No abstract

A major challenge in chemical synthesis is to develop catalytic systems that convert simple molecules to complex high-value products. Often these valuable compounds must be manufactured asymmetrically, as their biochemical properties can differ based on the chirality of the molecule. Of great interest are enantioenriched amine diastereomers, which are prevalent in pharmaceuticals and agrochemicals, 1 yet their preparation often relies on low-e ciency multi-step synthesis. 2 Herein, we report the discovery and characterisation of a multi-functional biocatalyst, which operates using a previously unreported conjugate reduction-reductive amination mechanism. This enzyme (pIR-120), identi ed within a metagenomic imine reductase (IRED) collection 3 and originating from an unclassi ed Pseudomonas species, possesses an unusual active site architecture that facilitates an amine-activated conjugate alkene reduction followed by reductive amination. This enzyme enables the coupling of a broad selection of α,β-unsaturated carbonyls with amines for the e cient preparation of enantioenriched amine diastereomers. Moreover, employing a racemic substrate partner or conjugated dienyl-ketone provides a means of controlling additional stereocentres using the single catalyst. Mechanistic and structural studies have been carried out to delineate the order of individual steps catalysed by pIR-120 which have led to a proposal for the overall catalytic cycle. This work shows that the IRED family can serve as a platform for facilitating the discovery of further enzymatic activities for application in synthetic biology and organic synthesis.

Enzymes are attractive tools for synthetic applications. To be viable for industrial use, enzymes need sufficient stability towards the desired reaction conditions such as high substrate and cosolvent concentration, non‐neutral pH and elevated temperatures. Thermal stability is an attractive feature not only because it allows for protein purification by thermal treatment and higher process temperatures but also due to the associated higher stability against other destabilising factors. Therefore, high‐throughput screening (HTS) methods are desirable for the identification of thermostable biocatalysts by discovery from nature or by protein engineering but current methods have low throughput and require time‐demanding purification of protein samples. We found that nanoscale differential scanning fluorimetry (nanoDSF) is a valuable tool to rapidly and reliably determine melting points of native proteins. To avoid intrinsic problems posed by crude protein extracts, hypotonic extraction of overexpressed protein from bacterial host cells resulted in higher sample quality and accurate manual determination of several hundred melting temperatures per day. We have probed the use of nanoDSF for HTS of a phylogenetically diverse aldolase library to identify novel thermostable enzymes from metagenomic sources and for the rapid measurements of variants from saturation mutagenesis. The feasibility of nanoDSF for the screening of synthetic reaction conditions was proved by studies of cosolvent tolerance, which showed protein melting temperature to decrease linearly with increasing cosolvent concentration for all combinations of six enzymes and eight water‐miscible cosolvents investigated, and of substrate affinity, which showed stabilisation of hexokinase by sugars in the absence of ATP cofactor. Enzymes Alcohol dehydrogenase (NADP+) (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC1/1/1/2.html), transketolase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/2/1/1.html), hexokinase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC2/7/1/1.html), 2‐deoxyribose‐5‐phosphate aldolase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC4/1/2/4.html), fructose‐6‐phosphate aldolase (http://www.chem.qmul.ac.uk/iubmb/enzyme/EC4/1/2.html.n).

SummarySystemic sclerosis is an autoimmune idiopathic connective tissue disease, characterized by vasculopathy, inflammation and fibrosis. There appears to be a link between inflammation and fibrosis, although the exact nature of the relationship is unknown. Serum amyloid A (SAA) is an acute-phase protein that is elevated up to 1000-fold in times of infection or inflammation. This acute-phase reactant, as well as being a marker of inflammation, may initiate signals in a cytokine-like manner, possibly through toll-like receptors (TLRs) promoting inflammation. This study addressed the role of SAA in initiating interleukin-6 (IL-6) production in dermal fibroblasts and the role of TLR2 in this system. We show that SAA induces IL-6 secretion in healthy dermal fibroblasts and that blockade of TLR2 with a neutralizing antibody to TLR2 or specific small interfering RNA attenuated the SAA-induced IL-6 secretion and that this was also mediated through the TLR adaptor protein IL-1 receptor-associated kinase 4. The effect is nuclear factor-jB-mediated because blockade of nuclear factor-jB reduced the induction. We also demonstrate that dermal fibroblasts express TLR2; this is functional and over-expressed in the fibroblasts of patients with systemic sclerosis. Taken together these data suggest that SAA is a danger signal that initiates IL-6 signalling in systemic sclerosis via enhanced TLR2 signalling.

The Covid-19 pandemic highlights the urgent need for cost-effective processes to rapidly manufacture antiviral drugs at scale. Here we report a concise biocatalytic process for Molnupiravir, a nucleoside analogue recently approved as an orally available treatment for SARS-CoV-2. Key to the success of this process was the development of an efficient biocatalyst for the production of N -hydroxy-cytidine through evolutionary adaption of the hydrolytic enzyme cytidine deaminase. This engineered biocatalyst performs >85 000 turnovers in less than 3 h, operates at 180 g/L substrate loading, and benefits from in situ crystallization of the N -hydroxy-cytidine product (85% yield), which can be converted to Molnupiravir by a selective 5′-acylation using Novozym 435.