R.B. Leveson-Gower

Benzoxazolinate is a rare bis-heterocyclic moiety that interacts with proteins and DNA. It was found that a putative acyl AMP-ligase mediates the last cyclization step to afford benzoxazolinate. The enzyme was then used as a probe for genome mining, which led to the discovery of a new class of benzoxazolinate-containing compounds in diverse bacteria.

Abstract

Benzoxazolinate is a rare bis-heterocyclic moiety that interacts with proteins and DNA and confers extraordinary bioactivities on natural products, such as C-1027. However, the biosynthetic gene responsible for the key cyclization step of benzoxazolinate remains unclear. Herein, we show a putative acyl AMP-ligase responsible for the last cyclization step. We used the enzyme as a probe for genome mining and discovered that the orphan benzobactin gene cluster in entomopathogenic bacteria prevails across Proteobacteria and Firmicutes. It turns out that Pseudomonas chlororaphis produces various benzobactins, whose biosynthesis is highlighted by a synergistic effect of two unclustered genes encoding enzymes on boosting benzobactin production; the formation of non-proteinogenic 2-hydroxymethylserine by a serine hydroxymethyltransferase; and the types I and II NRPS architecture for structural diversity. Our findings reveal the biosynthetic potential of a widespread benzobactin gene cluster.

Medium chain alcohol dehydrogenases usually perform a reversible 1,2-reduction of aldehydes to form the corresponding alcohols. Here we show how the active site of these dehydrogenases can be modified to perform 1,2-reduction of iminium moieties, as well 1,4-reduction of α,β-unsaturated iminium groups and 1,4-reduction of α,β-unsaturated aldehydes.

Abstract

Medium-chain alcohol dehydrogenases (ADHs) comprise a highly conserved enzyme family that catalyse the reversible reduction of aldehydes. However, recent discoveries in plant natural product biosynthesis suggest that the catalytic repertoire of ADHs has been expanded. Here we report the crystal structure of dihydroprecondylocarpine acetate synthase (DPAS), an ADH that catalyses the non-canonical 1,4-reduction of an α,β-unsaturated iminium moiety. Comparison with structures of plant-derived ADHs suggest the 1,4-iminium reduction does not require a proton relay or the presence of a catalytic zinc ion in contrast to canonical 1,2-aldehyde reducing ADHs that require the catalytic zinc and a proton relay. Furthermore, ADHs that catalysed 1,2-iminium reduction required the presence of the catalytic zinc and the loss of the proton relay. This suggests how the ADH active site can be modified to perform atypical carbonyl reductions, providing insight into how chemical reactions are diversified in plant metabolism.

Dinitroalkanes are powerful synthetic building blocks because of the versatility of the 1,3-dinitro motif. Here, we show that dinitroalkanes can be synthesized from alcohol substrates using a combination of biocatalysis and organocatalysis in a single-flask process. Alcohol oxidase oxidizes alcohol substrates to an intermediate aldehyde, which is sequentially converted to a nitroalcohol, then a nitroalkene, and finally, to a 1,3-dinitroalkane with a combination of phosphate buffer and lysine catalysis. Simultaneous addition of all reagents gives a maximal yield of 52%, whereas staggering the introduction of the amino acid catalyst and nitromethane substrate boosts the yield to 71% with near-quantitative conversion. Taken together, this work shows that biocatalysed oxidation can be coupled to multi-step catalytic cascades to expand the types of products available from bioprocesses.

Biocatalytic cascades can be used for the efficient, asymmetric synthesis of bioactive compounds. A substrate multiplexed screening (SUMS) based directed evolution approach was implemented to improve the substrate scope overlap between a transaldolase and a decarboxylase. The matched substrate scope of the enzymes was leveraged to produce a variety of enantiomerically pure 1,2-amino alcohols in a one-pot cascade from aldehydes or styrene oxides.

Abstract

Biocatalytic cascades are uniquely powerful for the efficient, asymmetric synthesis of bioactive compounds. However, high substrate specificity can hinder the scope of biocatalytic cascades because the constituent enzymes may have non-complementary activity. In this study, we implemented a substrate multiplexed screening (SUMS) based directed evolution approach to improve the substrate scope overlap between a transaldolase (ObiH) and a decarboxylase for the production of chiral 1,2-amino alcohols. To generate a promiscuous cascade, we engineered a tryptophan decarboxylase to act efficiently on β-OH amino acids while avoiding activity on l-threonine, which is needed for ObiH activity. We leveraged this exquisite selectivity with matched substrate scope to produce a variety of enantiopure 1,2-amino alcohols in a one-pot cascade from aldehydes or styrene oxides. This demonstration shows how SUMS can be used to guide the development of promiscuous, C−C bond forming cascades.

An artificial metalloenzyme was screened in vivo using a high-throughput microfluidics-based assay. Single E. coli cells expressing streptavidin were encapsulated in double emulsion droplets together with a fluorogenic substrate and a cofactor catalyzing the reaction to yield a fluorescent product. Using this method, a 400-member library was screened, and the same hits found previously in a 96-well plate assay were identified.

Abstract

The potential for ultrahigh-throughput compartmentalization renders droplet microfluidics an attractive tool for the directed evolution of enzymes. Importantly, it ensures maintenance of the phenotype-genotype linkage, enabling reliable identification of improved mutants. Herein, we report an approach for ultrahigh-throughput screening of an artificial metalloenzyme in double emulsion droplets (DEs) using commercially available fluorescence-activated cell sorters (FACS). This protocol was validated by screening a 400 double-mutant streptavidin library for ruthenium-catalyzed deallylation of an alloc-protected aminocoumarin. The most active variants, identified by next-generation sequencing, were in good agreement with hits obtained using a 96-well plate procedure. These findings pave the way for the systematic implementation of FACS for the directed evolution of (artificial) enzymes and will significantly expand the accessibility of ultrahigh-throughput DE screening protocols.

In this study, we describe the creation of an artificial protein cage housing a dual metal-tagged guest protein that catalyzes a linear, two-step sequential cascade reaction. The guest protein consists of a fusion protein of HaloTag and monomeric rhizavidin. Inside the protein capsid, we establish a ruthenium-catalyzed alloc-deprotection followed by a gold-catalyzed ring-closing hydroamination reaction that leads to indoles and phenanthridines with an overall yield of up to 67% in aqueous solutions. Furthermore, we show that the encapsulation stabilizes the metal catalysts against deactivation by air, proteins and cell lysate.

Nature, Published online: 21 September 2022; doi:10.1038/s41586-022-05342-4

Enantioselective [2+2]-cycloadditions with triplet photoenzymes

Nature, Published online: 21 September 2022; doi:10.1038/s41586-022-05335-3

A Designed Photoenzyme for Enantioselective [2+2]-Cycloadditions

Nature Catalysis, Published online: 20 September 2022; doi:10.1038/s41929-022-00836-w

Metabolic engineering of microbes constitutes a promising strategy to make the industrial production of chemicals more sustainable. This Review discusses recent advances of targeted high-throughput genome editing to construct next-generation cell factories for bioproduction.

Nature Chemical Biology, Published online: 15 September 2022; doi:10.1038/s41589-022-01127-y

Polyketides are assembled by modular polyketide synthases and undergo chemical tailoring reactions. A dehydratase domain variant catalyzes two sequential elimination reactions from thioester intermediates to produce conjugated diene modifications.

Biocatalytic Synthesis: This study focuses on the reversal of cofactor preference for short-chain dehydrogenases/reductases and the excellent NADH-dependent recombinant LfSDR1-V186A/G92V/E141L/G38D/T15A variant (Mu2) was obtained. Meanwhile, a co-expressed E. coli whole-cell biocatalyst containing the genes of Mu2 and PpFDH was developed to reduce ketone 1. Finally, ketone 1 was almost completely converted into the product (R)-2 with a space-time yield of 115.7 g⋅L⁻¹⋅d⁻¹ and a 98.8 % ee value.

Abstract

Switching cofactor preference of oxidoreductases from NADPH to NADH by rational engineering, replacing the expensive cofactor NADP⁺ with the cheap cofactor NAD⁺, is a focus of attention in the industrial application of oxidoreductases. This study focuses on the reversal of cofactor preference for short-chain dehydrogenases/reductases (SDRs). Combined with bioinformatics analyses and in silico analyses, a small and smart mutant library (Mu1-Mu3) of LfSDR1 was rationally designed and constructed. Thus, the excellent NADH-dependent recombinant LfSDR1-V186A/G92V/E141L/G38D/T15A variant (Mu2) was obtained. Meanwhile, novel enzymatic processes for synthesis of the key intermediates [(R)-2 and (S)-4] of telotristat ethyl and crizotinib were successfully created, which mainly relied on Mu2 coupled with an FDH-catalyzed cofactor regeneration system. A co-expressed E. coli whole-cell biocatalyst containing the genes of Mu2 and PpFDH was developed to reduce ketones 1 and 3. Finally, ketone 1 was almost completely converted into the product (R)-2 with a space-time yield of 115.7 g⋅L⁻¹⋅d⁻¹ and a 98.8 % ee value.

Mechanisms: A comprehensive review of the catalytic mechanisms of imine reductase (IRED)-catalyzed imine reduction (IR), reductive amination (RA), recently reported conjugate reduction (CR), and stepwise CR-RA. Mechanistic insights from protein engineering were also included.

Abstract

The synthesis of chiral amines is significant in the pharmaceutical industry. Imine reductase (IRED), a promising biocatalyst that was previously known to only catalyze asymmetric imine reduction (IR), was revealed to achieve direct asymmetric reductive amination (RA) of ketones with excess amines, producing secondary and tertiary amines. Moreover, conjugate reduction (CR) and RA activity by a single IRED has been reported for the preparation of valuable amine diastereomers. IREDs catalyzing these different reactions share the same standard quaternary structure, but possess varying active sites, indicating correlations and differences between their catalytic mechanisms. In this review, we trace the catalytic mechanisms reported for IRED-catalyzed IR, RA, and CR-RA. Insights obtained from structural and protein engineering in understanding the IRED-catalyzed asymmetric synthesis are also included. This review will help readers acquire comprehensive insights into the catalytic mechanism of IREDs and ultimately inspire the engineering of IREDs for industrial applications.

Nature Communications, Published online: 14 September 2022; doi:10.1038/s41467-022-33131-0

The biosynthesis of xanthones has not been well documented. Here, the authors report that monooxygenase FlsO1 catalyzes three successive oxidations – hydroxylation, epoxidation and Baeyer–Villiger oxidation—to form the xanthone scaffold in actinomycetes.

Selective functionalization of aliphatic C–H bonds, ubiquitous in molecular structures, could allow ready access to diverse chemical products. While enzymatic oxygenation of C–H bonds is well established, the analogous enzymatic nitrogen functionalization is still unknown; nature is reliant on pre-oxidized compounds for nitrogen incorporation. Likewise, synthetic methods for selective nitrogen derivatization of unbiased C–H bonds remain elusive. In this work, new-to-nature heme-containing nitrene transferases were used as starting points for the directed evolution of enzymes to selectively aminate and amidate unactivated C(sp3)–H sites. The desymmetrization of methyl- and ethylcyclohexane with divergent site selectivity is offered as demonstration. The evolved enzymes in these lineages are highly promiscuous and show activity towards a wide array of substrates, providing a foundation for further evolution of nitrene transferase function. Computational studies and kinetic isotope effects (KIEs) are consistent with a stepwise radical pathway involving an irreversible, enantiodetermining hydrogen atom transfer (HAT), followed by a lower-barrier diastereoselectivity determining radical rebound step. In-enzyme molecular dynamics (MD) simulations reveal a predominantly hydrophobic pocket with favorable dispersion interactions with the substrate. By offering a direct path from saturated precursors, these enzymes present a new biochemical logic for accessing nitrogen-containing compounds.

Nitrogenase catalyzes the multi-electron reduction of dinitrogen to ammonia. Electron transfer in the catalytic protein (MoFeP) proceeds through a unique [8Fe-7S] cluster (P-cluster) to the active site (FeMoco). In the reduced, all-ferrous (PN) state, the P-cluster is coordinated by six cysteine residues. Upon two-electron oxi-dation to the P2+ state, the P-cluster undergoes conformational changes in which a highly conserved oxygen-based residue (a Ser or a Tyr) and a backbone amide additionally ligate the cluster. Previous studies of Azotobacter vinelandii (Av) MoFeP revealed that when the oxygen-based residue, βSer188, was mutated to a non-ligating residue, Ala, the P-cluster became redox-labile and reversibly lost two of its eight Fe centers. Surprisingly, the Av strain with a MoFeP variant that lacked the serine ligand (Av βSer188Ala MoFeP) could still grow and fix nitrogen as quickly as wild-type Av MoFeP, calling into question the necessity of this conserved ligand for nitrogenase function. Based on these observations, we hypothesized that βSer188 plays a role in protecting the P-cluster under non-ideal conditions. Here, we investigated the protective role of βSer188 both in vivo and in vitro by characterizing the ability of Av βSer188Ala cells to grow under subop-timal conditions (high oxidative stress or Fe limitation) and by characterizing the ability of Av βSer188Ala MoFeP to be mismetallated in vitro. Our results demonstrate that βSer188 (1) increases Av cell survival upon exposure to oxidative stress in the form of hydrogen peroxide, (2) is necessary for efficient Av diazotrophic growth under Fe-limiting conditions, and (3) protects the P-cluster from metal exchange in vitro. Taken together, our findings suggest a structural adaptation of nitrogenase to protect the P-cluster via Ser ligation, which is a previously unidentified functional role of the Ser residue in redox proteins and adds to the ex-panding functional roles of non-Cys ligands to FeS clusters.

Bioremediation. The discovery of MG8, an efficient polyethylene terephthalate (PET) hydrolase enzyme from the human saliva metagenome, is reported by Worawan Bhanthumnavin, Chayasith Uttamapinant et al. in their Research Article (e202203061). Aside from its plastic degradation capability, MG8 was further engineered via genetic code expansion into a covalent binder of PET plastic and can be used to attach protein payloads to PET and other polyesters.

Two analogues of geranylgeranyl diphosphate with shifted double bond were enzymatically converted with twelve diterpene synthases. The double bond shift causes a change of reactivity that results in the formation of many diterpenes with novel skeletons. A total number of 28 new diterpenes is reported and their mechanism of formation is discussed.

Abstract

Two analogues of the diterpene precursor geranylgeranyl diphosphate with shifted double bonds, named iso-GGPP I and iso-GGPP II, were enzymatically converted with twelve diterpene synthases from bacteria, fungi and protists. The changed reactivity in the substrate analogues resulted in the formation of 28 new diterpenes, many of which exhibit novel skeletons.

Nature Chemistry, Published online: 05 September 2022; doi:10.1038/s41557-022-01021-z

Oligonucleotide catalysts such as ribozymes and DNAzymes can cleave RNA efficiently and specifically but are typically dependent on high concentrations of divalent cations, limiting their biological applications. A modular XNAzyme catalyst composed of 2′-deoxy-2′-fluoro-β-d-arabino nucleic acid (FANA) has now been developed that can cleave long (>5 kb), highly structured mRNAs under physiological conditions and enables allele-specific catalytic RNA knockdown inside cells.