Repurposing a Fully Reducing Polyketide Synthase toward 2-Methyl Guerbet-like Lipids.

In nature, thousands of diverse and bioactive polyketides are assembled by a family of multifunctional, "assembly line" enzyme complexes called polyketide synthases (PKS). Since the late 20th century, there have been several attempts to decode, rearrange, and "reprogram" the PKS assembly line to generate valuable materials such as biofuels and platform chemicals. Here, the first module from () PKS12, an unorthodox, "modularly iterative" PKS, was modified and repurposed toward the formation of 2-methyl Guerbet lipids, which have wide applications in industry.

Installation of an organocatalyst into a protein scaffold creates an artificial Stetterase.

Using a protein scaffold covalently functionalised with a thiamine-inspired N-heterocyclic carbene (NHC), we created an artificial Stetterase (ArtiSt) which catalyses a stereoselective, intramolecular Stetter reaction. We demonstrate that ArtiSt functions under ambient conditions with low catalyst loading. Furthermore, activity can be increased >20 fold by altering the protein scaffold.

Developing deprotectase biocatalysts for synthesis.

Organic synthesis often requires multiple steps where a functional group (FG) is concealed from reaction by a protecting group (PG). Common PGs include -carbobenzyloxy (Cbz or Z) of amines and -butyloxycarbonyl (OBu) of acids. An essential step is the removal of the PG, but this often requires excess reagents, extensive time and can have low % yield.

Collaborative regulation of yeast SPT-Orm2 complex by phosphorylation and ceramide.

The homeostatic regulation of serine palmitoyltransferase (SPT) activity in yeast involves N-terminal phosphorylation of Orm proteins, while higher eukaryotes lack these phosphorylation sites. Although recent studies have indicated a conserved ceramide-mediated feedback inhibition of the SPT-ORM/ORMDL complex in higher eukaryotes, its conservation and relationship with phosphorylation regulation in yeast remain unclear. Here, we determine the structure of the yeast SPT-Orm2 complex in a dephosphomimetic state and identify an evolutionarily conserved ceramide-sensing site.

Visualizing the Interface of Biotin and Fatty Acid Biosynthesis through SuFEx Probes.

Site-specific covalent conjugation offers a powerful tool to identify and understand protein-protein interactions. In this study, we discover that sulfur fluoride exchange (SuFEx) warheads effectively crosslink the acyl carrier protein (AcpP) with its partner BioF, a key pyridoxal 5'-phosphate (PLP)-dependent enzyme in the early steps of biotin biosynthesis by targeting a tyrosine residue proximal to the active site. We identify the site of crosslink by MS/MS analysis of the peptide originating from both partners.

Direct purification and immobilization of his-tagged enzymes using unmodified nickel ferrite NiFeO magnetic nanoparticles.

Purification of valuable engineered proteins and enzymes can be laborious, costly, and generating large amount of chemical waste. Whilst enzyme immobilization can enhance recycling and reuse of enzymes, conventional methods for immobilizing engineered enzymes from purified samples are also inefficient with multiple-step protocols, regarding both the carrier preparation and enzyme binding. Nickel ferrite magnetic nanoparticles (NiFeO MNPs) offer distinct advantages in both purification and immobilization of enzymes.

An improved, optimised and robust keratin azure assay for accurate assessment of keratinase activity.

Keratin, in the form of coarse sheep wool, has been identified as an undervalued natural resource, which with the appropriate tools ( a keratinase biocatalyst) can be repurposed for various textile and industrial biotechnology applications. For these purposes, we describe a novel method for identifying keratinase activity through the use of α-keratin azure (KA), an anthraquinone dyed substrate. A colourimetric method monitored the keratinase activity of Proteinase K (PK), which degrades the KA substrate and releases soluble products that are observed at 595 nm.

Versatile Chemo-Biocatalytic Cascade Driven by a Thermophilic and Irreversible C-C Bond-Forming α-Oxoamine Synthase.

We report a chemo-biocatalytic cascade for the synthesis of substituted pyrroles, driven by the action of an irreversible, thermostable, pyridoxal 5'-phosphate (PLP)-dependent, C-C bond-forming biocatalyst (AOS). The AOS catalyzes the Claisen-like condensation between various amino acids and acyl-CoA substrates to generate a range of α-aminoketones. These products are reacted with β-keto esters in an irreversible Knorr pyrrole reaction.

Coupled Natural Fusion Enzymes in a Novel Biocatalytic Cascade Convert Fatty Acids to Amines.

Tambjamine YP1 is a pyrrole-containing natural product. Analysis of the enzymes encoded in the "" biosynthetic gene cluster (BGC) identified a unique di-domain biocatalyst (TamH). Sequence and bioinformatic analysis predicts that TamH comprises an N-terminal, pyridoxal 5'-phosphate (PLP)-dependent transaminase (TA) domain fused to a NADH-dependent C-terminal thioester reductase (TR) domain.

Characterization of inositol lipid metabolism in gut-associated Bacteroidetes.

Inositol lipids are ubiquitous in eukaryotes and have finely tuned roles in cellular signalling and membrane homoeostasis. In Bacteria, however, inositol lipid production is relatively rare. Recently, the prominent human gut bacterium Bacteroides thetaiotaomicron (BT) was reported to produce inositol lipids and sphingolipids, but the pathways remain ambiguous and their prevalence unclear.

BioWF: A Naturally-Fused, Di-Domain Biocatalyst from Biotin Biosynthesis Displays an Unexpectedly Broad Substrate Scope.

The carbon backbone of biotin is constructed from the C di-acid pimelate, which is converted to an acyl-CoA thioester by an ATP-dependent, pimeloyl-CoA synthetase (PCAS, encoded by BioW). The acyl-thioester is condensed with ʟ-alanine in a decarboxylative, Claisen-like reaction to form an aminoketone (8-amino-7-oxononanoic acid, AON). This step is catalysed by the pyridoxal 5'-phosphate (PLP)-dependent enzyme (AON synthase, AONS, encoded by BioF).

Convergent evolution of bacterial ceramide synthesis.

The bacterial domain produces numerous types of sphingolipids with various physiological functions. In the human microbiome, commensal and pathogenic bacteria use these lipids to modulate the host inflammatory system. Despite their growing importance, their biosynthetic pathway remains undefined since several key eukaryotic ceramide synthesis enzymes have no bacterial homolog.

Creation of an Engineered Amide Synthetase Biocatalyst by the Rational Separation of a Two-Step Nitrile Synthetase.

The synthesis of amides through acid and amine coupling is one of the most commonly used reactions in medicinal chemistry, yet still requires atom-inefficient coupling reagents. There is a current demand to develop greener, biocatalytic approaches to amide bond formation. The nitrile synthetase (NS) enzymes are a small family of ATP-dependent enzymes which catalyse the transformation of a carboxylic acid into the corresponding nitrile via an amide intermediate.

Direct monitoring of biocatalytic deacetylation of amino acid substrates by H NMR reveals fine details of substrate specificity.

Amino acids are key synthetic building blocks that can be prepared in an enantiopure form by biocatalytic methods. We show that the l-selective ornithine deacetylase ArgE catalyses hydrolysis of a wide-range of N-acyl-amino acid substrates. This activity was revealed by 1H NMR spectroscopy that monitored the appearance of the well resolved signal of the acetate product.

The N-Acetyl Amino Acid Racemases (NAAARs); Native and evolved biocatalysts applied to the synthesis of canonical and non-canonical amino acids.

Amino acids are one of the most important synthons employed in the biotechnology, pharmaceutical and agrochemical industries for the preparation of active agents. Recently, the emerging use of these compounds as tools for protein engineering, has also been reported. Numerous chemo- and biocatalytic strategies have been developed for the stereoselective synthesis of these compounds.

Solution Structure and Conformational Dynamics of a Doublet Acyl Carrier Protein from Prodigiosin Biosynthesis.

The acyl carrier protein (ACP) is an indispensable component of both fatty acid and polyketide synthases and is primarily responsible for delivering acyl intermediates to enzymatic partners. At present, increasing numbers of multidomain ACPs have been discovered with roles in molecular recognition of trans-acting enzymatic partners as well as increasing metabolic flux. Further structural information is required to provide insight into their function, yet to date, the only high-resolution structure of this class to be determined is that of the doublet ACP (two continuous ACP domains) from mupirocin synthase.

Harnessing and engineering amide bond forming ligases for the synthesis of amides.

The amide functional group is ubiquitous in nature and one of the most important motifs in pharmaceuticals, agrochemicals, and other valuable products. While coupling amides and carboxylic acids is a trivial synthetic transformation, it often requires protective group manipulation, along with stoichiometric quantities of expensive and deleterious coupling reagents. Nature has evolved a range of enzymes to construct amide bonds, the vast majority of which utilize adenosine triphosphate to activate the carboxylic acid substrate for amine coupling.

Synthesis of -acyl amide natural products using a versatile adenylating biocatalyst.

Natural products are secondary metabolites produced by many different organisms such as bacteria, fungi and plants. These biologically active molecules have been widely exploited for clinical application. Here we investigate TamA, a key enzyme from the biosynthetic pathway of tambjamine YP1, an acylated bipyrrole that is produced by the marine microorganism .

Use of isotopically labeled substrates reveals kinetic differences between human and bacterial serine palmitoyltransferase.

Isotope labels are frequently used tools to track metabolites through complex biochemical pathways and to discern the mechanisms of enzyme-catalyzed reactions. Isotopically labeled l-serine is often used to monitor the activity of the first enzyme in sphingolipid biosynthesis, serine palmitoyltransferase (SPT), as well as labeling downstream cellular metabolites. Intrigued by the effect that isotope labels may be having on SPT catalysis, we characterized the impact of different l-serine isotopologues on the catalytic activity of recombinant SPT isozymes from humans and the bacterium Our data show that SPT activity displays a clear isotope effect with [2,3,3-D]l-serine, whereas the human SPT isoform does not.