Unusual Enzymatic C-C Bond Formation and Cleavage Reactions during Natural Product Biosynthesis.

Natural products from plants and microorganisms provide a valuable reservoir of pharmaceutical compounds. C-C bond formation and cleavage are crucial events during natural product biosynthesis, playing pivotal roles in generating diverse and intricate chemical structures that are essential for biological functions. This review summarizes our recent findings regarding biosynthetic enzymes that catalyze unconventional C-C bond formation and cleavage reactions during natural product biosynthesis.

Multiple C-C Bond Cleavage Reactions Catalyzed by Tolyporphin Tetrapyrrole Biosynthetic Enzymes.

Tolyporphin A is an unusual tetrapyrrole secondary metabolite containing pendant deoxysugars and unsubstituted pyrrole ß sites. Herein, we describe the biosynthesis of the tolyporphin aglycon core. HemF1 catalyzes the oxidative decarboxylation of two propionate side chains of coproporphyrinogen III, an intermediate in heme biosynthesis. HemF2 then processes the two remaining propionate groups to generate a tetravinyl intermediate. All four vinyl groups from the macrocycle are truncated by TolI via repeated C-C bond cleavages to generate the unsubstituted pyrrole ß sites of tolyporphins. This study illustrates how the unprecedented C-C bond cleavage reactions branch from canonical heme biosynthesis to produce tolyporphins.

Structural and Mechanistic Insights into the C-C Bond-Forming Rearrangement Reaction Catalyzed by Heterodimeric Hinokiresinol Synthase.

Hinokiresinol synthase (HRS) from Asparagus officinalis consists of two subunits, α and ß, and catalyzes an unusual decarboxylative rearrangement reaction of 4-coumaryl 4-coumarate to generate (Z)-hinokiresinol with complete stereoselectivity. Herein, we describe the mechanism of rearrangement catalysis and the role played by the heterodimeric HRS, through structural and computational analyses. Our results suggest that the HRS reaction is unlikely to proceed via the previously hypothesized Claisen rearrangement mechanism. Instead, we propose that the 4-coumaryl 4-coumarate substrate is first cleaved into coumarate and an extended p-quinone methide, which then recombine to generate a new C-C bond. These processes are facilitated by proton transfers mediated by the basic residues (α-Lys164, α-Arg169, ß-Lys168, and ß-Arg173) in the cavity at the heterodimer interface. The active site residues, α-Asp165, ß-Asp169, ß-Trp17, ß-Met136, and ß-Ala171, play crucial roles in controlling the regioselectivity of the coupling between the fragmented intermediates as well as the stereoselectivity of the decarboxylation step, leading to the formation of the (Z)-hinokiresinol product.

Mechanistic Analysis of Stereodivergent Nitroalkane Cyclopropanation Catalyzed by Nonheme Iron Enzymes.

BelL and HrmJ are α-ketoglutarate-dependent nonheme iron enzymes that catalyze the oxidative cyclization of 6-nitronorleucine, resulting in the formation of two diastereomeric 3-(2-nitrocyclopropyl)alanine (Ncpa) products containing trans-cyclopropane rings with (1'R,2'R) and (1'S,2'S) configurations, respectively. Herein, we investigate the catalytic mechanism and stereodivergency of the cyclopropanases. The results suggest that the nitroalkane moiety of the substrate is first deprotonated to produce the nitronate form. Spectroscopic analyses and biochemical assays with substrates and analogues indicate that an iron(IV)-oxo species abstracts proS-H from C4 to initiate intramolecular C-C bond formation. A hydroxylation intermediate is unlikely to be involved in the cyclopropanation reaction. Additionally, a genome mining approach is employed to discover new homologues that perform the cyclopropanation of 6-nitronorleucine to generate cis-configured Ncpa products with (1'R,2'S) or (1'S,2'R) stereochemistries. Sequence and structure comparisons of these cyclopropanases enable us to determine the amino acid residues critical for controlling the stereoselectivity of cyclopropanation.

Structure-based engineering of α-ketoglutarate dependent oxygenases in fungal meroterpenoid biosynthesis.

Non-heme iron- and α-ketoglutarate-dependent oxygenases (αKG OXs) are key enzymes that play a major role in diversifying the structure of fungal meroterpenoids. They activate a specific C-H bond of the substrate to first generate radical species, which is usually followed by oxygen rebound to produce cannonical hydroxylated products. However, in some cases remarkable chemistry induces dramatic structural changes in the molecular scaffolds, depending on the stereoelectronic characters of the substrate/intermediates and the resulting conformational changes/movements of the active site of the enzyme. Their molecular bases have been extensively investigated by crystallographic structural analyses and structure-based mutagenesis, which revealed intimate structural details of the enzyme reactions. This information facilitates the manipulation of the enzyme reactions to create unnatural, novel molecules for drug discovery. This review summarizes recent progress in the structure-based engineering of αKG OX enzymes, involved in the biosynthesis of polyketide-derived fungal meroterpenoids. The literature published from 2016 through February 2022 is reviewed.

Diverse enzymatic chemistry for propionate side chain cleavages in tetrapyrrole biosynthesis.

Tetrapyrroles represent a unique class of natural products that possess diverse chemical architectures and exhibit a broad range of biological functions. Accordingly, they attract keen attention from the natural product community. Many metal-chelating tetrapyrroles serve as enzyme cofactors essential for life, while certain organisms produce metal-free porphyrin metabolites with biological activities potentially beneficial for the producing organisms and for human use. The unique properties of tetrapyrrole natural products derive from their extensively modified and highly conjugated macrocyclic core structures. Most of these various tetrapyrrole natural products biosynthetically originate from a branching point precursor, uroporphyrinogen III, which contains propionate and acetate side chains on its macrocycle. Over the past few decades, many modification enzymes with unique catalytic activities, and the diverse enzymatic chemistries employed to cleave the propionate side chains from the macrocycles, have been identified. In this review, we highlight the tetrapyrrole biosynthetic enzymes required for the propionate side chain removal processes and discuss their various chemical mechanisms. ONE-SENTENCE SUMMARY: This mini-review describes various enzymes involved in the propionate side chain cleavages during the biosynthesis of tetrapyrrole cofactors and secondary metabolites.

Studies of lincosamide formation complete the biosynthetic pathway for lincomycin A.

The structure of lincomycin A consists of the unusual eight-carbon thiosugar core methyllincosamide (MTL) decorated with a pendent N-methylprolinyl moiety. Previous studies on MTL biosynthesis have suggested GDP-ᴅ-erythro-α-ᴅ-gluco-octose and GDP-ᴅ-α-ᴅ-lincosamide as key intermediates in the pathway. However, the enzyme-catalyzed reactions resulting in the conversion of GDP-ᴅ-erythro-α-ᴅ-gluco-octose to GDP-ᴅ-α-ᴅ-lincosamide have not yet been elucidated. Herein, a biosynthetic subpathway involving the activities of four enzymes-LmbM, LmbL, CcbZ, and CcbS (the LmbZ and LmbS equivalents in the closely related celesticetin pathway)-is reported. These enzymes catalyze the previously unknown biosynthetic steps including 6-epimerization, 6,8-dehydration, 4-epimerization, and 6-transamination that convert GDP-ᴅ-erythro-α-ᴅ-gluco-octose to GDP-ᴅ-α-ᴅ-lincosamide. Identification of these reactions completes the description of the entire lincomycin biosynthetic pathway. This work is significant since it not only resolves the missing link in octose core assembly of a thiosugar-containing natural product but also showcases the sophistication in catalytic logic of enzymes involved in carbohydrate transformations.

Photoinduced Reductive Dehalogenation of Phenacyl Bromides with Pyridoxal 5'-Phosphate.

We describe the photoinduced reductive debromination of phenacyl bromides using pyridoxal 5'-phosphate (PLP). The reaction requires irradiation with cyan or blue light in an anaerobic atmosphere. Mechanistic analysis reveals the formation of the phenacyl radical as an intermediate in the reaction, implying a single electron transfer to phenacyl bromides from a PLP-derived species resulting from excitation by illumination.

Stereoselectivity and Substrate Specificity of the Fe(II)/α-Ketoglutarate-Dependent Oxygenase TqaL.

Non-heme iron enzymes are versatile catalysts in the biosynthesis of medicinal natural products and have attracted increasing attention as practical catalytic tools in chemical synthesis due to their ability to perform chemically challenging transformations. The Fe(II)/α-ketoglutarate-dependent oxygenase TqaL catalyzes unusual aziridine formation from l-Val via cleavage of the unactivated Cß-H bond. However, the mechanistic details as well as the synthetic potential of TqaL-catalyzed ring closure remain unclear. Herein, we show that the TqaL-catalyzed aziridination of l-Val proceeds with an atypical, mixed stereochemical course involving both the retention and inversion of the C3(Cß) stereocenter. It is also demonstrated that TqaL accepts l-Ile and l-allo-Ile to generate the same diastereomeric pairs of aziridine products via an enzyme-controlled, stereoconvergent process. Our mutagenesis studies reveal that the reaction type (aziridination versus hydroxylation) and the stereochemical outcome are regulated by Ile343 and Phe345. Proper substitutions of Ile343 or Phe345 also make TqaL highly active toward the oxidation of α-amino acid substrates. This work provides mechanistic insights into the stereoselectivity and substrate specificity of the TqaL reactions.

Stereodivergent Nitrocyclopropane Formation during Biosynthesis of Belactosins and Hormaomycins.

Belactosins and hormaomycins are peptide natural products containing 3-(2-aminocyclopropyl)alanine and 3-(2-nitrocyclopropyl)alanine residues, respectively, with opposite stereoconfigurations of the cyclopropane ring. Herein we demonstrate that the heme oxygenase-like enzymes BelK and HrmI catalyze the N-oxygenation of l-lysine to generate 6-nitronorleucine. The nonheme iron enzymes BelL and HrmJ then cyclize the nitroalkane moiety to the nitrocyclopropane ring with the desired stereochemistry found in the corresponding natural products. We also show that both cyclopropanases remove the 4-proS-H of 6-nitronorleucine during the cyclization, establishing the inversion and retention of the configuration at C4 during the BelL and HrmJ reactions, respectively. This study reveals the unique strategy for stereocontrolled cyclopropane synthesis in nature.

Identification of the Enzymes Mediating the Maturation of the Seryl-tRNA Synthetase Inhibitor SB-217452 during the Biosynthesis of Albomycins.

Albomycin δ2 is a sulfur-containing sideromycin natural product that shows potent antibacterial activity against clinically important pathogens. The l-serine-thioheptose dipeptide partial structure, known as SB-217452, has been found to be the active seryl-tRNA synthetase inhibitor component of albomycin δ2 . Herein, it is demonstrated that AbmF catalyzes condensation between the 6'-amino-4'-thionucleoside with the d-ribo configuration and seryl-adenylate supplied by the serine adenylation activity of AbmK. Formation of the dipeptide is followed by C3'-epimerization to produce SB-217452 with the d-xylo configuration, which is catalyzed by the radical S-adenosyl-l-methionine enzyme AbmJ. Gene deletion suggests that AbmC is involved in peptide assembly linking SB-217452 with the siderophore moiety. This study establishes how the albomycin biosynthetic machinery generates its antimicrobial component SB-217452.

Biosynthetic Origin of the Atypical Stereochemistry in the Thioheptose Core of Albomycin Nucleoside Antibiotics.

Albomycins are peptidyl thionucleoside natural products that display antimicrobial activity against clinically important pathogens. Their structures are characterized by a thioheptose with atypical stereochemistry including a d-xylofuranose ring modified with a d-amino acid moiety. Herein it is demonstrated that AbmH is a pyridoxal 5'-phosphate (PLP)-dependent transaldolase that catalyzes a threo-selective aldol-type reaction to generate the thioheptose core with a d-ribofuranose ring and an l-amino acid moiety. The conversion of l-to d-amino acid configuration is catalyzed by the PLP-dependent epimerase AbmD. The d- ribo to d- xylo conversion of the thiofuranose ring appears according to gene deletion experiments to be mediated by AbmJ, which is annotated as a radical S-adenosyl-l-methionine (SAM) enzyme. These studies establish several key steps in the assembly of the thioheptose core during the biosynthesis of albomycins.

Changes in Regioselectivity of H Atom Abstraction during the Hydroxylation and Cyclization Reactions Catalyzed by Hyoscyamine 6ß-Hydroxylase.

Hyoscyamine 6ß-hydroxylase (H6H) is an αKG-dependent nonheme iron oxidase that catalyzes the oxidation of hyoscyamine to scopolamine via two separate reactions: hydroxylation followed by oxidative cyclization. Both of these reactions are expected to involve H atom abstraction from each of two adjacent carbon centers (C6 vs C7) in the substrate. During hydroxylation, there is a roughly 85:1 preference for H atom abstraction from C6 versus C7; however, this inverts to a 1:16 preference during cyclization. Furthermore, 18O incorporation experiments in the presence of deuterated substrate are consistent with the catalytic iron(IV)-oxo complex being able to support the coordination of an additional ligand during hydroxylation. These observations suggest that subtle differences in the substrate binding configuration can have significant consequences for the catalytic cycle of H6H.

Substrate Conformation Correlates with the Outcome of Hyoscyamine 6ß-Hydroxylase Catalyzed Oxidation Reactions.

Hyoscyamine 6ß-hydroxylase (H6H) is an α-ketoglutarate dependent mononuclear nonheme iron enzyme that catalyzes C6-hydroxylation of hyoscyamine and oxidative cyclization of the resulting product to give the oxirane natural product scopolamine. Herein, the chemistry of H6H is investigated using hyoscyamine derivatives with modifications at the C6 or C7 position as well as substrate analogues possessing a 9-azabicyclo[3.3.1]nonane core. Results indicate that hydroxyl rebound is unlikely to take place during the cyclization reaction and that the hydroxylase versus oxidative cyclase activity of H6H is correlated with the presence of an exo-hydroxy group having syn-periplanar geometry with respect to the adjacent H atom to be abstracted.

A Fluorinated Cobalt(III) Porphyrin Complex for Hydroalkoxylation of Alkynes.

A fluorinated cobalt(III) porphyrin complex [Co(TPFPP)NTf2·2C2H5OH, where TPFPP=5,10,15,20-tetrakis(pentafluorophenyl)porphyrin, Tf=CF3SO2] promotes hydroalkoxylation of alkynes to give acetals in good to excellent yields. The acetals can be directly functionalized with nucleophiles in a one-pot procedure.

Characterization of Enzymes Catalyzing Transformations of Cysteine S-Conjugated Intermediates in the Lincosamide Biosynthetic Pathway.

Lincosamides such as lincomycin A, celesticetin, and Bu-2545, constitute an important group of antibiotics. These natural products are characterized by a thiooctose linked to a l-proline residue, but they differ with regards to modifications of the thioacetal moiety, the pyrrolidine ring, and the octose core. Here we report that the pyridoxal 5'-phosphate-dependent enzyme CcbF (celesticetin biosynthetic pathway) is a decarboxylating deaminase that converts a cysteine S-conjugated intermediate into an aldehyde. In contrast, the homologous enzyme LmbF (lincomycin biosynthetic pathway) catalyzes C-S bond cleavage of the same intermediate to afford a thioglycoside. We show that Ccb4 and LmbG (downstream methyltransferases) convert the aldehyde and thiol intermediates into a variety of methylated lincosamide compounds including Bu-2545. The substrates used in these studies are the ß-anomers of the natural substrates. The findings not only provide insight into how the biosynthetic pathway of lincosamide antibiotics can bifurcate to generate different lincosamides, but also reveal the promiscuity of the enzymes involved.

Three-membered ring formation catalyzed by α-ketoglutarate-dependent nonheme iron enzymes.

Epoxides, aziridines, and cyclopropanes are found in various medicinal natural products, including polyketides, terpenes, peptides, and alkaloids. Many classes of biosynthetic enzymes are involved in constructing these ring structures during their biosynthesis. This review summarizes our current knowledge regarding how α-ketoglutarate-dependent nonheme iron enzymes catalyze the formation of epoxides, aziridines, and cyclopropanes in nature, with a focus on enzyme mechanisms.

Functions and mechanisms of enzymes assembling lignans and norlignans.

Lignans and norlignans are distributed throughout the plant kingdom and exhibit diverse chemical structures and biological properties that offer potential for therapeutic use. Originating from the phenylpropanoid biosynthesis pathway, their characteristic carbon architectures are formed through unique enzyme catalysis, featuring regio- and stereoselective C-C bond forming processes. Despite extensive research on these plant natural products, their biosynthetic pathways, and enzyme mechanisms remain enigmatic. This review highlights recent advancements in elucidating the functions and mechanisms of the biosynthetic enzymes responsible for constructing the distinct carbon frameworks of lignans and norlignans.

Characterization of Aziridine-Forming α-Ketoglutarate-Dependent Oxygenase in l-Isovaline Biosynthesis.

l-Isovaline biosynthesis by TqaLFM-ti from Tolypocladium inflatum was demonstrated in vitro. The biochemical analysis of the α-ketoglutarate-dependent oxygenase TqaL-ti revealed that it produces (2S,3S)-3-ethyl-3-methylaziridine-2-carboxylic acid from l-isoleucine, thus exhibiting a stereoselectivity different from those of the reported homologues. Remarkably, a single mutation on I295 in TqaL-ti completely exchanged its stereoselectivity to produce the C-3 stereoisomer. TqaFM-ti generates d-isovaline from (2S,3R)-aziridine-2-carboxylic acid, suggesting that the stereochemistry of the TqaL product defines that of isovaline.

Hydration of terminal alkynes catalyzed by water-soluble cobalt porphyrin complexes.

Water-soluble cobalt(III) porphyrin complexes were found to promote the hydration of terminal alkynes to give methyl ketones. The alkyne hydration proceeded in good to excellent yield with 0.1 to 2 mol % cobalt catalyst 1 and was compatible with the presence of acid/base- or redox-sensitive functional groups such as alkyl silyl ethers; allyl ethers; trityl ethers; benzyl ethers; carboxylic esters; boronic esters; carboxamides; nitriles; and nitro, iodo, and acetal groups. Some of the alkyne substrates tested here are otherwise difficult to hydrate. The alkyne hydration can be performed on a gram scale, and the catalyst can be recovered by aqueous workup.