Byproduct formation during the biosynthesis of spinosyn A and evidence for an enzymatic interplay to prevent its formation.

Biosynthesis of spinosyn A in involves a 1,4-dehydration followed by an intramolecular [4 + 2]-cycloaddition catalyzed by SpnM and SpnF, respectively. The cycloaddition also takes place in the absence of SpnF leading to questions regarding its mechanism of catalysis and biosynthetic role. Substrate analogs were prepared with an unactivated dienophile or an acyclic structure and found to be unreactive consistent with the importance of these features for cyclization.

Evidence for an Enzyme-Catalyzed Rauhut-Currier Reaction during the Biosynthesis of Spinosyn A.

The catalog of enzymes known to catalyze the nucleophile-assisted formation of C-C bonds is extremely small, and there is presently no definitive example of a biological Rauhut-Currier reaction. Biosynthesis of the polyketide insecticide spinosyn A in involves a [4 + 2]-cycloaddition and a subsequent intramolecular C-C bond formation catalyzed by SpnF and SpnL, respectively. Isotope tracer experiments and kinetic isotope effects, however, imply that the SpnL-catalyzed reaction proceeds without initial deprotonation of the substrate.

Switchable CAR-T Cells Outperformed Traditional Antibody-Redirected Therapeutics Targeting Breast Cancers.

Various antibody-redirected immunotherapeutic approaches, including antibody-drug conjugates (ADCs), bispecific antibodies (bsAbs), and chimeric antigen receptor-T (CAR-T) cells, have been devised to produce specific activity against various cancer types. Using genetically encoded unnatural amino acids, we generated a homogeneous Her2-targeted ADC, a T cell-redirected bsAb, and a FITC-modified antibody capable of redirecting anti-FITC CAR-T (switchable CAR-T; sCAR-T) cells to target different Her2-expressing breast cancers. sCAR-T cells showed activity against Her2-expressing tumor cells comparable to that of conventional anti-Her2 CAR-T cells and superior to that of ADC- and bsAb-based approaches.

Design of Switchable Chimeric Antigen Receptor T Cells Targeting Breast Cancer.

Chimeric antigen receptor T (CAR-T) cells have demonstrated promising results against hematological malignancies, but have encountered significant challenges in translation to solid tumors. To overcome these hurdles, we have developed a switchable CAR-T cell platform in which the activity of the engineered cell is controlled by dosage of an antibody-based switch. Herein, we apply this approach to Her2-expressing breast cancers by engineering switch molecules through site-specific incorporation of FITC or grafting of a peptide neo-epitope (PNE) into the anti-Her2 antibody trastuzumab (clone 4D5).

Versatile strategy for controlling the specificity and activity of engineered T cells.

The adoptive transfer of autologous T cells engineered to express a chimeric antigen receptor (CAR) has emerged as a promising cancer therapy. Despite impressive clinical efficacy, the general application of current CAR-T--cell therapy is limited by serious treatment-related toxicities. One approach to improve the safety of CAR-T cells involves making their activation and proliferation dependent upon adaptor molecules that mediate formation of the immunological synapse between the target cancer cell and T-cell.

Exploring the potential impact of an expanded genetic code on protein function.

With few exceptions, all living organisms encode the same 20 canonical amino acids; however, it remains an open question whether organisms with additional amino acids beyond the common 20 might have an evolutionary advantage. Here, we begin to test that notion by making a large library of mutant enzymes in which 10 structurally distinct noncanonical amino acids were substituted at single sites randomly throughout TEM-1 β-lactamase. A screen for growth on the β-lactam antibiotic cephalexin afforded a unique p-acrylamido-phenylalanine (AcrF) mutation at Val-216 that leads to an increase in catalytic efficiency by increasing kcat, but not significantly affecting KM.

An anti-B cell maturation antigen bispecific antibody for multiple myeloma.

The development of immunotherapies for multiple myeloma is critical to provide new treatment strategies to combat drug resistance. We report a bispecific antibody against B cell maturation antigen (BiFab-BCMA), which potently and specifically redirects T cells to lyse malignant multiple myeloma cells. BiFab-BCMA lysed target BCMA-positive cell lines up to 20-fold more potently than a CS1-targeting bispecific antibody (BiFab-CS1) developed in an analogous fashion.

Mechanistic Investigation of the Radical S-Adenosyl-L-methionine Enzyme DesII Using Fluorinated Analogues.

DesII is a radical S-adenosyl-l-methionine (SAM) enzyme that can act as a deaminase or a dehydrogenase depending on the nature of its TDP-sugar substrate. Previous work has implicated a substrate-derived, C3-centered α-hydroxyalkyl radical as a key intermediate during catalysis. Although deprotonation of the α-hydroxyalkyl radical has been shown to be important for dehydrogenation, much less is known regarding the course of the deamination reaction.

Mechanistic studies of the radical S-adenosylmethionine enzyme DesII with TDP-D-fucose.

DesII is a radical S-adenosylmethionine (SAM) enzyme that catalyzes the C4-deamination of TDP-4-amino-4,6-dideoxyglucose through a C3 radical intermediate. However, if the C4 amino group is replaced with a hydroxy group (to give TDP-quinovose), the hydroxy group at C3 is oxidized to a ketone with no C4-dehydration. It is hypothesized that hyperconjugation between the C4 C-N/O bond and the partially filled p orbital at C3 of the radical intermediate modulates the degree to which elimination competes with dehydrogenation.

Chemoenzymatic synthesis of spinosyn A.

Following the biosynthesis of polyketide backbones by polyketide synthases (PKSs), post-PKS modifications result in a significantly elevated level of structural complexity that renders the chemical synthesis of these natural products challenging. We report herein a total synthesis of the widely used polyketide insecticide spinosyn A by exploiting the prowess of both chemical and enzymatic methods. As more polyketide biosynthetic pathways are characterized, this chemoenzymatic approach is expected to become readily adaptable to streamlining the synthesis of other complex polyketides with more elaborate post-PKS modifications.

Genetic incorporation of multiple unnatural amino acids into proteins in mammalian cells.

enables the expression of proteins in mammalian cells incorporating one unnatural amino acid (UAA) into multiple sites, as well as two different UAAs into distinct sites in a protein of interest. The utility of this technology was demonstrated by generating a full-length antibody, site-specifically conjugated to a drug and a fluorophore, and characterizing its activity in vitro (see picture). Picture was cropped to fit the available space.

Bispecific small molecule-antibody conjugate targeting prostate cancer.

Bispecific antibodies, which simultaneously target CD3 on T cells and tumor-associated antigens to recruit cytotoxic T cells to cancer cells, are a promising new approach to the treatment of hormone-refractory prostate cancer. Here we report a site-specific, semisynthetic method for the production of bispecific antibody-like therapeutics in which a derivative of the prostate-specific membrane antigen-binding small molecule DUPA was selectively conjugated to a mutant αCD3 Fab containing the unnatural amino acid, p-acetylphenylalanine, at a defined site. Homogeneous conjugates were generated in excellent yields and had good solubility.

EPR-kinetic isotope effect study of the mechanism of radical-mediated dehydrogenation of an alcohol by the radical SAM enzyme DesII.

The radical S-adenosyl-L-methionine enzyme DesII from Streptomyces venezuelae is able to oxidize the C3 hydroxyl group of TDP-D-quinovose to the corresponding ketone via an α-hydroxyalkyl radical intermediate. It is unknown whether electron transfer from the radical intermediate precedes or follows its deprotonation, and answering this question would offer considerable insight into the mechanism by which the small but important class of radical-mediated alcohol dehydrogenases operate. This question can be addressed by measuring steady-state kinetic isotope effects (KIEs); however, their interpretation is obfuscated by the degree to which the steps of interest limit catalysis.

Analysis of UDP-D-apiose/UDP-D-xylose synthase-catalyzed conversion of UDP-D-apiose phosphonate to UDP-D-xylose phosphonate: implications for a retroaldol-aldol mechanism.

UDP-D-apiose/UDP-D-xylose synthase (AXS) catalyzes the conversion of UDP-D-glucuronic acid to UDP-D-apiose and UDP-D-xylose. An acetyl-protected phosphonate analogue of UDP-D-apiose was synthesized and used in an in situ HPLC assay to demonstrate for the first time the ability of AXS to interconvert the two reaction products. Density functional theory calculations provided insight into the energetics of this process and the apparent inability of AXS to catalyze the conversion of UDP-D-xylose to UDP-D-apiose.

A fluoro analogue of UDP-α-D-glucuronic acid is an inhibitor of UDP-α-D-apiose/UDP-α-D-xylose synthase.

UDP-2F-glucuronic acid was synthesized and analyzed as a mechanistic probe to investigate the ring contraction step catalyzed by UDP-d-apiose/UDP-d-xylose synthase (AXS).

Enzyme-catalysed [4+2] cycloaddition is a key step in the biosynthesis of spinosyn A.

The Diels-Alder reaction is a [4+2] cycloaddition reaction in which a cyclohexene ring is formed between a 1,3-diene and an electron-deficient alkene via a single pericyclic transition state. This reaction has been proposed as a key transformation in the biosynthesis of many cyclohexene-containing secondary metabolites. However, only four purified enzymes have thus far been implicated in biotransformations that are consistent with a Diels-Alder reaction, namely solanapyrone synthase, LovB, macrophomate synthase, and riboflavin synthase.

Mechanistic studies of the radical S-adenosyl-L-methionine enzyme DesII: EPR characterization of a radical intermediate generated during its catalyzed dehydrogenation of TDP-D-quinovose.

DesII, a radical S-adenosyl-l-methionine (SAM) enzyme from Streptomyces venezuelae, catalyzes the deamination of TDP-4-amino-4,6-dideoxy-D-glucose to TDP-3-keto-4,6-dideoxy-D-glucose in the desosamine biosynthetic pathway. DesII can also catalyze the dehydrogenation of TDP-D-quinovose to the corresponding 3-keto sugar. Similar to other radical SAM enzymes, DesII catalysis has been proposed to proceed via a radical mechanism.

Stoichiometry of the redox neutral deamination and oxidative dehydrogenation reactions catalyzed by the radical SAM enzyme DesII.

DesII from Streptomyces venezuelae is a radical SAM (S-adenosyl-l-methionine) enzyme that catalyzes the deamination of TDP-4-amino-4,6-dideoxy-d-glucose to form TDP-3-keto-4,6-dideoxy-d-glucose in the biosynthesis of TDP-d-desosamine. DesII also catalyzes the dehydrogenation of the nonphysiological substrate TDP-D-quinovose to TDP-3-keto-6-deoxy-d-glucose. These properties prompted an investigation of how DesII handles SAM in the redox neutral deamination versus the oxidative dehydrogenation reactions.

Characterization and mechanistic studies of DesII: a radical S-adenosyl-L-methionine enzyme involved in the biosynthesis of TDP-D-desosamine.

D-desosamine (1) is a 3-(N,N-dimethylamino)-3,4,6-trideoxyhexose found in a number of macrolide antibiotics including methymycin (2), neomethymycin (3), pikromycin (4), and narbomycin (5) produced by Streptomyces venezuelae . It plays an essential role in conferring biological activities to its parent aglycones. Previous genetic and biochemical studies of the biosynthesis of desosamine in S.