R.B. Leveson-Gower

Molecular catalysts based on abundant elements that function in neutral water represent an essential component of sustainable hydrogen production. Artificial hydrogenases based on protein-inorganic hybrids have emerged as an intriguing class of catalysts for this purpose. We have prepared a novel artificial hydrogenase based on cobaloxime bound to a de novo three alpha-helical protein, α3C, via a pyridyl-based unnatural amino acid. The functionalized de novo protein was characterized by UV-visible, CD, and EPR spectroscopy, as well as MALDI spectrometry, which confirmed the presence and ligation of cobaloxime to the protein. The new de novo protein produced hydrogen under electrochemical, photochemical and reductive chemical conditions in neutral water solution. A change in hydrogen evolution capability of the de novo enzyme compared with native cobaloxime was observed, with tunover numbers around 80% for that of cobaloxime, and hydrogen evolution rates of 40% of that of cobaloxime. We discuss these findings in the context of existing literature, and our study contributes important information about the functionality of cobaloxime HER catalysts in protein environments, and the feasibility of artificial enzymes to the field of artificial metalloenzymes. Small de novo proteins as enzyme scaffolds have the potential to function as upscaleable bioinspired catalysts thanks to their efficient atom economy, and the findings presented here show that this type of novel enzymes are a possible product.

Nature, Published online: 13 April 2023; doi:10.1038/d41586-023-01286-5

Supporters of Susanne Täuber say her dismissal is a blow for academic freedom, with thousands signing a petition demanding she be allowed to return to work.

Publication date: 13 July 2023

Source: Chem, Volume 9, Issue 7

Author(s): Yu Fu, Xiaohong Liu, Yan Xia, Xuzhen Guo, Juan Guo, Junshuai Zhang, Weining Zhao, Yuzhou Wu, Jiangyun Wang, Fangrui Zhong

The ability to a site-selectively modify equivalent functional groups in a molecule has the potential to streamline syntheses and increase product yields by lowering step counts. Enzymes catalyze site-selective transformations throughout primary and secondary metabolism, but leveraging this capability for non-native substrates and reactions requires a detailed understanding of the potential and limitations of enzyme catalysis and how these bounds can be extended by protein engineering. In this review, we discuss representative examples of site-selective enzyme catalysis involving functional group manipulation and C-H bond functionalization. We include illustrative examples of native catalysis, but our focus is on cases involving non-native substrates and reactions often using engineered enzymes. We then discuss the use of these enzymes for chemoenzymatic transformations and target-oriented synthesis and conclude with a survey of tools and techniques that could expand the scope of non-native site-selective enzyme catalysis.

We demonstrate a novel nCAS9-mutagenic polymerase-based continuous evolution platform for improvement of enzymatic activity that functions at ultra-high throughput. By cycling cells between growth, mutagenesis, and microfluidics-based sorting, we mimic natural evolution but at a pace that is orders of magnitude faster, yielding an alditol oxidase variant with 10.5-fold improved catalytic efficiency for waste product, glycerol as a substrate.

Abstract

Enzymes are highly specific catalysts delivering improved drugs and greener industrial processes. Naturally occurring enzymes must typically be optimized which is often accomplished through directed evolution; however, this is still a labor- and capital-intensive process, due in part to multiple molecular biology steps including DNA extraction, in vitro library generation, transformation, and limited screening throughput. We present an effective and broadly applicable continuous evolution platform that enables controlled exploration of fitness landscape to evolve enzymes at ultrahigh throughput based on direct measurement of enzymatic activity. This drop-based microfluidics platform cycles cells between growth and mutagenesis followed by screening with minimal human intervention, relying on the nCas9 chimera with mutagenesis polymerase to produce in vivo gene diversification using sgRNAs tiled along the gene. We evolve alditol oxidase to change its substrate specificity towards glycerol, turning a waste product into a valuable feedstock. We identify a variant with a 10.5-fold catalytic efficiency.

Visible light absorbing iridium(III) complexes have been widely employed as photocatalysts in organic synthesis. Their catalytic reactivity and selectivity are generally optimized by modifying the structures of coordinated ligands to obtain desired photophysical properties. Artificial metalloenzymes (ArMs) can combine the unique features of both metal complexes and enzymes by incorporating a cofactor within a protein scaffold, which offers another strategy to improve the performance of metal catalysts. Herein, we describe a panel of Ir(III)-ArMs constructed by covalently embedding iridium(III) polypyridyl complexes into a prolyl oligopeptidase scaffold. A series of spectroscopic methods were used to examine how properties of the resulting ArMs are influenced by structural variation of the cyclometalated ligands and the protein scaffold. Visible light photocatalysis by these hybrid catalysts was also examined, leading to the finding that they catalyze inter/intra-molecular [2+2] photocycloaddition in aqueous solution and indicating that they can serve as new bio-photocatalysts for further exploration.

Nature, Published online: 30 March 2023; doi:10.1038/d41586-023-00928-y

Crocodiles and Komodo dragons provide evidence to support the idea of a scaly cover over the teeth of dinosaur Tyrannosaurus rex.

Science, Volume 379, Issue 6639, Page 1358-1363, March 2023.

Terpenoids are applied in various ways, in flavors and fragrances as well as in pharmaceuticals and plant protection. Through diversification of the carbon scaffold, non-natural terpenoids can be generated and screened for improved properties. The identification and engineering of methyltransferases for late-stage C-methylation of unactivated alkenes with high selectivity provided access to methylated derivatives of readily available terpenoids.

Abstract

Terpenoids are built from isoprene building blocks and have numerous biological functions. Selective late-stage modification of their carbon scaffold has the potential to optimize or transform their biological activities. However, the synthesis of terpenoids with a non-natural carbon scaffold is often a challenging endeavor because of the complexity of these molecules. Herein we report the identification and engineering of (S)-adenosyl-l-methionine-dependent sterol methyltransferases for selective C-methylation of linear terpenoids. The engineered enzyme catalyzes selective methylation of unactivated alkenes in mono-, sesqui- and diterpenoids to produce C ₁₁, C ₁₆ and C ₂₁ derivatives. Preparative conversion and product isolation reveals that this biocatalyst performs C−C bond formation with high chemo- and regioselectivity. The alkene methylation most likely proceeds via a carbocation intermediate and regioselective deprotonation. This method opens new avenues for modifying the carbon scaffold of alkenes in general and terpenoids in particular.

An unprecedented β-C−H functionalization reaction that is enabled by ene reductases is reported. When the reaction is coupled with photocatalysis, various 6-azabicyclo[3.2.1]octan-3-ones can be synthesized in a straightforward manner from readily available cyclohexenones and N-phenylglycines. This chemoenzymatic reaction can be carried out on a gram scale, and the product can be further selectively derivatized.

Abstract

Herein we report that ene reductases (EREDs) can facilitate an unprecedented intramolecular β-C−H functionalization reaction for the synthesis of bridged bicyclic nitrogen heterocycles containing the 6-azabicyclo[3.2.1]octane scaffold. To streamline the synthesis of these privileged motifs, we developed a gram-scale one-pot chemoenzymatic cascade by combining iridium photocatalysis with EREDs, using readily available N-phenylglycines and cyclohexenones that can be obtained from biomass. Further derivatization using enzymatic or chemical methods can convert 6-azabicyclo[3.2.1]octan-3-one into 6-azabicyclo[3.2.1]octan-3α-ols, which can be potentially utilized for the synthesis of azaprophen and its analogues for drug discovery. Mechanistic studies revealed the reaction requires oxygen, presumably to produce oxidized flavin, which can selectively dehydrogenate the 3-substituted cyclohexanone derivatives to form the α,β-unsaturated ketone, which subsequently undergoes spontaneous intramolecular aza-Michael addition under basic conditions.

The biomimetic regeneration system for S-adenosylmethionine (SAM) and SAM analogues presented is based on the salvage of the adenine moiety and in situ supply of d-ribose and polyphosphate. It is compatible with a broad range of SAM-dependent enzymes including aminopropyl transferases, and is shown to support ethylation reactions with both conventional and radical SAM methyltransferases.

Abstract

S-Adenosylmethionine (SAM) is an enzyme cofactor involved in methylation, aminopropyl transfer, and radical reactions. This versatility renders SAM-dependent enzymes of great interest in biocatalysis. The usage of SAM analogues adds to this diversity. However, high cost and instability of the cofactor impedes the investigation and usage of these enzymes. While SAM regeneration protocols from the methyltransferase (MT) byproduct S-adenosylhomocysteine are available, aminopropyl transferases and radical SAM enzymes are not covered. Here, we report a set of efficient one-pot systems to supply or regenerate SAM and SAM analogues for all three enzyme classes. The systems’ flexibility is showcased by the transfer of an ethyl group with a cobalamin-dependent radical SAM MT using S-adenosylethionine as a cofactor. This shows the potential of SAM (analogue) supply and regeneration for the application of diverse chemistry, as well as for mechanistic studies using cofactor analogues.

Engineered biocatalysis: Here we show that the scope of reductive aminases (RedAms) can be extended to allow selective reductive aminations of cyclic secondary amines, such as piperidines and morpholines, with both aldehydes and ketones. These biotransformations provide access to important motifs found in active pharmaceutical ingredients and other bioactive molecules.

Abstract

Reductive aminases (RedAms) have recently emerged as promising biocatalysts for the synthesis of chiral secondary amines by coupling primary amines with aldehydes/ketones. However, access to tertiary amines remains more problematic, particularly when coupling ketones with secondary amines. Here we show that the scope of these enzymes can be extended to allow selective reductive aminations of cyclic secondary amines, such as piperidines and morpholines, with both aldehydes and ketones. These biotransformations provide access to important motifs found in active pharmaceutical ingredients and other bioactive molecules. RedAm-361, discovered from a metagenomic library, was engineered via directed evolution to allow efficient coupling of cyclic amines with carbonyl partners, including dynamic kinetic resolutions of α-functionalized aldehydes and enantioselective amination of ketones. These RedAms now serve as valuable scaffolds for the engineering of industrial biocatalysts to produce key pharmaceutical intermediates.

Anaerobic flow biocatalysis: An engineered cytochrome c enzyme, capable of carbon-silicon bond formation, was immobilised for the first time using the SpyTag/SpyCatcher bioconjugation system. When used in a continuous flow reactor, the immobilised enzyme was able to produce organosilicons with up to 6-fold increased turnover numbers compared to the free enzyme in conventional batch experiments.

Abstract

The recent development of tailored cytochrome enzymes has enabled “new-to-nature” reactivities, such as the biocatalytic formation of carbon-silicon bonds using the cytochrome c from Rhodothermus marinus. To maximise the potential of this remarkable biocatalyst by increasing its turnover numbers (TON) and to enable its reusability in continuous processes, we report the use of the SpyTag/SpyCatcher bioconjugation system to immobilise this enzyme. We successfully modified the enzyme with a SpyTag without significant effects on its catalytic activity. Even after immobilization on microparticles the enzyme retained 60 % activity. When the immobilized enzyme was used in sequential batch or continuous flow to produce an organosilicon, we observed up to 6-fold higher turnover numbers over a total period of 10 days compared to the free enzyme reaction, however we observed a drop in stereoselectivity under these conditions. This is the first report on the successful immobilisation of a carbon-silicon bond forming enzyme for the continuous, biocatalytic production of organosilicons.

Proceedings of the National Academy of Sciences, Volume 120, Issue 12, March 2023.

The role of peptides is nowadays relevant in fields such as drug discovery and biotechnology. Computational analyses are required to study their properties and gain insights into rational design strategies. Both natural and modified peptides containing non-natural amino acids require customized tools to run sequence and structure-based studies. PepFun 2.0 is a new version of the python package for the study of natural and modified peptides using a set of modules to analyze the sequence and structure of the molecules. PepFun 2.0 comprises five main modules for different tasks such as sequence alignments, prediction of properties, generation of conformers, modification of structures, detection of protein-peptide interactions, and extra functions to include peptides containing non-natural amino acids. The code and tutorial are available at: https://github.com/rochoa85/PepFun2

Nature, Published online: 20 March 2023; doi:10.1038/s41586-023-05950-8

Enantioconvergent Cu-catalyzed N-alkylation of aliphatic amines

Nature, Published online: 17 March 2023; doi:10.1038/d41586-023-00831-6

Editors threaten to resign over ‘no-reject’ model that others see as the future of research journals.