R.B. Leveson-Gower

Nature Communications, Published online: 06 May 2023; doi:10.1038/s41467-023-38328-5

Recently, a pipeline for the design of protein-binding proteins using only the structure of the target protein was reported. Here, the authors report that the incorporation of deep learning methods into the original pipeline increases experimental success rate by ten-fold.

Nature, Published online: 03 May 2023; doi:10.1038/s41586-023-06027-2

The α-diazoester azaserine can be produced by Streptomyces albus engineered with a biosynthetic gene cluster and act as the carbene precursor for coupling with intracellularly produced styrene to generate unnatural amino acids containing a cyclopropyl group.

Separation not required: Mass spectrometry without prior chromatographic separation, carried out on a single-quadrupole HPLC-MS, can be used for the qualitative and quantitative analysis of diverse biotransformations. This flow-injection analysis mass spectrometry (FIA-MS) approach represents an attractive alternative to more traditional photometric, fluorometric, and chromatographic methods for screening enzymatic reactions.

Abstract

Mass spectrometry-based high-throughput screening methods combine the advantages of photometric or fluorometric assays and analytical chromatography, as they are reasonably fast (throughput ≥1 sample/min) and broadly applicable, with no need for labelled substrates or products. However, the established MS-based screening approaches require specialised and expensive hardware, which limits their broad use throughout the research community. We show that a more common instrumental platform, a single-quadrupole HPLC-MS, can be used to rapidly analyse diverse biotransformations by flow-injection mass spectrometry (FIA-MS), that is, by automated infusion of samples to the ESI-MS detector without prior chromatographic separation. Common organic buffers can be employed as internal standard for quantification, and the method provides readily validated activity and selectivity information with an analytical run time of one minute per sample. We report four application examples that cover a broad range of analyte structures and concentrations (0.1–50 mM before dilution) and diverse biocatalyst preparations (crude cell lysates and whole microbial cells). Our results establish FIA-MS as a versatile and reliable alternative to more traditional methods for screening enzymatic reactions.

Sequence-based functional annotation of enzymes is an essential step in the discovery and development of new biocatalysts. The vinylglycine ketimine (VGK) subfamily of pyridoxal-phosphate dependent enzymes plays critical roles in sulfur metabolism and is home to a growing range of secondary metabolic enzymes that synthesize noncanonical amino acids. However, discovery of useful new enzymes has been slowed because functional assignments within the VGK sub-family are convoluted by pervasive mis-annotation. Here, we used a whole-cell substrate multiplexed screening approach to rapidly measure catalytic activities of 40 homologs in the VGK subfamily. This strategy gives direct information on enzyme specificity without having to purify each enzyme or measure individual kinetic constants. We identified a thermostable cystathionine γ-lyase from Thermobifida fusca and performed mechanistic and structural studies. For biocatalytic applications, we identified a well-behaved, thermostable, and promiscuous amino acid γ-synthase from Caldicellulosiruptor hydrothermalis (CahyGS). We showed CahyGS can catalyze a stereoselective γ-addition into L-allylglycine, providing preparative-scale access to a unique set of γ-branched amino acids. High resolution crystal structures of CahyGS show an open-closed transition associated with ligand binding and provide a basis for subsequent engineering. Together, these data show how multiplexed screening approaches aid in the rapid deconvolution of enzyme function and identify enzymes with useful properties for enzymology and biocatalysis.

A bioinspired approach using enzyme electrocatalysis for the efficient direct reduction of CO₂ at low concentrations to formate using Carbonic Anhydrase co-immobilized with Formate Dehydrogenase in a mesoporous indium tin oxide electrode is described.

Abstract

The electrolysis of dilute CO₂ streams suffers from low concentrations of dissolved substrate and its rapid depletion at the electrolyte-electrocatalyst interface. These limitations require first energy-intensive CO₂ capture and concentration, before electrolyzers can achieve acceptable performances. For direct electrocatalytic CO₂ reduction from low-concentration sources, we introduce a strategy that mimics the carboxysome in cyanobacteria by utilizing microcompartments with nanoconfined enzymes in a porous electrode. A carbonic anhydrase accelerates CO₂ hydration kinetics and minimizes substrate depletion by making all dissolved carbon available for utilization, while a highly efficient formate dehydrogenase reduces CO₂ cleanly to formate; down to even atmospheric concentrations of CO₂. This bio-inspired concept demonstrates that the carboxysome provides a viable blueprint for the reduction of low-concentration CO₂ streams to chemicals by using all forms of dissolved carbon.

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.