Substrate Trapping in Polyketide Synthase Thioesterase Domains: Structural Basis for Macrolactone Formation.

Emerging antibiotic resistance requires continual improvement in the arsenal of antimicrobial drugs, especially the critical macrolide antibiotics. Formation of the macrolactone scaffold of these polyketide natural products is catalyzed by a modular polyketide synthase (PKS) thioesterase (TE). The TE accepts a linear polyketide substrate from the termina PKS acyl carrier protein to generate an acyl-enzyme adduct that is resolved by attack of a substrate hydroxyl group to form the macrolactone. Our limited mechanistic understanding of TE selectivity for a substrate nucleophile and/or water has hampered development of TEs as biocatalysts that accommodate a variety of natural and non-natural substrates. To understand how TEs direct the substrate nucleophile for macrolactone formation, acyl-enzyme intermediates were trapped as stable amides by substituting the natural serine OH with an amino group. Incorporation of the unnatural amino acid, 1,3-diaminopropionic acid (DAP), was tested with five PKS TEs. DAP-modified TEs (TE DAP ) from the pikromycin and erythromycin pathways were purified and tested with six full-length polyketide intermediates from three pathways. The erythromycin TE had permissive substrate selectivity, whereas the pikromycin TE was selective for its native hexaketide and heptaketide substrates. In a crystal structure of a native substrate trapped in pikromycin TE DAP , the linear heptaketide was curled in the active site with the nucleophilic hydroxyl group positioned 4 Å from the amide-enzyme linkage. The curled heptaketide displayed remarkable shape complementarity with the TE acyl cavity. The strikingly different shapes of acyl cavities in TEs of known structure, including those reported here for juvenimicin, tylosin and fluvirucin biosynthesis, provide new insights to facilitate TE engineering and optimization.

Discovering type I cis-AT polyketides through computational mass spectrometry and genome mining with Seq2PKS.

Type 1 polyketides are a major class of natural products used as antiviral, antibiotic, antifungal, antiparasitic, immunosuppressive, and antitumor drugs. Analysis of public microbial genomes leads to the discovery of over sixty thousand type 1 polyketide gene clusters. However, the molecular products of only about a hundred of these clusters are characterized, leaving most metabolites unknown. Characterizing polyketides relies on bioactivity-guided purification, which is expensive and time-consuming. To address this, we present Seq2PKS, a machine learning algorithm that predicts chemical structures derived from Type 1 polyketide synthases. Seq2PKS predicts numerous putative structures for each gene cluster to enhance accuracy. The correct structure is identified using a variable mass spectral database search. Benchmarks show that Seq2PKS outperforms existing methods. Applying Seq2PKS to Actinobacteria datasets, we discover biosynthetic gene clusters for monazomycin, oasomycin A, and 2-aminobenzamide-actiphenol.

Metabologenomics reveals strain-level genetic and chemical diversity of Microcystis secondary metabolism.

Microcystis spp. are renowned for producing the hepatotoxin microcystin in freshwater cyanobacterial harmful algal blooms around the world, threatening drinking water supplies and public and environmental health. However, Microcystis genomes also harbor numerous biosynthetic gene clusters (BGCs) encoding the biosynthesis of other secondary metabolites, including many with toxic properties. Most of these BGCs are uncharacterized and currently lack links to biosynthesis products. However, recent field studies show that many of these BGCs are abundant and transcriptionally active in natural communities, suggesting potentially important yet unknown roles in bloom ecology and water quality. Here, we analyzed 21 xenic Microcystis cultures isolated from western Lake Erie to investigate the diversity of the biosynthetic potential of this genus. Through metabologenomic and in silico approaches, we show that these Microcystis strains contain variable BGCs, previously observed in natural populations, and encode distinct metabolomes across cultures. Additionally, we find that the majority of metabolites and gene clusters are uncharacterized, highlighting our limited understanding of the chemical repertoire of Microcystis spp. Due to the complex metabolomes observed in culture, which contain a wealth of diverse congeners as well as unknown metabolites, these results underscore the need to deeply explore and identify secondary metabolites produced by Microcystis beyond microcystins to assess their impacts on human and environmental health.IMPORTANCEThe genus Microcystis forms dense cyanobacterial harmful algal blooms (cyanoHABs) and can produce the toxin microcystin, which has been responsible for drinking water crises around the world. While microcystins are of great concern, Microcystis also produces an abundance of other secondary metabolites that may be of interest due to their potential for toxicity, ecological importance, or pharmaceutical applications. In this study, we combine genomic and metabolomic approaches to study the genes responsible for the biosynthesis of secondary metabolites as well as the chemical diversity of produced metabolites in Microcystis strains from the Western Lake Erie Culture Collection. This unique collection comprises Microcystis strains that were directly isolated from western Lake Erie, which experiences substantial cyanoHAB events annually and has had negative impacts on drinking water, tourism, and industry.

N-to-S Acyl Transfer as an Enabling Strategy in Asymmetric and Chemoenzymatic Synthesis.

The observation of thioester-mediated acyl transfer processes in nature has inspired the development of novel protein synthesis and functionalization methodologies. The chemoselective transfer of an acyl group from S-to-N is the basis of several powerful ligation strategies. In this work, we sought to apply the reverse process, the transfer of an acyl group from N-to-S, as a method to convert stable chiral amides into more reactive thioesters. To this end, we developed a novel cysteine-derived oxazolidinone that serves as both a chiral imide auxiliary and an acyl transfer agent. This auxiliary combines the desirable features of rigid chiral imides as templates for asymmetric transformations with the synthetic applicability of thioesters. We demonstrate that the auxiliary can be applied in a range of highly selective asymmetric transformations. Subsequent intramolecular N-to-S acyl transfer of the chiral product and in situ trapping of the resulting thioester provides access to diverse carboxylic acid derivatives under mild conditions. The oxazolidinone thioester products can also be isolated and used in Pd-mediated transformations to furnish highly valuable chiral scaffolds, such as noncanonical amino acids, cyclic ketones, tetrahydropyrones, and dihydroquinolinones. Finally, we demonstrate that the oxazolidinone thioesters can also serve as a surrogate for SNAC-thioesters, enabling their seamless use as non-native substrates in biocatalytic transformations.

Structure-Activity Relationships of Natural and Semisynthetic Plecomacrolides Suggest Distinct Pathways for HIV-1 Immune Evasion and Vacuolar ATPase-Dependent Lysosomal Acidification.

The human immunodeficiency virus (HIV)-encoded accessory protein Nef enhances pathogenicity by reducing major histocompatibility complex I (MHC-I) cell surface expression, protecting HIV-infected cells from immune recognition. Nef-dependent downmodulation of MHC-I can be reversed by subnanomolar concentrations of concanamycin A (1), a well-known inhibitor of vacuolar ATPase, at concentrations below those that interfere with lysosomal acidification or degradation. We conducted a structure-activity relationship study that assessed 76 compounds for Nef inhibition, 24 and 72 h viability, and lysosomal neutralization in Nef-expressing primary T cells. This analysis demonstrated that the most potent compounds were natural concanamycins and their derivatives. Comparison against a set of new, semisynthetic concanamycins revealed that substituents at C-8 and acylation of C-9 significantly affected Nef potency, target cell viability, and lysosomal neutralization. These findings provide important progress toward understanding the mechanism of action of these compounds and the identification of an advanced lead anti-HIV Nef inhibitory compound.

Structure of a modular polyketide synthase reducing region.

The chemical scaffolds of numerous therapeutics are polyketide natural products, many formed by bacterial modular polyketide synthases (PKS). The large and flexible dimeric PKS modules have distinct extension and reducing regions. Structures are known for all individual enzyme domains and several extension regions. Here, we report the structure of the full reducing region from a modular PKS, the ketoreductase (KR), dehydratase (DH), and enoylreductase (ER) domains of module 5 of the juvenimicin PKS. The modular PKS-reducing region has a different architecture than the homologous fatty acid synthase (FAS) and iterative PKS systems in its arrangement of domains and dimer interface. The structure reveals a critical role for linker peptides in the domain interfaces, leading to discovery of key differences in KR domains dependent on module composition. Finally, our studies provide insight into the mechanism underlying modular PKS intermediate shuttling by carrier protein (ACP) domains.

Expression of Microcystis Biosynthetic Gene Clusters in Natural Populations Suggests Temporally Dynamic Synthesis of Novel and Known Secondary Metabolites in Western Lake Erie.

Microcystis spp. produce diverse secondary metabolites within freshwater cyanobacterial harmful algal blooms (cyanoHABs) around the world. In addition to the biosynthetic gene clusters (BGCs) encoding known compounds, Microcystis genomes harbor numerous BGCs of unknown function, indicating a poorly understood chemical repertoire. While recent studies show that Microcystis produces several metabolites in the lab and field, little work has focused on analyzing the abundance and expression of its broader suite of BGCs during cyanoHAB events. Here, we use metagenomic and metatranscriptomic approaches to track the relative abundance of Microcystis BGCs and their transcripts throughout the 2014 western Lake Erie cyanoHAB. The results indicate the presence of several transcriptionally active BGCs that are predicted to synthesize both known and novel secondary metabolites. The abundance and expression of these BGCs shifted throughout the bloom, with transcript abundance levels correlating with temperature, nitrate, and phosphorus concentrations and the abundance of co-occurring predatory and competitive eukaryotic microorganisms, suggesting the importance of both abiotic and biotic controls in regulating expression. This work highlights the need for understanding the chemical ecology and potential risks to human and environmental health posed by secondary metabolites that are produced but often unmonitored. It also indicates the prospects for identifying pharmaceutical-like molecules from cyanoHAB-derived BGCs. IMPORTANCE Microcystis spp. dominate cyanobacterial harmful algal blooms (cyanoHABs) worldwide and pose significant threats to water quality through the production of secondary metabolites, many of which are toxic. While the toxicity and biochemistry of microcystins and several other compounds have been studied, the broader suite of secondary metabolites produced by Microcystis remains poorly understood, leaving gaps in our understanding of their impacts on human and ecosystem health. We used community DNA and RNA sequences to track the diversity of genes encoding synthesis of secondary metabolites in natural Microcystis populations and assess patterns of transcription in western Lake Erie cyanoHABs. Our results reveal the presence of both known gene clusters that encode toxic secondary metabolites as well as novel ones that may encode cryptic compounds. This research highlights the need for targeted studies of the secondary metabolite diversity in western Lake Erie, a vital freshwater source to the United States and Canada.

An NmrA-like enzyme-catalysed redox-mediated Diels-Alder cycloaddition with anti-selectivity.

The Diels-Alder cycloaddition is one of the most powerful approaches in organic synthesis and is often used in the synthesis of important pharmaceuticals. Yet, strictly controlling the stereoselectivity of the Diels-Alder reactions is challenging, and great efforts are needed to construct complex molecules with desired chirality via organocatalysis or transition-metal strategies. Nature has evolved different types of enzymes to exquisitely control cyclization stereochemistry; however, most of the reported Diels-Alderases have been shown to only facilitate the energetically favourable diastereoselective cycloadditions. Here we report the discovery and characterization of CtdP, a member of a new class of bifunctional oxidoreductase/Diels-Alderase, which was previously annotated as an NmrA-like transcriptional regulator. We demonstrate that CtdP catalyses the inherently disfavoured cycloaddition to form the bicyclo[2.2.2]diazaoctane scaffold with a strict α-anti-selectivity. Guided by computational studies, we reveal a NADP+/NADPH-dependent redox mechanism for the CtdP-catalysed inverse electron demand Diels-Alder cycloaddition, which serves as the first example of a bifunctional Diels-Alderase that utilizes this mechanism.

Molecular Dynamics Simulations Guide Chimeragenesis and Engineered Control of Chemoselectivity in Diketopiperazine Dimerases.

In the biosynthesis of the tryptophan-linked dimeric diketopiperazines (DKPs), cytochromes P450 selectively couple DKP monomers to generate a variety of intricate and isomeric frameworks. To determine the molecular basis for selectivity of these biocatalysts we obtained a high-resolution crystal structure of selective Csp2 -N bond forming dimerase, AspB. Overlay of the AspB structure onto C-C and C-N bond forming homolog NzeB revealed no significant structural variance to explain their divergent chemoselectivities. Molecular dynamics (MD) simulations identified a region of NzeB with increased conformational flexibility relative to AspB, and interchange of this region along with a single active site mutation led to a variant that catalyzes exclusive C-N bond formation. MD simulations also suggest that intermolecular C-C or C-N bond formation results from a change in mechanism, supported experimentally through use of a substrate mimic.

Data Science-Driven Analysis of Substrate-Permissive Diketopiperazine Reverse Prenyltransferase NotF: Applications in Protein Engineering and Cascade Biocatalytic Synthesis of (-)-Eurotiumin A.

Prenyltransfer is an early-stage carbon-hydrogen bond (C-H) functionalization prevalent in the biosynthesis of a diverse array of biologically active bacterial, fungal, plant, and metazoan diketopiperazine (DKP) alkaloids. Toward the development of a unified strategy for biocatalytic construction of prenylated DKP indole alkaloids, we sought to identify and characterize a substrate-permissive C2 reverse prenyltransferase (PT). As the first tailoring event within the biosynthesis of cytotoxic notoamide metabolites, PT NotF catalyzes C2 reverse prenyltransfer of brevianamide F. Solving a crystal structure of NotF (in complex with native substrate and prenyl donor mimic dimethylallyl S-thiolodiphosphate (DMSPP)) revealed a large, solvent-exposed active site, intimating NotF may possess a significantly broad substrate scope. To assess the substrate selectivity of NotF, we synthesized a panel of 30 sterically and electronically differentiated tryptophanyl DKPs, the majority of which were selectively prenylated by NotF in synthetically useful conversions (2 to >99%). Quantitative representation of this substrate library and development of a descriptive statistical model provided insight into the molecular origins of NotF's substrate promiscuity. This approach enabled the identification of key substrate descriptors (electrophilicity, size, and flexibility) that govern the rate of NotF-catalyzed prenyltransfer, and the development of an "induced fit docking (IFD)-guided" engineering strategy for improved turnover of our largest substrates. We further demonstrated the utility of NotF in tandem with oxidative cyclization using flavin monooxygenase, BvnB. This one-pot, in vitro biocatalytic cascade enabled the first chemoenzymatic synthesis of the marine fungal natural product, (-)-eurotiumin A, in three steps and 60% overall yield.

Chemoenzymatic synthesis of fluorinated polyketides.

Modification of polyketides with fluorine offers a promising approach to develop new pharmaceuticals. While synthetic chemical methods for site-selective incorporation of fluorine in complex molecules have improved in recent years, approaches for the biosynthetic incorporation of fluorine in natural compounds are still rare. Here, we report a strategy to introduce fluorine into complex polyketides during biosynthesis. We exchanged the native acyltransferase domain of a polyketide synthase, which acts as the gatekeeper for the selection of extender units, with an evolutionarily related but substrate tolerant domain from metazoan type I fatty acid synthase. The resulting polyketide-synthase/fatty-acid-synthase hybrid can utilize fluoromalonyl coenzyme A and fluoromethylmalonyl coenzyme A for polyketide chain extension, introducing fluorine or fluoro-methyl units in polyketide scaffolds. We demonstrate the feasibility of our approach in the chemoenzymatic synthesis of fluorinated 12- and 14-membered macrolactones and fluorinated derivatives of the macrolide antibiotics YC-17 and methymycin.

Metagenomic and Metatranscriptomic Insights into Population Diversity of Microcystis Blooms: Spatial and Temporal Dynamics of mcy Genotypes, Including a Partial Operon That Can Be Abundant and Expressed.

Cyanobacterial harmful algal blooms (cyanoHABs) degrade freshwater ecosystems globally. Microcystis aeruginosa often dominates cyanoHABs and produces microcystin (MC), a class of hepatotoxins that poses threats to human and animal health. Microcystin toxicity is influenced by distinct structural elements across a diversity of related molecules encoded by variant mcy operons. However, the composition and distribution of mcy operon variants in natural blooms remain poorly understood. Here, we characterized the variant composition of mcy genes in western Lake Erie Microcystis blooms from 2014 and 2018. Sampling was conducted across several spatial and temporal scales, including different bloom phases within 2014, extensive spatial coverage on the same day (2018), and frequent, autonomous sampling over a 2-week period (2018). Mapping of metagenomic and metatranscriptomic sequences to reference sequences revealed three Microcystis mcy genotypes: complete (all genes present [mcyA-J]), partial (truncated mcyA, complete mcyBC, and missing mcyD-J), and absent (no mcy genes). We also detected two different variants of mcyB that may influence the production of microcystin congeners. The relative abundance of these genotypes was correlated with pH and nitrate concentrations. Metatranscriptomic analysis revealed that partial operons were, at times, the most abundant genotype and expressed in situ, suggesting the potential biosynthesis of truncated products. Quantification of genetic divergence between genotypes suggests that the observed strains are the result of preexisting heterogeneity rather than de novo mutation during the sampling period. Overall, our results show that natural Microcystis populations contain several cooccurring mcy genotypes that dynamically shift in abundance spatiotemporally via strain succession and likely influence the observed diversity of the produced congeners. IMPORTANCE Cyanobacteria are responsible for producing microcystins (MCs), a class of potent and structurally diverse toxins, in freshwater systems around the world. While microcystins have been studied for over 50 years, the diversity of their chemical forms and how this variation is encoded at the genetic level remain poorly understood, especially within natural populations of cyanobacterial harmful algal blooms (cyanoHABs). Here, we leverage community DNA and RNA sequences to track shifts in mcy genes responsible for producing microcystin, uncovering the relative abundance, expression, and variation of these genes. We studied this phenomenon in western Lake Erie, which suffers annually from cyanoHAB events, with impacts on drinking water, recreation, tourism, and commercial fishing.

Engineering sequence and selectivity of late-stage C-H oxidation in the MycG iterative cytochrome P450.

MycG is a multifunctional P450 monooxygenase that catalyzes sequential hydroxylation and epoxidation or a single epoxidation in mycinamicin biosynthesis. In the mycinamicin-producing strain Micromonospora griseorubida A11725, very low-level accumulation of mycinamicin V generated by the initial C-14 allylic hydroxylation of MycG is observed due to its subsequent epoxidation to generate mycinamicin II, the terminal metabolite in this pathway. Herein, we investigated whether MycG can be engineered for production of the mycinamicin II intermediate as the predominant metabolite. Thus, mycG was subject to random mutagenesis and screening was conducted in Escherichia coli whole-cell assays. This enabled efficient identification of amino acid residues involved in reaction profile alterations, which included MycG R111Q/V358L, W44R, and V135G/E355K with enhanced monohydroxylation to accumulate mycinamicin V. The MycG V135G/E355K mutant generated 40-fold higher levels of mycinamicin V compared to wild-type M. griseorubida A11725. In addition, the E355K mutation showed improved ability to catalyze sequential hydroxylation and epoxidation with minimal mono-epoxidation product mycinamicin I compared to the wild-type enzyme. These approaches demonstrate the ability to selectively coordinate the catalytic activity of multifunctional P450s and efficiently produce the desired compounds.

Biosynthetic Cyclization Catalysts for the Assembly of Peptide and Polyketide Natural Products.

Many biologically active natural products are synthesized by nonribosomal peptide synthetases (NRPSs), polyketide synthases (PKSs) and their hybrids. These megasynthetases contain modules possessing distinct catalytic domains that allow for substrate initiation, chain extension, processing and termination. At the end of a module, a terminal domain, usually a thioesterase (TE), is responsible for catalyzing the release of the NRPS or PKS as a linear or cyclized product. In this review, we address the general cyclization mechanism of the TE domain, including oligomerization and the fungal C-C bond forming Claisen-like cyclases (CLCs). Additionally, we include examples of cyclization catalysts acting within or at the end of a module. Furthermore, condensation-like (CT) domains, terminal reductase (R) domains, reductase-like domains that catalyze Dieckmann condensation (RD), thioesterase-like Dieckmann cyclases, trans-acting TEs from the penicillin binding protein (PBP) enzyme family, product template (PT) domains and others will also be reviewed. The studies summarized here highlight the remarkable diversity of NRPS and PKS cyclization catalysts for the production of biologically relevant, complex cyclic natural products and related compounds.

Structural diversification of hapalindole and fischerindole natural products via cascade biocatalysis.

Hapalindoles and related compounds (ambiguines, fischerindoles, welwitindolinones) are a diverse class of indole alkaloid natural products. They are typically isolated from the Stigonemataceae order of cyanobacteria and possess a broad scope of biological activities. Recently the biosynthetic pathway for assembly of these metabolites has been elucidated. In order to generate the core ring system, L-tryptophan is converted into the cis-indole isonitrile subunit before being prenylated with geranyl pyrophosphate at the C-3 position. A class of cyclases (Stig) catalyzes a three-step process including a Cope rearrangement, 6-exo-trig cyclization and electrophilic aromatic substitution to create a polycyclic core. Formation of the initial alkaloid is followed by diverse late-stage tailoring reactions mediated by additional biosynthetic enzymes to give rise to the wide array of structural variations observed in this compound class. Herein, we demonstrate the versatility and utility of the Fam prenyltransferase and Stig cyclases toward core structural diversification of this family of indole alkaloids. Through synthesis of cis-indole isonitrile subunit derivatives, and aided by protein engineering and computational analysis, we have employed cascade biocatalysis to generate a range of derivatives, and gained insights into the basis for substrate flexibility in this system.

Ribosome-binding and anti-microbial studies of the mycinamicins, 16-membered macrolide antibiotics from Micromonospora griseorubida.

Macrolides have been effective clinical antibiotics for over 70 years. They inhibit protein biosynthesis in bacterial pathogens by narrowing the nascent protein exit tunnel in the ribosome. The macrolide class of natural products consist of a macrolactone ring linked to one or more sugar molecules. Most of the macrolides used currently are semi-synthetic erythromycin derivatives, composed of a 14- or 15-membered macrolactone ring. Rapidly emerging resistance in bacterial pathogens is among the most urgent global health challenges, which render many antibiotics ineffective, including next-generation macrolides. To address this threat and advance a longer-term plan for developing new antibiotics, we demonstrate how 16-membered macrolides overcome erythromycin resistance in clinically isolated Staphylococcus aureus strains. By determining the structures of complexes of the large ribosomal subunit of Deinococcus radiodurans (D50S) with these 16-membered selected macrolides, and performing anti-microbial studies, we identified resistance mechanisms they may overcome. This new information provides important insights toward the rational design of therapeutics that are effective against drug resistant human pathogens.

Recent advances in hapalindole-type cyanobacterial alkaloids: biosynthesis, synthesis, and biological activity.

Covering: 1984 up to the end of 2020Hapalindoles, fischerindoles, ambiguines and welwitindolinones are all members of a class of indole alkaloid natural products that have been isolated from the Stigonematales order of cyanobacteria. These compounds possess a polycyclic ring system, unique functional groups and various stereo- and regiochemical isomers. Since their initial isolation in 1984, they have been explored as potential therapeutics due to their wide variety of biological activities. Although numerous groups have pursued total syntheses of these densely functionalized structures, hapalindole biosynthesis has only recently been unveiled. Several groups have uncovered a wide range of novel enzymes that catalyze formation and tailoring of the hapalindole-type metabolites. In this article, we provide an overview of these natural products, their biological activities, highlight general synthetic routes, and provide an extensive review on the surprising biosynthetic processes leading to these structurally diverse metabolites.

Computationally-guided exchange of substrate selectivity motifs in a modular polyketide synthase acyltransferase.

Polyketides, one of the largest classes of natural products, are often clinically relevant. The ability to engineer polyketide biosynthesis to produce analogs is critically important. Acyltransferases (ATs) of modular polyketide synthases (PKSs) catalyze the installation of malonyl-CoA extenders into polyketide scaffolds. ATs have been targeted extensively to site-selectively introduce various extenders into polyketides. Yet, a complete inventory of AT residues responsible for substrate selection has not been established, limiting the scope of AT engineering. Here, molecular dynamics simulations are used to prioritize ~50 mutations within the active site of EryAT6 from erythromycin biosynthesis, leading to identification of two previously unexplored structural motifs. Exchanging both motifs with those from ATs with alternative extender specificities provides chimeric PKS modules with expanded and inverted substrate specificity. Our enhanced understanding of AT substrate selectivity and application of this motif-swapping strategy are expected to advance our ability to engineer PKSs towards designer polyketides.

An overview of the cytochrome P450 enzymes that catalyze the same-site multistep oxidation reactions in biotechnologically relevant selected actinomycete strains.

Cytochrome P450 enzymes (P450s) are one of the major factors responsible for the diversity of metabolites produced through many biosynthetic and biodegradative processes in actinomycetes. P450s typically catalyze a single oxidative modification; however, several P450s have been identified with the unique ability to iteratively oxidize the same-site of the substrate. These P450s are capable of forming diverse compounds that affect biological processes, including alcohols, ketones, aldehydes, and carboxylic acids. Although further structural and functional studies are needed to elucidate the mechanisms that allow multistep oxidative modification, recent studies have revealed the enzymatic properties and reaction mechanisms of these P450s. This mini-review covers the current knowledge of P450s that catalyze the multistep oxidation reactions and contribute to the production of a wide variety of metabolites by selected actinomycete strains, along with insights into their application and utility. Understanding the characteristics of these remarkable enzymes will facilitate their utilization in biotechnological applications to create biologically active and other high-value compounds. KEY POINTS: • The multistep oxidation by P450s plays a key role in the diversity of metabolites. • The mechanisms that enable P450s to catalyze iterative oxidation remains unknown. • The effective use of P450s that iteratively oxidize the same-site is discussed.

Engineering P450 TamI as an Iterative Biocatalyst for Selective Late-Stage C-H Functionalization and Epoxidation of Tirandamycin Antibiotics.

Iterative P450 enzymes are powerful biocatalysts for selective late-stage C-H oxidation of complex natural product scaffolds. These enzymes represent useful tools for selectivity and cascade reactions, facilitating direct access to core structure diversification. Recently, we reported the structure of the multifunctional bacterial P450 TamI and elucidated the molecular basis of its substrate binding and strict reaction sequence at distinct carbon atoms of the substrate. Here, we report the design and characterization of a toolbox of TamI biocatalysts, generated by mutations at Leu101, Leu244, and/or Leu295, that alter the native selectivity, step sequence, and number of reactions catalyzed, including the engineering of a variant capable of catalyzing a four-step oxidative cascade without the assistance of the flavoprotein and oxidative partner TamL. The tuned enzymes override inherent substrate reactivity, enabling catalyst-controlled C-H functionalization and alkene epoxidation of the tetramic acid-containing natural product tirandamycin. Five bioactive tirandamycin derivatives (6-10) were generated through TamI-mediated enzymatic synthesis. Quantum mechanics calculations and MD simulations provide important insights into the basis of altered selectivity and underlying biocatalytic mechanisms for enhanced continuous oxidation of the iterative P450 TamI.