AGPAT2 interaction with CDP-diacylglycerol synthases promotes the flux of fatty acids through the CDP-diacylglycerol pathway

AGPATs (1-acylglycerol-3-phosphate O-acyltransferases) catalyze the acylation of lysophosphatidic acid to form phosphatidic acid (PA), a key step in the glycerol-3-phosphate pathway for the synthesis of phospholipids and triacylglycerols. AGPAT2 is the only AGPAT isoform whose loss-of-function mutations cause a severe form of human congenital generalized lipodystrophy. Paradoxically, AGPAT2 deficiency is known to dramatically increase the level of its product, PA. Here, we find that AGPAT2 deficiency impairs the biogenesis and growth of lipid droplets. We show that AGPAT2 deficiency compromises the stability of CDP-diacylglycerol (DAG) synthases (CDSs) and decreases CDS activity in both cell lines and mouse liver. Moreover, AGPAT2 and CDS1/2 can directly interact and form functional complexes, which promote the metabolism of PA along the CDP-DAG pathway of phospholipid synthesis. Our results provide key insights into the regulation of metabolic flux during lipid synthesis and suggest substrate channelling at a major branch point of the glycerol-3-phosphate pathway.

A moonlighting role for enzymes of glycolysis in the co-localization of mitochondria and chloroplasts

Glycolysis is one of the primordial pathways of metabolism, playing a pivotal role in energy metabolism and biosynthesis. Glycolytic enzymes are known to form transient multi-enzyme assemblies. Here we examine the wider protein-protein interactions of plant glycolytic enzymes and reveal a moonlighting role for specific glycolytic enzymes in mediating the co-localization of mitochondria and chloroplasts. Knockout mutation of phosphoglycerate mutase or enolase resulted in a significantly reduced association of the two organelles. We provide evidence that phosphoglycerate mutase and enolase form a substrate-channelling metabolon which is part of a larger complex of proteins including pyruvate kinase. These results alongside a range of genetic complementation experiments are discussed in the context of our current understanding of chloroplast-mitochondrial interactions within photosynthetic eukaryotes.

A Spatially Orthogonal Hierarchically Porous Acid-Base Catalyst for Cascade and Antagonistic Reactions

<p>Complex organic molecules are of great importance to academic and industrial chemistry and typically synthesised from smaller building blocks by multistep reactions. The ability to perform multiple (distinct) transformations in a single reactor would greatly reduce the number of manipulations required for chemical manufacturing, and hence the development of multifunctional catalysts for such one-pot reactions is highly desirable. Here we report the synthesis of a hierarchically porous framework, in which the macropores are selectively functionalised with a sulfated zirconia solid acid coating, while the mesopores are selectively functionalised with MgO solid base nanoparticles. Active site compartmentalisation and substrate channelling protects base catalysed triacylglyceride transesterification from poisoning by free fatty acid impurities (even at 50 mol%), and promotes the efficient two-step cascade deacetylation-Knoevenagel condensation of dimethyl acetals to cyanoates.<br></p>

A Spatially Orthogonal Hierarchically Porous Acid-Base Catalyst for Cascade and Antagonistic Reactions

<p>Complex organic molecules are of great importance to academic and industrial chemistry and typically synthesised from smaller building blocks by multistep reactions. The ability to perform multiple (distinct) transformations in a single reactor would greatly reduce the number of manipulations required for chemical manufacturing, and hence the development of multifunctional catalysts for such one-pot reactions is highly desirable. Here we report the synthesis of a hierarchically porous framework, in which the macropores are selectively functionalised with a sulfated zirconia solid acid coating, while the mesopores are selectively functionalised with MgO solid base nanoparticles. Active site compartmentalisation and substrate channelling protects base catalysed triacylglyceride transesterification from poisoning by free fatty acid impurities (even at 50 mol%), and promotes the efficient two-step cascade deacetylation-Knoevenagel condensation of dimethyl acetals to cyanoates.<br></p>

A Spatially Orthogonal Hierarchically Porous Acid-Base Catalyst for Cascade and Antagonistic Reactions

<p>Complex organic molecules are of great importance to academic and industrial chemistry and typically synthesised from smaller building blocks by multistep reactions. The ability to perform multiple (distinct) transformations in a single reactor would greatly reduce the number of manipulations required for chemical manufacturing, and hence the development of multifunctional catalysts for such one-pot reactions is highly desirable. Here we report the synthesis of a hierarchically porous framework, in which the macropores are selectively functionalised with a sulfated zirconia solid acid coating, while the mesopores are selectively functionalised with MgO solid base nanoparticles. Active site compartmentalisation and substrate channelling protects base catalysed triacylglyceride transesterification from poisoning by free fatty acid impurities (even at 50 mol%), and promotes the efficient two-step cascade deacetylation-Knoevenagel condensation of dimethyl acetals to cyanoates.<br></p>

Passing the Baton: Substrate Channelling in Respiratory Metabolism

Despite species-specific differences in the pathways of respiratory metabolism are remarkably conserved across the kingdoms of life with glycolysis, the tricarboxylic acid cycle, and mitochondrial electron transport chain representing the major components of the process in the vast majority of organisms. In addition to being of critical importance in fueling life itself these pathways serve as interesting case studies for substrate channelling with research on this theme having been carried out for over 40 years. Here we provide a cross-kingdom review of the ample evidence for protein-protein interaction and enzyme assemblies within the three component pathways as well as describing the scarcer available evidence for substrate channelling itself.

Self-assembling all-enzyme hydrogels for biocatalytic flow processes

We describe the construction of binary self-assembling all-enzyme hydrogels that are comprised entirely of two tetrameric globular enzymes, the stereoselective dehydrogenase LbADH and the cofactor-regenerating glucose 1-dehydrogenase GDH. The enzymes were genetically fused with a SpyTag or SpyCatcher domain, respectively, to generate two complementary homo-tetrameric building blocks that polymerise under physiological conditions into porous hydrogels. The biocatalytic gels were used for the highly stereoselective reduction of a prochiral diketone substrate where they showed the typical behaviour of the coupled kinetics of coenzyme regenerating reactions in the substrate channelling regime. They effectively sequestrate the NADPH cofactor even under continuous flow conditions. Owing to their sticky nature, the gels can be readily mounted in simple microfluidic reactors without the need for supportive membranes. The reactors revealed extraordinary high space-time yields with nearly quantitative conversion (>95%), excellent stereoselectivity (d.r. > 99:1), and total turnover numbers of the expensive cofactor NADP(H) that are amongst the highest values ever reported.