R.B. Leveson-Gower

Most flavin-dependent enzymes contain a dissociable flavin cofactor. We present a new approach for installing a covalent bond between a flavin cofactor and its hosting protein. By using a flavin transferase and carving a flavinylation motif in target proteins, we demonstrate that ‘dissociable’ flavoproteins can be turned into covalent flavoproteins. Specifically, three different FMN-containing proteins were engineered to undergo covalent flavinylation: a light-oxygen-voltage (LOV) domain protein, a mini singlet-oxygen-generator (miniSOG), and a nitroreductase (BtNR). Optimizing the flavinylation motif and expression conditions led to covalent flavinylation of all three flavoproteins. The engineered covalent flavoproteins retained function and often exhibited improved performance such as higher thermostability or catalytic performance. Crystal structures of all three covalent flavoproteins confirmed the designed threonyl-phosphate linkage. The targeted flavoproteins differ in fold and function, indicating that this method of introducing a covalent flavin-protein bond is a powerful new method to create flavoproteins which cannot lose their cofactor, boosting their performance.

Artificial metalloenzymes based on a cobalt cofactor show higher activity than the free cobalt complex for the oxidation of benzylic C(sp³)−H bonds in aqueous medium. The cobalt cofactor is presumably stabilized by the hydrophobic cleft provided by the protein NB4.

Abstract

Artificial metalloenzymes have emerged as biohybrid catalysts that allow to combine the reactivity of a metal catalyst with the flexibility of protein scaffolds. This work reports the artificial metalloenzymes based on the β-barrel protein nitrobindin NB4, in which a cofactor [Co^IIX(Me₃TACD-Mal)]⁺X⁻ (X=Cl, Br; Me₃TACD=N,N^',N''-trimethyl-1,4,7,10-tetraazacyclododecane, Mal=CH₂CH₂CH₂NC₄H₂O₂) was covalently anchored via a Michael addition reaction. These biohybrid catalysts showed higher efficiency than the free cobalt complexes for the oxidation of benzylic C(sp³)−H bonds in aqueous media. Using commercially available oxone (2KHSO₅ ⋅ KHSO₄ ⋅ K₂SO₄) as oxidant, a total turnover number of up to 220 and 97 % ketone selectivity were achieved for tetralin. As catalytically active intermediate, a mononuclear terminal cobalt(IV)-oxo species [Co(IV)=O]²⁺ was generated by reacting the cobalt(II) cofactor with oxone in aqueous solution and characterized by ESI-TOF MS.

A novel insect-derived flavoenzyme from the honeybee Apis mellifera (beeHMFO) can selectively oxidize 5-hydroxymethylfurfural (HMF) to the corresponding dialdehyde 2,5-diformylfuran (DFF), which is an interesting bio-based polymer precursor. Moreover, activity toward a number of other aromatic alcohols is observed. The predicted structure of beeHMFO shows high similarity to other flavoprotein oxidases capable of oxidizing HMF.

Abstract

The chemical 5-hydroxymethylfurfural (HMF) can be derived from lignocellulose and is an interesting bio-based platform chemical as it has the potential to be transformed into numerous valuable building blocks such as the polymer-precursor 2,5-diformylfuran (DFF). To date, only a few oxidases acting on HMF are known and by sampling atypical species, we discovered a novel flavin-dependent oxidoreductase from the honeybee Apis mellifera (beeHMFO). The enzyme can perform the chemoselective oxidation of HMF to DFF but can also readily accept other aromatic alcohols as substrates. The function of the enzyme may well be the antimicrobial generation of hydrogen peroxide using HMF, which is very abundant in honey. The discovery of this insect-derived flavoprotein oxidase holds promising potential in the synthesis of renewable products and demonstrates that insects can be an interesting source of novel biocatalysts.

NRPSs are an important source of pharmaceutically valuable natural products. The large sequence space of NRPS variant libraries requires a robust, high-throughput sorting and screening platform. Here we present a novel high-throughput microfluidic screening platform for the investigation of mutants of NRP producing bacteria in a highly parallel manner. Our results demonstrate the power of this platform for studying large libraries of NRPS variants.

Abstract

Nonribosomal peptide synthetases (NRPSs) are giant enzymatic assembly lines that deliver many pharmaceutically valuable natural products, including antibiotics. As the search for new antibiotics motivates attempts to redesign nonribosomal metabolic pathways, more robust and rapid sorting and screening platforms are needed. Here, we establish a microfluidic platform that reliably detects production of the model nonribosomal peptide gramicidin S. The detection is based on calcein-filled sensor liposomes yielding increased fluorescence upon permeabilization. From a library of NRPS mutants, the sorting platform enriches the gramicidin S producer 14.5-fold, decreases internal stop codons 250-fold, and generates enrichment factors correlating with enzyme activity. Screening for NRPS activity with a reliable non-binary sensor will enable more sophisticated structure-activity studies and new engineering applications in the future.

A nucleotidoprotein engineered via facile “green synthesis” exhibits strong electrostatic attraction and hydrogen bonding with complementary base-modified polyethyleneimine to form salt-resistant nanocomplexes with robust cytosolic delivery efficiency. The acidic endolysosomal environment enables traceless restoration of the nucleotidoprotein and consequently promotes the intracellular release of native protein.

Abstract

Protein therapeutics targeting intracellular machineries hold profound potential for disease treatment, and hence robust cytosolic protein delivery technologies are imperatively demanded. Inspired by the super-negatively charged, nucleotide-enriched structure of nucleic acids, adenylated pro-proteins (A-proteins) with dramatically enhanced negative surface charges have been engineered for the first time via facile green synthesis. Then, thymidine-modified polyethyleneimine is developed, which exhibits strong electrostatic attraction, complementary base pairing, and hydrophobic interaction with A-proteins to form salt-resistant nanocomplexes with robust cytosolic delivery efficiencies. The acidic endolysosomal environment enables traceless restoration of the A-proteins and consequently promotes the intracellular release of the native proteins. This strategy shows high efficiency and universality for a variety of proteins with different molecular weights and isoelectric points in mammalian cells. Moreover, it enables highly efficient delivery of CRISPR-Cas9 ribonucleoproteins targeting fusion oncogene EWSR1-FLI1, leading to pronounced anti-tumor efficacy against Ewing sarcoma. This study provides a potent and versatile platform for cytosolic protein delivery and gene editing, and may benefit the development of protein pharmaceuticals.

Science, Volume 382, Issue 6666, Page 19-20, October 2023.

Cylindrocyclophanes A and F and merocyclophanes A and D were synthesized by a chemoenzymatic approach. The synthesis features an enzymatic Friedel–Crafts alkylation, reagent-controlled lithiation–borylation chemistry, cobalt-catalyzed asymmetric hydroboration, and Ni- or Pd-catalyzed alkyl–alkyl cross-coupling.

Abstract

Incorporating enzymatic reactions into natural product synthesis can significantly improve synthetic efficiency and selectivity. In contrast to the increasing applications of biocatalytic functional-group interconversions, the use of enzymatic C−C bond formation reactions in natural product synthesis is underexplored. Herein, we report a concise and efficient approach for the synthesis of [7.7]paracyclophane natural products, a family of polyketides with diverse biological activities. By using enzymatic Friedel–Crafts alkylation, cylindrocyclophanes A and F and merocyclophanes A and D were synthesized in six to eight steps in the longest linear sequence. This study demonstrates the power of combining enzymatic reactions with contemporary synthetic methodologies and provides opportunities for the structure–activity relationship studies of [7.7]paracyclophane natural products.

Nature Chemical Biology, Published online: 28 September 2023; doi:10.1038/s41589-023-01426-y

The flavoenzyme nicotine oxidoreductase degrades nicotine in the bloodstream. Now, genetic selection in bacteria has been used to improve the catalytic performance of nicotine oxidoreductase, isolating variants with increased O2 reactivity that were more effective at degrading nicotine in the blood of rats.

The creation of enzymes containing non-biological functionalities with activation modes outside of Nature’s canon paves the way towards fully programmable biocatalysis. Here, we present a fully genetically encoded boronic acid containing designer enzyme with organocatalytic reactivity not achievable with natural or engineered biocatalysts. This boron enzyme catalyzes the kinetic resolution of hydroxyketones by oxime formation where crucial interactions with the protein scaffold assist in the catalysis. A directed evolution campaign lead to a variant with natural enzyme like enantioselectivities for a number of different substrates. The unique activation mode of the boron enzyme was studied via X-ray crystallography, high resolution mass spectrometry and 11B NMR spectroscopy and opens up the possibility for a new class of boron dependent biocatalysts.

Saturated N-heterocycles constitute a vital scaffold for pharmaceutical chemistry, but are challenging to access synthetically, particularly in asymmetric mode. Here we demonstrate how imine reductases can achieve annulation through tandem inter and intramolecular reductive amination processes. Imine reductases were used in combination with further enzymes to access un-substituted, α-substituted and α,α’-disubstituted N-heterocycles from simple starting materials, in one pot and under benign conditions. The work was exemplified in regard to product scope and a new route to the valuable natural product nicotine was demonstrated.

Nature, Published online: 20 September 2023; doi:10.1038/s41586-023-06529-z

Heterogeneous geminal-atom catalysts, which pair single-atom sites in specific coordination and spatial proximity, offer a new avenue for the sustainable manufacture of fine chemicals.

In this study, we describe a light-driven photocatalytic decarboxylation of palmitic acid and related fatty acids using Chlorella variabilis fatty acid photodecarboxylase (CvFAP). By utilizing violet LED light (50 W; 397 nm), we achieved a remarkable conversion efficiency of 99% within just 4 minutes, surpassing the previous 79% conversion achieved in 60 minutes using blue LED light (300 W; 439 nm). Importantly, the use of 50 W violet LED light also resulted with lower enzyme photoinactivation rate when compared to 300 W blue LED. Comparing the whole-cell biocatalyst with the enzymatic extract, we found that the former demonstrated superior catalytic performance and reduced susceptibility to photoinactivation. Furthermore, through fed-batch reactions using three pulses of 13 mM palmitic acid, we achieved the production of 39 mM of pentadecane within 1 hour, highlighting a promising strategy for enhanced productivity. These findings represent a significant advancement in CvFAP photodecarboxylation processes, utilizing an alternative light source, with potential implications for biofuel production.

Efficient Mn artificial transfer hydrogenases (ATHases) were developed using the biotin-streptavidin technology, which exhibits high activity and enantioselectivity for the transfer hydrogenation of a wide range of aryl ketones. The S112Y-K121 M double mutation and the appropriate chemical structure of the Mn cofactor play critical roles in the reactivity and enantioselectivity of the enzymes.

Abstract

Artificial (transfer) hydrogenases have been developed for organic synthesis, but they rely on precious metals. Native hydrogenases use Earth-abundant metals, but these cannot be applied for organic synthesis due, in part, to their substrate specificity. Herein, we report the design and development of manganese transfer hydrogenases based on the biotin-streptavidin technology. By incorporating bio-mimetic Mn(I) complexes into the binding cavity of streptavidin, and through chemo-genetic optimization, we have obtained artificial enzymes that hydrogenate ketones with nearly quantitative yield and up to 98 % enantiomeric excess (ee). These enzymes exhibit broad substrate scope and high functional-group tolerance. According to QM/MM calculations and X-ray crystallography, the S112Y mutation, combined with the appropriate chemical structure of the Mn cofactor plays a critical role in the reactivity and enantioselectivity of the artificial metalloenzyme (ArMs). Our work highlights the potential of ArMs incorporating base-meal cofactors for enantioselective organic synthesis.

Sulfur trioxide acts as a strong oxidizer towards halides as well as pseudohalides. Therefore, reactions of sulfur trioxide with cyanide anions do usually not lead to the formation of cyanido-sulfates anion but to oxidation of the cyanide ion. This problem can be avoided if a sulfur trioxide-pyridine complex is used instead of neat sulfur trioxide. In this way, the oxidation power is sufficiently reduced, and the pyridine molecule can be replaced by the cyanide anion under formation of the cyanido-sulfate anion, SO₃CN⁻. Thus sulfur trioxide gets rid of the badly smelling pyridine molecule, however, at the expense of gaining the toxic cyanide anion. More information can be found in the Research Article by M. S. Wickleder and co-workers (DOI: 10.1002/chem.202301761).

Generating artificial metalloenzymes (ArMs) by incorporating a non-natural metallocofactor within a protein host is an attractive strategy to complement homogeneous catalysts and enzymes. In an effort to achieve Co(TAML) catalyzed asymmetric radical-type oxygen atom transfer catalysis, we anchored a biotinylated Co(TAML) cofactor into streptavidin. In the presence of iodosylbenzene and α-methylstyrene, the Co(TAML) based ArM led to the corresponding enantioenriched oxirane, thereby expanding the scope of ArMs that facilitate radical-type transformations. Screening of the Sav library—that included (double) mutations at positions S112 and K121—enabled improvement of both the activity and the selectivity of the Co-TAML-catalyzed epoxidation reaction. Evaluation of the secondary coordination sphere around the Co(TAML) cofactor by analysis of the X-ray structures of two different ArMs: Co(TAML) · Sav WT and Co(TAML) · Sav S112Y, suggested how mutations may affect the catalytic properties.

Proceedings of the National Academy of Sciences, Volume 120, Issue 37, September 2023.

Nature Catalysis, Published online: 14 September 2023; doi:10.1038/s41929-023-01024-0

Non-natural photobiocatalysis is attractive but usually involves UV light activation or the formation of electron donor–acceptor complexes. Now direct visible-light excitation of flavin-dependent ene-reductases allows stereocontrolled intermolecular radical hydroarylation of alkenes initiated by single-electron oxidation.