PKSs conserved in pathogenic mycobacterial species.
