In contrast to previous SAR studies of aza-compounds (23vs.24 and 25), the present study using analogues (26a–26c, 27c, and 28a–28c) of salacinol (1) revealed an essential role of the thiosugar ring in effectively inhibiting α-glucosidase.

We describe activity-based protein profiling for analyzing the adenylation domains of non-ribosomal peptide synthetases (ABPP-NRPS) in bacterial proteomes. Using a range of non-proteoinogenic amino acid sulfamoyladenosines, the competitive format of ABPP-NRPS provided substrate tolerance toward non-proteinogenic amino acids. When coupled with precursor-directed biosynthesis, a non-proteinogenic amino acid (O-allyl-L-serine) was successfully incorporated into gramicidin S.

Non-ribosomal peptide synthetases (NRPSs) biosynthesize many pharmaceuticals and virulence factors. The biosynthesis of these natural peptide products from biosynthetic gene clusters depends on complex regulations in bacteria. However, our current knowledge of NRPSs is based on enzymological studies using full NRPS systems and/or a single NRPS domain in heterologous hosts. Chemical and/or biochemical strategies to capture the endogenous activities of NRPSs facilitate studies on NRPS cell biology in bacterial cells. Here, we describe a chemical scaffold for the rapid and selective photoaffinity labelling of NRPSs in purified systems, crude biological samples and living bacterial cells. We synthesized photoaffinity labelling probes coupled with 5'-O-N-(phenylalanyl)sulfamoyladenosine with clickable alkyl diazirine or trifluoromethyl phenyl diazirine. We found that a trifluoromethyl phenyl diazirine-based probe cross-linked the Phe-activating domain of a GrsA-NRPS with high selectivity and sensitivity at shorter ultraviolet (UV) irradiation times (less than 5 min) relative to a prototypical benzophenone-based probe. Our results demonstrated that this quick labelling protocol can prevent damage to proteins and cells caused by long UV irradiation times, providing a mild photoaffinity labelling method for biological samples. This article is part of the theme issue 'Reactivity and mechanism in chemical and synthetic biology'.

A major challenge in fluorescence imaging experiments, which are essential to determine protein activity, expression, and localization, is the penetration of small-molecule probes through the outer membrane permeability barrier of bacteria. Here, we describe a novel strategy for small-molecule probe-based fluorescence protein labeling and imaging in the Gram-negative bacterium Escherichia coli. We targeted a siderophore enterobactin biosynthetic enzyme EntE in E. coli. When coupled with an efflux pump inhibitor carbonyl cyanide m-chlorophenylhydrazone, small-molecule probes were able to efficiently enter the cells, leading to the fluorescence labeling and imaging of overproduced EntE in E. coli. This study demonstrates that the combination of small-molecule probes with appropriate efflux pump inhibitors may substantially enhance their interaction with the target proteins in live bacteria.

Much of our current knowledge on nonribosomal peptide synthetases (NRPSs) is based on studies in which the full NRPS system or each protein domain is expressed in heterologous hosts. Consequently, methods to detect the endogenous activity of NRPSs, under natural cellular conditions, are needed for the study of NRPS cell biology. Here, we describe the in vivo activity-based protein profiling (ABPP) for endogenous NRPSs and its applications to the study of their activities in bacteria. Remarkably, in vitro and in vivo ABPP in the context of the surfactin producer Bacillus subtilis enabled the visualization, tracking, and imaging of an endogenous SrfAB-NRPS with remarkable selectivity and sensitivity. Furthermore, in vivo, ABPP allowed the discovery of the degradation processes of the endogenous SrfAB-NRPS in the context of its native producer bacteria. Overall, this study deepens our understanding of the properties of NRPSs that cannot be addressed by conventional methods.

The recent discovery that an ERK signaling modulator [ACA-28 (2a)] preferentially kills human melanoma cell lines by inducing ERK-dependent apoptosis has generated significant interest in the field of anti-cancer therapy. In the first SAR study on 2a, here, we successfully developed candidates (2b, 2c) both of which induce more potent and selective apoptosis towards ERK-active melanoma cells than 2a, thus revealing the structural basis for inducing the ERK-dependent apoptosis and proposing the therapeutic prospect of these candidates against ERK-dependent cancers represented by melanoma.

Adenylation (A) domains act as the gatekeepers of non-ribosomal peptide synthetases (NRPSs), ensuring the activation and thioesterification of the correct amino acid/aryl acid building blocks. Aryl acid building blocks are most commonly observed in iron-chelating siderophores, but are not limited to them. Very little is known about the reprogramming of aryl acid A-domains. We show that a single asparagine-to-glycine mutation in an aryl acid A-domain leads to an enzyme that tolerates a wide range of non-native aryl acids. The engineered catalyst is capable of activating non-native aryl acids functionalized with nitro, cyano, bromo, and iodo groups, even though no enzymatic activity of wild-type enzyme was observed toward these substrates. Co-crystal structures with non-hydrolysable aryl-AMP analogues revealed the origins of this expansion of substrate promiscuity, highlighting an enlargement of the substrate binding pocket of the enzyme. Our findings may be exploited to produce diversified aryl acid containing natural products and serve as a template for further directed evolution in combinatorial biosynthesis.

Acyltransferases (ATs) are responsible for the selection and incorporation of acyl building blocks in the biosynthesis of various polyketide natural products. The trans-AT modular polyketide synthases have a discrete trans-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 trans-AT assembly lines, the dynamic actions of ACPs and trans-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 trans-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 trans-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 trans-acting ATs and provides detailed mechanistic insights into their ability to recognize ACPs.

Structural and activity studies have revealed the dynamic and transient actions of carrier protein (CP) activity in primary and secondary metabolic pathways. CP-mediated interactions play a central role in nonribosomal peptide biosynthesis, as they serve as covalent tethers for amino acid and aryl acid substrates and enable the growth of peptide intermediates. Strategies are therefore required to study protein-protein interactions efficiently. Herein, we describe activity-based probes used to demonstrate the protein-protein interactions between aryl CP (ArCP) and aryl acid adenylation (A) domains as well as the substrate specificities of the aryl acid Adomains. If coupled with in-gel fluorescence imaging, this strategy allows visualization of the protein-protein interactions required to recognize and transfer the substrate to the partner ArCP. This technique has potential for the analysis of protein-protein interactions within these biosynthetic enzymes at the molecular level and for use in the combinatorial biosynthesis of new nonribosomal peptides.

Phosphopantetheinylation is an essential post-translational protein modification to primary and secondary metabolic pathways that ensures bacterial cell viability and virulence, and it is used in the production of many pharmaceuticals. Traditional methods have not provided a comprehensive understanding of these modifications. By using chemical proteomic probes for adenylation and thiolation domains in nonribosomal peptide synthetases (NRPSs), chemoproteomics has been applied to survey and validate the cellular activity of 4-[3-chloro-5-(trifluoromethyl)pyridin-2-yl]-N-(4-methoxypyridin-2-yl)piperazine-1-carbothioamide (ML267), which is a potent and selective small-molecule 4-phosphopantetheinyl transferase (PPTase) inhibitor that attenuates secondary metabolism and viability of bacterial cells. ML267 inhibited Sfp-type PPTase and antagonized phosphopantetheinylation in cells, which resulted in a decrease in phosphopantetheinylated NRPSs and the attenuation of Sfp-PPTase-dependent metabolite production. These results indicate that this chemoproteomics platform should enable a precise interpretation of the cellular activities of Sfp-type PPTase inhibitors.

The first total syntheses of the geranylated pyranocoumarins, mameasins C (1) and D (2), aromatase inhibitors isolated from the flowers of Mammea siamensis, were accomplished in five steps, starting from phloroglucinol 3. In this strategy, the characteristic pyran ring-fused coumarin core of 1 and 2 was effectively constructed by Friedel-Crafts acylation of 3, followed by Reformatsky reaction of the resultant ketone to give a key coumarin intermediate 9. Compound 9 was converted to targets 1 and 2 in a stepwise manner by successive C-acylation and O-geranylation, followed by a [1,3]-sigmatropic geranyl shift. Furthermore, screening of intermediates obtained in the synthetic pathway to 1 and 2 revealed that de-geranylated pyranocoumarins (10 and 11) show superior aromatase inhibitory activity as compared to the natural products 1 and 2. (C) 2017 Elsevier Ltd. All rights reserved.

Andrimid (Adm) synthase, which belongs to the type II system of enzymes, produces Adm in Pantoea agglomerans. The adm biosynthetic gene cluster lacks canonical acyltransferases (ATs) to load the malonyl group to acyl carrier proteins (ACPs), thus suggesting that a malonyl-CoA ACP transacylase (MCAT) from the fatty acid synthase (FAS) complex provides the essential AT activity in Adm biosynthesis. Here we report that an MCAT is essential for catalysis of the transacylation of malonate from malonyl-CoA to AdmA polyketide synthase (PKS) ACP in vitro. Catalytic self-malonylation of AdmA (PKS ACP) was not observed in reactions without MCAT, although many type II PKS ACPs are capable of catalyzing self-acylation. This lack of self-malonylation was explained by amino acid sequence analysis of the AdmA PKS ACP and the type II PKS ACPs. The results show that MCAT from the organism's FAS complex can provide the missing AT activity in trans, thus suggesting a protein-protein interaction between the fatty acid and polyketide synthases in the Adm assembly line.

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, beta-elimination, aldol, and decarboxylation reactions. Although both antibodies were generated with the same hapten, phosphonate diester 1, they showed different catalytic activity. When phenylacetic acid 4 was used as the catalytic component, 25E2 efficiently catalyzed the elimination reaction of beta-haloketone 2, whereas 27C1 showed no catalytic activity. In this work, we focused on the beta-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 beta-elimination, Arg(H52) of 27C1 would form a salt bridge with the carboxylate of 4, thus destroying reactivity. Therefore, we utilized site-directed chemical mutation to change the charge properties of the catalytic components. When amine components 7-10 were used, 27C1 efficiently catalyzed the beta-elimination reaction. It is noteworthy that chemical mutation with secondary amine 8 provided extremely high activity, with a rate acceleration [ (k(cat)/Km(2))/kuncat] 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 pKa of the amine is perturbed and shifts to a lower pKa, which efficiently abstracts the alpha-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.

We describe a proof-of-concept study of a competitive enzyme-linked immunosorbent assay (ELISA) system for the adenylation (A) domains of nonribosomal peptide synthetases (NRPSs) with active-site-directed probes coupled to a 5-O-N-(aminoacyl)sulfamoyladenosine scaffold. A biotin functionality immobilizes the probes onto a streptavidin-coated solid support. Dissociation constants were determined with a series of ligands, including enzyme substrates and a library of sulfamoyloxy-linked aminoacyl/aryl-AMP analogues. As it enables direct readout of protein-ligand interaction, the competitive ELISA technique provided information on comparative structure- activity relationships and insights into the enzyme active-site architecture of NRPS A-domains. These studies indicate that the ELISA technique can accelerate the discovery of small-molecule inhibitors of the A-domains with new scaffolds that perturb the production of NRPS-related virulence factors.

Derivatization of a 5 '-(vinylsulfonylaminodeoxy) adenosine scaffold with a clickable functionality provided an activity-based probe that was used to label native carrier protein (CP) motifs in nonribosomal peptide synthetases (NRPSs). When coupled with a fluorescent tag, this probe selectively targeted phosphopantetheinylated CPs (holo-form) from recombinant NRPS enzyme systems and in whole proteomes.

Genetic approaches have greatly contributed to our understanding of nonribosomal peptide biosynthetic machinery; however, proteomic investigations are limited. Here, we developed a highly sensitive detection strategy for multidomain nonribosomal peptide synthetases (NRPSs) by using a multiple-labeling technique with active-site-directed probes for adenylation domains. When applied to gramicidin S-producing and -nonproducing strains of Aneurinibacillus migulanus (DSM 5759 and DSM 2895, respectively), the multiple technique sensitively detected an active multidomain NRPS (GrsB) in lysates obtained from the organisms. This functional proteomics method revealed an unknown inactive precursor (or other inactive form) of GrsB in the nonproducing strain. This method provides a new option for the direct detection, functional analysis, and high-resolution identification of low-abundance active NRPS enzymes in native proteomic environments.

A significant, gap exists between protein engineering and enzymes used for the biosynthesis of natural, products, largely because 1;, there is a paucity of strategies:that rapidly detect. active site phenotypes the enzymes with desired activities. Herein, we describe a proof-oft : concept study of an enzyme-linked immunosorbe4-asaay (ELISA) system for the adenylation (A) domains in nonribosomal peptide synthetase (NRPSs)' using a,combination of active site-directed probes coupled a 5'-O-N-(aminoacyl)sulfamoyladenosine scaffold with a biotin functionality, that, immobilizes probe xriOlectles onto a Streptavidin-coated solid Support. The recombinant NRPSs have a C-terrinnal His tag motif that is targeted by an anti-6xHis 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 domains in the crude cell-free homogenates from the Escherichia,colt 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-dihydroxybenxoic 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 k(cat)/K-m value with the noncognate substrate Sal and a corresponding 48 fold decrease in the k(cat)/K-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.

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'-O-N-(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.

Microorganisms produce a large number of peptide-based natural products that display a broad range of biologically interesting properties, including antimicrobial, immunosuppressant, and anticancer activities, as well as behaving as virulence factors and signaling molecules. These peptide natural products are composed of proteinogenic amino acids, as well as a number of other compound classes, including non-proteinogenic amino acids, aryl acids, fatty acids, hydroxyl acids, heterocyclic rings, and sugars, which provide a complex level of chemical diversity. Many of these natural products are biosynthesized by large, highly versatile multifunctional megasynthetases, which are known as nonribosomal peptide synthetases (NRPSs). The adenylation (A) domains found in all NRPS modules are essential catalytic components and function as gatekeepers to select the appropriate amino acid building blocks during nonribosomal peptide (NRP) biosynthesis. The results of extensive periods of genetic, biochemical, and bioinformatic investigations have provided a detailed understanding of the functional characteristics and molecular basis underpinning the A domain enzymology in NRP biosynthesis. This review will therefore focus on the recent discoveries and breakthroughs in the structural elucidation, molecular mechanism, and chemical biology underlying the A domains within NRPS enzymes.

We describe competitive activity-based protein profiling (ABPP) to accelerate the functional prediction and assessment of adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) in proteomic environments. Using a library of sulfamoyloxy-linked aminoacyl-AMP analogs, the competitive ABPP technique offers a simple and rapid assay system for adenylating enzymes and provides insight into enzyme substrate candidates and enzyme active-site architecture.

We describe a general strategy for selective chemical labeling of individual adenylation (A) domains in nonribosomal peptide synthetases (NRPSs) using active site-directed proteomic probes coupled to the 5 '-O-N-(aminoacyl) sulfamoyladenosine (AMS) scaffold with a clickable benzophenone functionality. These proteomic tools can greatly facilitate the molecular identification, functional characterization, and profiling of virtually any kind of A domains of NRPS enzymes in complex biological systems.

A putative 7-dimethylallyl tryptophan synthase (DMATS) gene from a fungal Neosartorya sp. was cloned and overexpressed as a soluble His(6)-fusion protein in Escherichia coli. The enzyme was found to catalyze the prenylation of L-tryptophan at the C7 position of the indole moiety in the presence of dimethylallyl diphosphate; thus, it functions as a 7-DMATS. In this study, we describe the biochemical characterization of 7-DMATS from Neosartorya sp., referred to as 7-DMATS(Neo), and the structural basis of the regioselective prenylation of L-tryptophan at the C7 position by comparison of the three-dimensional structural models of 7-DMATS(Neo) with FgaPT2 (4-DMATS) from Aspergillus fumigatus. (C) 2014 Elsevier Ltd. All rights reserved.

We targeted the development of an affinity probe for adenylation (A) domains that can facilitate enrichment, identification, and quantification of A domain-containing modules in nonribosomal peptide synthetase (NRPS)-polyketide synthase (PKS) hybrids and NRPSs. A 5'-O-sulfamoyladenosine (AMS) non-hydrolyzable analogue of adenosine monophosphate (AMP) has been reported as a scaffold for the design of inhibitors exhibiting tight binding of adenylation enzymes. Here we describe the application of an affinity probe for A domains. Our synthetic probe, a biotinylated L-Phe-AMS (L-Phe-AMS-biotin) specifically targets the A domains in NRPS modules that activates L-Phe to an aminoacyladenylate intermediate in both recombinant NRPS enzyme systems and whole proteomes. (C) 2014 Elsevier Ltd. All rights reserved.

Acyl carrier protein (ACP) transports the growing fatty acid chain between enzymatic domains of fatty acid synthase (FAS) during biosynthesis(1). Because FAS enzymes operate on ACP-bound acyl groups, ACP must stabilize and transport the growing lipid chain(2). ACPs have a central role in transporting starting materials and intermediates throughout the fatty acid biosynthetic pathway(3-5). The transient nature of ACP-enzyme interactions impose major obstacles to obtaining high-resolution structural information about fatty acid biosynthesis, and a new strategy is required to study protein-protein interactions effectively. Here we describe the application of a mechanism-based probe that allows active site-selective covalent crosslinking of AcpP to FabA, the Escherichia coli ACP and fatty acid 3-hydroxyacyl-ACP dehydratase, respectively. We report the 1.9 angstrom crystal structure of the crosslinked AcpP-FabA complex as a homodimer in which AcpP exhibits two different conformations, representing probable snapshots of ACP in action: the 4'-phosphopantetheine group of AcpP first binds an arginine-rich groove of FabA, then an AcpP helical conformational change locks AcpP and FabA in place. Residues at the interface of AcpP and FabA are identified and validated by solution nuclear magnetic resonance techniques, including chemical shift perturbations and residual dipolar coupling measurements. These not only support our interpretation of the crystal structures but also provide an animated view of ACP in action during fatty acid dehydration. These techniques, in combination with molecular dynamics simulations, show for the first time that FabA extrudes the sequestered acyl chain from the ACP binding pocket before dehydration by repositioning helix III. Extensive sequence conservation among carrier proteins suggests that the mechanistic insights gleaned from our studies may be broadly applicable to fatty acid, polyketide and non-ribosomal biosynthesis. Here the foundation is laid for defining the dynamic action of carrier-protein activity in primary and secondary metabolism, providing insight into pathways that can have major roles in the treatment of cancer, obesity and infectious disease.

Catalytic antibody 27C1 bears binding sites for both a substrate- and a functionalized small nonprotein component in the active site. We investigated the possibility of exploiting imine and enamine intermediates using a primary amine molecule into the active site of antibody 27C1. The antibody catalyzed β-keto acid decarboxylation with a rate enhancement (kcat/K m/kuncat) of 140,000, as well as highly regioselective cross-aldol reactions of ketones and p-nitrobenzaldehyde. These studies provide new strategies for the generation of catalytic antibodies possessing binding sites for functionalized components. © 2013 Elsevier Ltd. All rights reserved.

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 beta-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 crypto-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.

We targeted the development of a dehydratase (DH)-specific reactive probe that can facilitate detection, enrichment, and identification of DH enzymes in fatty acid synthases (FASs) and polyketide synthases (PKSs). The first reported mechanism-based inactivator, 3-decynoyl-N-acetylcysteamine (3-decynoyl-NAC), while active against the Escherichia coli beta-hydroxydecanoyl thiol ester DH FabA, translates poorly to an activity-based probe because of nonspecific reactivity of the thioester moiety. Here we describe the design, synthesis, and utility of a DH-specific probe that contains a sulfonyl 3-alkyne reactive warhead engineered to avoid hydrolysis or nonenzymatic inactivation. When coupled with a fluorescent tag, this probe targets DH enzymes from recombinant type I and type II FAS and PKS enzyme systems and in whole proteomes. Activity studies, including FabA inactivation and antibiotic susceptibility, suggest that this sulfonyl 3-alkyne scaffold selectively targets a common DH mechanism. These studies indicate that the DH-specific mechanism-based probe can greatly accelerate both the functional characterization and molecular identification of virtually any type of FAS and PKS in complex proteomes.

A single antibody catalyzes multiple chemical transformations upon replacement of the funcctionalized small nonportein components.