Biosynthetic Origins of the Epoxyquinone Skeleton in Epoxyquinols A and B

The biosynthetic origins of epoxyquinols A (<b>1</b>) and B (<b>2</b>) produced by an unidentified fungus have attracted considerable interest because these compounds could be assembled from a biosynthetic precursor, epoxycyclohexenone aldehyde (<b>3</b>), via an electrocyclization/intermolecular Diels–Alder dimerization cascade reaction. Furthermore, very little is known about the biosynthetic origins of naturally occurring epoxyquinone moieties. We herein describe the incorporation of ¹³C at specific positions within the structure of a shunt product, epoxycyclohexenone (<b>4</b>), using stable isotope feeding experiments with sodium [1-¹³C]-acetate and sodium [1,2-¹³C₂]-acetate. The results of these experiments strongly suggest that the epoxyquinone skeleton is assembled by a polyketide synthase

Accurate Detection of Adenylation Domain Functions in Nonribosomal Peptide Synthetases by an Enzyme-linked Immunosorbent Assay System Using Active Site-directed Probes for Adenylation Domains

A significant gap exists between protein engineering and enzymes used for the biosynthesis of natural products, largely because there is a paucity of strategies that rapidly detect active-site phenotypes of the enzymes with desired activities. Herein, we describe a proof-of-concept study of an enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using a combination of active site-directed probes coupled to a 5′-<i>O</i>-<i>N</i>-(aminoacyl)­sulfamoyladenosine scaffold with a biotin functionality that immobilizes probe molecules onto a streptavidin-coated solid support. The recombinant NRPSs have a C-terminal His-tag motif that is targeted by an anti-6×His mouse antibody as the primary antibody and a horseradish peroxidase-linked goat antimouse antibody as the secondary antibody. These probes can selectively capture the cognate A domains by ligand-directed targeting. In addition, the ELISA technique detected A domains in the crude cell-free homogenates from the <i>Escherichia coli</i> expression systems. When coupled with a chromogenic substrate, the antibody-based ELISA technique can visualize probe–protein binding interactions, which provides accurate readouts of the A-domain functions in NRPS enzymes. To assess the ELISA-based engineering of the A domains of NRPSs, we reprogramed 2,3-dihydroxybenzoic acid (DHB)-activating enzyme EntE toward salicylic acid (Sal)-activating enzymes and investigated a correlation between binding properties for probe molecules and enzyme catalysts. We generated a mutant of EntE that displayed negligible loss in the <i>k</i>_cat/<i>K</i>_m value with the noncognate substrate Sal and a corresponding 48-fold decrease in the <i>k</i>_cat/<i>K</i>_m value with the cognate substrate DHB. The resulting 26-fold switch in substrate specificity was achieved by the replacement of a Ser residue in the active site of EntE with a Cys toward the nonribosomal codes of Sal-activating enzymes. Bringing a laboratory ELISA technique and adenylating enzymes together using a combination of active site-directed probes for the A domains in NRPSs should accelerate both the functional characterization and manipulation of the A domains in NRPSs

Profiling Nonribosomal Peptide Synthetase Activities Using Chemical Proteomic Probes for Adenylation Domains

Nonribosomal peptide synthetases (NRPSs) and polyketide synthases are large diverse families of biosynthetic enzymes that catalyze the synthesis of natural products that display biologically important activities. Genetic investigations have greatly contributed to our understanding of these biosynthetic enzymes; however, proteomic studies are limited. Here we describe the application of active site-directed proteomic probes for adenylation (A) domains to profile the activity of NRPSs directly in native proteomic environments. Derivatization of a 5′-<i>O</i>-<i>N</i>-(aminoacyl)­sulfamoyladenosine appended clickable benzophenone functionality enabled activity-based protein profiling of the A-domains in NRPSs in proteomic extracts. These probes were used to identify natural product producing microorganisms, optimize culture conditions, and profile the activity dynamics of NRPSs. Our proteomic approach offers a simple and versatile method to monitor NRPS expression at the protein level and will facilitate the identification of orphan enzymatic pathways involved in secondary metabolite production

Sulfonyl 3‑Alkynyl Pantetheinamides as Mechanism-Based Cross-Linkers of Acyl Carrier Protein Dehydratase

Acyl carrier proteins (ACPs) play a central role in acetate biosynthetic pathways, serving as tethers for substrates and growing intermediates. Activity and structural studies have highlighted the complexities of this role, and the protein–protein interactions of ACPs have recently come under scrutiny as a regulator of catalysis. As existing methods to interrogate these interactions have fallen short, we have sought to develop new tools to aid their study. Here we describe the design, synthesis, and application of pantetheinamides that can cross-link ACPs with catalytic β-hydroxy-ACP dehydratase (DH) domains by means of a 3-alkynyl sulfone warhead. We demonstrate this process by application to the Escherichia coli fatty acid synthase and apply it to probe protein–protein interactions with noncognate carrier proteins. Finally, we use solution-phase protein NMR spectroscopy to demonstrate that sulfonyl 3-alkynyl pantetheinamide is fully sequestered by the ACP, indicating that the <i>crypto</i>-ACP closely mimics the natural DH substrate. This cross-linking technology offers immediate potential to lock these biosynthetic enzymes in their native binding states by providing access to mechanistically cross-linked enzyme complexes, presenting a solution to ongoing structural challenges

Structural Basis of Protein–Protein Interactions between a <i>trans</i>-Acting Acyltransferase and Acyl Carrier Protein in Polyketide Disorazole Biosynthesis

Acyltransferases (ATs) are responsible for the selection and incorporation of acyl building blocks in the biosynthesis of various polyketide natural products. The <i>trans</i>-AT modular polyketide synthases have a discrete <i>trans</i>-acting AT for the loading of an acyl unit onto the acyl carrier protein (ACP) located within each module. Despite the importance of protein–protein interactions between ATs and ACPs in <i>trans</i>-AT assembly lines, the dynamic actions of ACPs and <i>trans</i>-acting ATs remain largely uncharacterized because of the inherently transient nature of ACP–enzyme interactions. Herein, we report the crystal structure of the AT–ACP complex of disorazole <i>trans</i>-AT polyketide synthase. We used a bromoacetamide pantetheine cross-linking probe in combination with a Cys mutation to trap the transient AT–ACP complex, allowing the determination of the crystal structure of the disorazole AT–ACP complex at 2.03 Å resolution. On the basis of the cross-linked AT–ACP complex structure, ACP residues recognized by <i>trans</i>-acting AT were identified and validated by mutational studies, which demonstrated that the disorazole AT recognizes the loop 1 and helix III′ residues of disorazole ACP. The disorazole AT–ACP complex structure presents a foundation for defining the dynamic processes associated with <i>trans</i>-acting ATs and provides detailed mechanistic insights into their ability to recognize ACPs

Site-Directed Chemical Mutations on Abzymes: Large Rate Accelerations in the Catalysis by Exchanging the Functionalized Small Nonprotein Components

Taking advantage of antibody molecules to generate tailor-made binding sites, we propose a new class of protein modifications, termed as “site-directed chemical mutation.” In this modification, chemically synthesized catalytic components with a variety of steric and electronic properties can be noncovalently and nongenetically incorporated into specific sites in antibody molecules to induce enzymatic activity. Two catalytic antibodies, 25E2 and 27C1, possess antigen-combining sites which bind catalytic components and act as apoproteins in catalytic reactions. By simply exchanging these components, antibodies 25E2 and 27C1 can catalyze a wide range of chemical transformations including acyl-transfer, β-elimination, aldol, and decarboxylation reactions. Although both antibodies were generated with the same hapten, phosphonate diester <b>1</b>, they showed different catalytic activity. When phenylacetic acid <b>4</b> was used as the catalytic component, 25E2 efficiently catalyzed the elimination reaction of β-haloketone <b>2</b>, whereas 27C1 showed no catalytic activity. In this work, we focused on the β-elimination reaction and examined the site-directed chemical mutation of 27C1 to induce activity and elucidate the catalytic mechanism. Molecular models showed that the cationic guanidyl group of Arg^H52 in 27C1 makes a hydrogen bond with the PO oxygen in the hapten. This suggested that during β-elimination, Arg^H52 of 27C1 would form a salt bridge with the carboxylate of <b>4</b>, thus destroying reactivity. Therefore, we utilized site-directed chemical mutation to change the charge properties of the catalytic components. When amine components <b>7</b>–<b>10</b> were used, 27C1 efficiently catalyzed the β-elimination reaction. It is noteworthy that chemical mutation with secondary amine <b>8</b> provided extremely high activity, with a rate acceleration [(<i>k</i>_cat/<i>K</i>_m <b>2</b>)/<i>k</i>_uncat] of 1 000 000. This catalytic activity likely arises from the proximity effect, plus general-base catalysis associated the electrostatic interactions. In 27C1, the cationic guanidyl group of Arg^H52 is spatially close to the nitrogen of the amine components. In this microenvironment, the intrinsic p<i>K</i>_a of the amine is perturbed and shifts to a lower p<i>K</i>_a, which efficiently abstracts the α-proton during the reaction. This mechanism is consistent with the observed kinetic isotope effect (E2 or E1cB mechanism). Thus, site-directed chemical mutation provides a better understanding of enzyme functions and opens new avenues in biocatalyst research

Diastereoselective Synthesis of Salacinol-Type α‑Glucosidase Inhibitors

A facile and highly diastereoselective approach toward the synthesis of potent salacinol-type α-glucosidase inhibitors, originally isolated from plants of the genus “<i>Salacia</i>”, was developed using the <i>S</i>-alkylation of thiosugars with epoxides in HFIP (∼90%, dr, α/β = ∼ 26/1). The dr ratio of the product was significantly improved by the protocol as compared to that of the conventional <i>S</i>-alkylation of thiosugars (dr, α/β = ∼ 8/1). The protocol could be used for gram scale synthesis of the desired compounds. The 3′-<i>O</i>-benzylated salacinol analogs, which are the most potent <i>in vitro</i> inhibitors to date, were synthesized and evaluated <i>in vivo</i>; all analogs suppressed blood glucose levels in maltose–loaded mice, at levels comparable to those of the antidiabetic agent, voglibose

Total Synthesis of γ‑Alkylidenebutenolides, Potent Melanogenesis Inhibitors from Thai Medicinal Plant <i>Melodorum fruticosum</i>

A hitherto unreported member of γ-alkylidenebutenolides in <i>Melodorum fruticosum</i> (Annonaceae), (4<i>E</i>)-6-benzoyloxy-7-hydroxy-2,4-heptadiene-4-olide, named as isofruticosinol (<b>4</b>) was isolated from the methanol extract of flowers, along with the known related butenolides, namely, the (4<i>Z</i>)-isomer (<b>3</b>) of <b>4</b>, melodrinol (<b>1</b>), and its (4<i>E</i>)-isomer (<b>2</b>). To unambiguously determine the absolute configuration at the C-6 position in these butenolides, the first total syntheses of both enantiomers of <b>2</b>–<b>4</b> were achieved over 6–7 steps from commercially available D- or L-ribose (<b>D-</b> and <b>L-5</b>). Using the same protocol, both enantiomers of <b>1</b> were also synthesized. Based on chiral HPLC analysis of all synthetic compounds (<i><b>S</b></i><b>-</b> and <i><b>R</b></i><b>-1–4</b>), all naturally occurring butenolides were assigned as partial racemic mixtures with respect to the chiral center at C-6 (enantiomeric ratio, 6<i>S</i>/6<i>R</i> = ∼83/17). Furthermore, the melanogenesis inhibitory activities of <i><b>S</b></i><b>-</b> and <i><b>R</b></i><b>-1</b>–<b>4</b> were evaluated, with all shown to be potent inhibitors with IC₅₀ values in the range 0.29–2.9 μM, regardless of differences in the stereochemistry at C-6. In particular, <i><b>S</b></i><b>-4</b> (IC₅₀ = 0.29 μM) and <i><b>R</b></i><b>-4</b> (0.39 μM) showed potent inhibitory activities compared with that of reference standard arbutin (174 μM)

Total Synthesis of γ‑Alkylidenebutenolides, Potent Melanogenesis Inhibitors from Thai Medicinal Plant <i>Melodorum fruticosum</i>

A hitherto unreported member of γ-alkylidenebutenolides in <i>Melodorum fruticosum</i> (Annonaceae), (4<i>E</i>)-6-benzoyloxy-7-hydroxy-2,4-heptadiene-4-olide, named as isofruticosinol (<b>4</b>) was isolated from the methanol extract of flowers, along with the known related butenolides, namely, the (4<i>Z</i>)-isomer (<b>3</b>) of <b>4</b>, melodrinol (<b>1</b>), and its (4<i>E</i>)-isomer (<b>2</b>). To unambiguously determine the absolute configuration at the C-6 position in these butenolides, the first total syntheses of both enantiomers of <b>2</b>–<b>4</b> were achieved over 6–7 steps from commercially available D- or L-ribose (<b>D-</b> and <b>L-5</b>). Using the same protocol, both enantiomers of <b>1</b> were also synthesized. Based on chiral HPLC analysis of all synthetic compounds (<i><b>S</b></i><b>-</b> and <i><b>R</b></i><b>-1–4</b>), all naturally occurring butenolides were assigned as partial racemic mixtures with respect to the chiral center at C-6 (enantiomeric ratio, 6<i>S</i>/6<i>R</i> = ∼83/17). Furthermore, the melanogenesis inhibitory activities of <i><b>S</b></i><b>-</b> and <i><b>R</b></i><b>-1</b>–<b>4</b> were evaluated, with all shown to be potent inhibitors with IC₅₀ values in the range 0.29–2.9 μM, regardless of differences in the stereochemistry at C-6. In particular, <i><b>S</b></i><b>-4</b> (IC₅₀ = 0.29 μM) and <i><b>R</b></i><b>-4</b> (0.39 μM) showed potent inhibitory activities compared with that of reference standard arbutin (174 μM)