Phosphopantetheinyl Transferase Research Articles

The Impacts of Sortase A and the 4'-Phosphopantetheinyl Transferase Homolog Sfp on Streptococcus mutans Extracellular Membrane Vesicle Biogenesis.

Extracellular membrane vesicles (EMVs) are produced by many Gram-positive organisms, but information regarding vesiculogenesis is incomplete. We used single gene deletions to evaluate the impacts on Streptococcus mutans EMV biogenesis of Sortase A (SrtA), which affects S. mutans EMV composition, and Sfp, a 4′-phosphopantetheinyl transferase that affects Bacillus subtilis EMV stability. ΔsrtA EMVs were notably larger than Δsfp and wild-type (WT) EMVs. EMV proteins identified from all three strains are known to be involved in cell wall biogenesis and cell architecture, bacterial adhesion, biofilm cell density and matrix development, and microbial competition. Notably, the AtlA autolysin was not processed to its mature active form in the ΔsrtA mutant. Proteomic and lipidomic analyses of all three strains revealed multiple dissimilarities between vesicular and corresponding cytoplasmic membranes (CMs). A higher proportion of EMV proteins are predicted substrates of the general secretion pathway (GSP). Accordingly, the GSP component SecA was identified as a prominent EMV-associated protein. In contrast, CMs contained more multi-pass transmembrane (TM) protein substrates of co-translational transport machineries than EMVs. EMVs from the WT, but not the mutant strains, were enriched in cardiolipin compared to CMs, and all EMVs were over-represented in polyketide flavonoids. EMVs and CMs were rich in long-chain saturated, monounsaturated, and polyunsaturated fatty acids, except for Δsfp EMVs that contained exclusively polyunsaturated fatty acids. Lipoproteins were less prevalent in EMVs of all three strains compared to their CMs. This study provides insight into biophysical characteristics of S. mutans EMVs and indicates discrete partitioning of protein and lipid components between EMVs and corresponding CMs of WT, ΔsrtA, and Δsfp strains.

Improved Butanol Production Using FASII Pathway in E. coli.

n-Butanol is often considered a potential substitute for gasoline due to its physicochemical properties being closely related to those of gasoline. In this study, we extend our earlier work to convert endogenously producing butyrate via the FASII pathway using thioesterase TesBT to its corresponding alcohol, i.e., butanol. We first assembled pathway genes, i.e., car encoding carboxylic acid reductase from Mycobacterium marinum, sfp encoding phosphopantetheinyl transferase from Bacillus subtilis, and adh2 encoding alcohol dehydrogenase from S. cerevisiae, responsible for bioconversion of butyrate to butanol in three different configurations (Operon, Pseudo-Operon, and Monocistronic) to achieve optimum expression of each gene and compared with the clostridial solventogenic pathway for in vivo conversion of butyrate to butanol under aerobic conditions. An E. coli strain harboring car, sfp, and adh2 in pseudo-operon configuration was able to convert butyrate to butanol with 100% bioconversion efficiency when supplemented with 1 g/L of butyrate. Further, co-cultivation of an upstream strain (butyrate-producing) with a downstream strain (butyrate to butanol converting) at different inoculation ratios was investigated, and an optimized ratio of 1:4 (upstream strain: downstream strain) was found to produce ∼2 g/L butanol under fed-batch fermentation. Further, a mono-cultivation approach was applied by transforming a plasmid harboring tesBT gene into the downstream strain. This approach produced 0.42 g/L in a test tube and ∼2.9 g/L butanol under fed-batch fermentation. This is the first report where both mono- and co-cultivation approaches were tested and compared for butanol production, and butanol titers achieved using both strategies are the highest reported values in recombinant E. coli utilizing FASII pathway.

Fluorolabeling of the PPTase-Related Chemical Tags: Comparative Study of Different Membrane Receptors and Different Fluorophores in the Labeling Reactions.

The set-up of an advanced imaging experiment requires a careful selection of suitable labeling strategies and fluorophores for the tagging of the molecules of interest. Here we provide an experimental workflow to allow evaluation of fluorolabeling performance of the chemical tags target of phosphopantetheinyl transferase enzymes (PPTases), once inserted in the sequence of different proteins of interest. First, S6 peptide tag was fused to three different single-pass transmembrane proteins (the tyrosine receptor kinases TrkA and VEGFR2 and the tumor necrosis factor receptor p75NTR), providing evidence that all of them can be conveniently albeit differently labeled. Moreover, we chose the S6-tagged TrkA construct to test eight different organic fluorophores for the PPTase labeling of membrane receptors in living cells. We systematically compared their non-specific internalization when added to a S6-tag negative cell culture, the percentage of S6-TrkA expressing cells effectively labeled and the relative mean fluorescence intensity, their photostability upon conjugation, and ratio of specific (cellular) versus background (glass-adhered) signal. This allowed to identify which fluorophores are actually recommended for these labeling reactions. Finally, we compared the PPTase labeling of a purified, YBBR-tagged Nerve Growth Factor with two differently charged organic dyes. We detected some batch-to-batch variability in the labeling yield, regardless of the fluorophore used. However, upon purification of the fluorescent species and incubation with living primary DRG neurons, no significant difference could be appreciated in both internalization and axonal transport of the labeled neurotrophins.

Enzyme-Directed Functionalization of Designed, Two-Dimensional Protein Lattices.

The design and construction of crystalline protein arrays to selectively assemble ordered nanoscale materials have potential applications in sensing, catalysis, and medicine. Whereas numerous designs have been implemented for the bottom-up construction of protein assemblies, the generation of artificial functional materials has been relatively unexplored. Enzyme-directed post-translational modifications are responsible for the functional diversity of the proteome and, thus, could be harnessed to selectively modify artificial protein assemblies. In this study, we describe the use of phosphopantetheinyl transferases (PPTases), a class of enzymes that covalently modify proteins using coenzyme A (CoA), to site-selectively tailor the surface of designed, two-dimensional (2D) protein crystals. We demonstrate that a short peptide (ybbR) or a molecular tag (CoA) can be covalently tethered to 2D arrays to enable enzymatic functionalization using Sfp PPTase. The site-specific modification of two different protein array platforms is facilitated by PPTases to afford both small molecule- and protein-functionalized surfaces with no loss of crystalline order. This work highlights the potential for chemoenzymatic modification of large protein surfaces toward the generation of sophisticated protein platforms reminiscent of the complex landscape of cell surfaces.

The discovery of potential phosphopantetheinyl transferase Ppt2 inhibitors against drug-resistant Candida albicans.

With the high-frequency use or abuse of antifungal drugs, the crisis of drug-resistant fungi continues to increase worldwide; in particular, the infection of drug-resistant Candida albicans brings the great challenge to the clinical treatment. Therefore, to decelerate the spread of this resistance, it is extremely urgent to facilitate the new antifungal targets with novel drugs. Phosphopantetheinyl transferases PPTases (Ppt2 in Candida albicans) had been identified in bacterium and fungi and mammals, effects as a vital enzyme in the metabolism of organisms in C. albicans. Ppt2 transfers the phosphopantetheinyl group of coenzyme A to the acyl carrier protein Acp1 in mitochondria for the synthesis of lipoic acid that is essential for fungal respiration, so making Ppt2 an ideal target for antifungal drugs. In this study, 110 FDA-approved drugs were utilized to investigate the Ppt2 inhibition against drug-resistant Candida albicans by the improved fluorescence polarization experiments, which have enough druggability and structural variety under the novel strategy of drug repurposing. Thereinto, eight agents revealed the favourable Ppt2 inhibitory activities. Further, broth microdilution assay of incubating C. albicans with these eight drugs showed that pterostilbene, procyanidine, dichlorophen and tea polyphenol had the superior MIC values. In summary, these findings provide more valuable insight into the treatment of drug-resistant C. albicans.

Understanding the Structure and Function of Type I Acyl Carrier Protein and its Interaction with Growing Acyl Chain

Fatty acids are central to the formation of biological membranes and play important roles in metabolism. There are two major classes of fatty acid synthases (FAS); Type I present in mammals and yeast that utilizes large multifunctional enzyme complexes, while type II, harbored by prokaryotes and plants, utilizing discrete monofunctional enzymes. Acyl Carrier Protein (ACP) is a universal and highly conserved player in both the pathways. ACP is modified from apo to holo form by enzyme 4′‐Phosphopantetheinyl Transferase (PPT). PPT enzyme is crucial for growth of many pathogens and is therefore regarded as a potential drug target. Structural and biochemical studies on type II ACP highlight various aspects of ACP‐PPT and ACP‐acyl chain interaction. However, acyl chain binding and dynamic interactions of type I ACP are not well understood. We therefore intend to understand the function of type I ACP, their interaction with bound acyl chain and cognate and non cognate transferases. Literature highlights the importance of type I FAS in the survival and pathogenicity of fungal pathogens like C. albicans and C. parasilopsis. For our studies, we employed type I ACP from Saccharomyces cereviceae and its putative PPT enzyme. Using NMR spectroscopy we show that the growing acyl chain was sequestered up to a certain carbon chain length in the hydrophobic core of the protein employing a unique motif. In conclusion, our studies provide some interesting insights into the structure and function of type I ACP and the molecular mechanism it employs to protect the growing acyl chain. Our findings can provide new avenues for structure based drug designing.Support or Funding InformationDepartment of Biotechnology, India

Conformational flexibility of coenzyme A and its impact on the post-translational modification of acyl carrier proteins by 4'-phosphopantetheinyl transferases.

One central question surrounding the biosynthesis of fatty acids and polyketide-derived natural products is how the 4'-phosphopantetheinyl transferase (PPTase) interrogates the essential acyl carrier protein (ACP) domain to fulfill the initial activation step. The triggering factor of this study was the lack of structural information on PPTases at physiological pH, which could bias our comprehension of the mechanism of action of these important enzymes. Structural and functional studies on the family II PPTase PptAb of Mycobacteriumabscessus show that pH has a profound effect on the coordination of metal ions and on the conformation of endogenously bound coenzyme A (CoA). The observed conformational flexibility of CoA at physiological pH is accompanied by a disordered 4'-phosphopantetheine (Ppant) moiety. Finally, structural and dynamical information on an isolated mycobacterial ACP domain, in its apo form and in complex with the activator PptAb, suggests an alternate mechanism for the post-translational modification of modular megasynthases.

Proximicins F and G and Diproximicin A: Aminofurans from the Marine-Derived Verrucosispora sp. SCSIO 40062 by Overexpression of PPtase Genes.

Overexpression of phosphopantetheinyl transferase (PPtase)-encoding genes sfp and svp in the marine-derived Verrucosispora sp. SCSIO 40062 led to the production of two new aminofuran monomers, proximicin F (1) and proximicin G (3) and a new dimer diproximicin A (2), along with two known compounds, proximicins B (4) and C (5). Their structures were unambiguously elucidated on the basis of detailed NMR spectroscopic analysis and high-resolution electrospray ionization mass spectrometry (HRESIMS) data. Proximicin B (4) showed moderate antibacterial activities against Staphylococcus aureus, methicillin-resistant S. aureus, and Bacillus subtilis.

Efficient production of surfactin from xylose-rich corncob hydrolysate using genetically modified Bacillus subtilis 168.

As one of the most powerful biosurfactants, surfactin has extensive application prospects in numerous industrial fields. Bacillus subtilis 168 was genetically modified to produce surfactin by increasing the supply of the precursor fatty acyl-CoA by overexpressing 4' phosphopantetheinyl transferase, medium-chain acyl-acyl carrier protein (ACP) thioesterase and fatty acyl CoA ligase (encoded by sfp, bte, and yhfL, respectively), and knocking out acyl-CoA dehydrogenase (encoded by fadE). The resulting recombinant strain BSFX022 produced 2203mg/L surfactin with xylose as carbon source. The lower accumulation of organic acids with xylose as carbon source made it possible to maintain surfactin production in a non-buffered fermentation system, and the yield reached 2074mg/L. Furthermore, to reduce the costs, waste biomass such as corncob hydrolysate and monosodium glutamate wastewater (MGW) were used, and 2032mg/L of surfactin was produced in the optimal waste-based medium. To our best knowledge, this is the first report of surfactin production using genetically modified Bacillus subtilis 168 with xylose as carbon source.

Silencing of a phosphopantetheinyl transferase gene Ss-Ppt1 affects multiple developmental pathways and pathogenicity in Sclerotinia sclerotiorum

In the genome sequence of the fungal pathogen Sclerotinia sclerotiorum, a phosphopantetheinyl transferase gene homolog Ss-Ppt1 (Accession XM_ 001596461.1) with 1 SCOP and 1 ACPS module was identified. And the transcription of Ss-Ppt1 was increased significantly at the sclerotial maturation stage. Silenced mutant strains of Ss-Ppt1 were generated by RNA interference method. The mutant strains were screened and characterized. Compared to the wild-type strain, the hyphal growth of the mutant strains was significantly decreased and the branching pattern was altered. Aberrant sclerotia were produced by the mutant strains with larger volume but reduced number. Moreover, the virulence of the mutant strains was attenuated, and the mutant strains failed to generate necrotic disease symptoms as the wide-type strain. These results indicated that Ss-Ppt1 protein plays an important role in hyphal growth, sclerotial development and pathogenicity of S. sclerotiorum.

Structural insights into phosphopantetheinyl hydrolase PptH from Mycobacterium tuberculosis.

The amidinourea 8918 was recently reported to inhibit the type II phosphopantetheinyl transferase (PPTase) of Mycobacterium tuberculosis (Mtb), PptT, a potential drug-target that activates synthases and synthetases involved in cell wall biosynthesis and secondary metabolism. Surprisingly, high-level resistance to 8918 occurred in Mtb harboring mutations within the gene adjacent to pptT, rv2795c, highlighting the role of the encoded protein as a potentiator of the bactericidal action of the amidinourea. Those studies revealed that Rv2795c (PptH) is a phosphopantetheinyl (PpT) hydrolase, possessing activity antagonistic with respect to PptT. We have solved the crystal structure of Mtb's phosphopantetheinyl hydrolase, making it the first phosphopantetheinyl (carrier protein) hydrolase structurally characterized. The 2.5 Å structure revealed the hydrolases' four-layer (α/β/β/α) sandwich fold featuring a Mn-Fe binuclear center within the active site. A structural similarity search confirmed that PptH most closely resembles previously characterized metallophosphoesterases (MPEs), particularly within the vicinity of the active site, suggesting that it may utilize a similar catalytic mechanism. In addition, analysis of the structure has allowed for the rationalization of the previously reported PptH mutations associated with 8918-resistance. Notably, differences in the sequences and predicted structural characteristics of the PpT hydrolases PptH of Mtb and E. coli's acyl carrier protein hydrolase (AcpH) indicate that the two enzymes evolved convergently and therefore are representative of two distinct PpT hydrolase families.

Labeling surface proteins with high specificity: Intrinsic limitations of phosphopantetheinyl transferase systems.

ObjectiveFluorescent labeling of specific cell-surface proteins enables a manifold of techniques to study their function in health and disease. A frequently cited family of methods employs phosphopantetheinyl transferases (PPTases) to attach probes, provided as conjugates of Coenzyme A. This method appears attractive, as only short peptide tags genetically fused to the protein of interest are needed as conjugation sites. Here, we describe observations we made when evaluating such protocols for delicate single-molecule applications where we require a particular combination of dyes, low background binding or low labeling of other proteins, and a high degree of labeling.ResultsWhen we tested a PPTase-acceptor peptide couple with several experimental protocols and various CoA conjugates for labeling of a protein on the cell surface, we noticed substantial non-specific labeling. For the first time, we provide here a quantification of the non-specific fraction of the signals obtained using appropriate controls. We further present evidence that this background is due to CoA-dye conjugates entering the cell, where they may be covalently attached to endogenous proteins. However, when studying cell-surface proteins, most fluorescent readouts require that labeling is strictly limited to the protein of interest located at the cell surface. While such data have so far been missing in the literature, they suggest that for applications where labeling of unwanted molecules would affect the conclusions, researchers need to be aware of this potential non-specificity of PPTase methods when selecting a labeling strategy. We show, again by quantitative comparison, that the HaloTag is a viable alternative.

Microbial synthesis of the type I polyketide 6-methylsalicylate with Corynebacterium glutamicum.

Type I polyketide synthases (PKSs) are large multi-domain proteins converting simple acyl-CoA thioesters such as acetyl-CoA and malonyl-CoA to a large diversity of biotechnologically interesting molecules. Such multi-step reaction cascades are of particular interest for applications in engineered microbial cell factories, as the introduction of a single protein with many enzymatic activities does not require balancing of several individual enzymatic activities. However, functional introduction of type I PKSs into heterologous hosts is very challenging as the large polypeptide chains often do not fold properly. In addition, PKS usually require post-translational activation by dedicated 4'-phosphopantetheinyl transferases (PPTases). Here, we introduce an engineered Corynebacterium glutamicum strain as a novel microbial cell factory for type I PKS-derived products. Suitability of C. glutamicum for polyketide synthesis could be demonstrated by the functional introduction of the 6-methylsalicylic acid synthase ChlB1 from Streptomyces antibioticus. Challenges related to protein folding could be overcome by translation fusion of ChlB1Sa to the C-terminus of the maltose-binding protein MalE from Escherichia coli. Surprisingly, ChlB1Sa was also active in the absence of a heterologous PPTase, which finally led to the discovery that the endogenous PPTase PptACg of C. glutamicum can also activate ChlB1Sa. The best strain, engineered to provide increased levels of acetyl-CoA and malonyl-CoA, accumulated up to 41mg/L (0.27mM) 6-methylsalicylic acid within 48h of cultivation. Further experiments showed that PptACg of C. glutamicum can also activate nonribosomal peptide synthetases (NRPSs), rendering C. glutamicum a promising microbial cell factory for the production of several fine chemicals and medicinal drugs.

Heterologous Expression of Sfp-Type Phosphopantetheinyl Transferase is Indispensable in the Biosynthesis of Lipopeptide Biosurfactant.

Phosphopantetheinyl transferases are of tremendous enthusiasm inferable from their fundamental parts in activating polyketide, fatty acid, and non-ribosomal peptide synthetase enzymes and additionally an increasing number of biotechnological applications. The present study reports the identification of sfp gene from the Paenibacillus sp. D9, which encompasses 693bp encoding a 230-amino acid protein with a molecular weight of 25.3kDa. The amino acid sequence Paenibacillus sp. D9 Sfp revealed more than 90% sequence identity to other Sfp proteins from other Paenibacillus. The sfp gene was cloned and recovered efficiently using affinity chromatography with maximal specific phosphopantetheinyl transferase activity at an optimal pH of 8.0 and temperature of 30°C. The enzyme also exhibited stability under a wide-ranging pH and temperature. The presence of Zn2+, Cu2+, and Fe2+ ions improved the enzymatic activity, while other metals such as Ni2+, Co2+, and Mg2+ had inhibitory effects. The introduction of EDTA also displayed no inhibition. Kinetic parameters were obtained having values of 4.52mg/mL, 35.33 U/mg, 3.64s-1, and 0.104mM-1 s-1 for Km, Vmax, kcat, and kcat/Km, respectively. The biosurfactant synthesized by the recombinant BioSp was found to be surface active, reducing the surface tension to 33.7 mN/m on the glucose substrate after 5days of incubation at 37°C. The recombinant Escherichia coli strain also exhibited an improvement in biosurfactant yield (1.11g/L) when contrasted with 0.52g/L from Paenibacillus sp. D9. High esterase activity of 2.55IU/mL using p-nitrophenyl acetate was observed on the recombinant strain, as the protein connected with the release of the biosurfactant was observed to be an esterase. The characteristics of improved biosurfactant and esterase synthesis by hyper-producing recombinant strain possess numerous values from biotechnology standpoint.

De novo production of aromatic m-cresol in Saccharomyces cerevisiae mediated by heterologous polyketide synthases combined with a 6-methylsalicylic acid decarboxylase

As a flavor and platform chemical, m-cresol (3-methylphenol) is a valuable industrial compound that currently is mainly synthesized by chemical methods from fossil resources. In this study, we present the first biotechnological de novo production of m-cresol from sugar in complex yeast extract-peptone medium with the yeast Saccharomyces cerevisiae. A heterologous pathway based on the decarboxylation of the polyketide 6-methylsalicylic acid (6-MSA) was introduced into a CEN.PK yeast strain. For synthesis of 6-MSA, expression of different variants of 6-MSA synthases (MSASs) were compared. Overexpression of codon-optimized MSAS from Penicillium patulum together with activating phosphopantetheinyl transferase npgA from Aspergillus nidulans resulted in up to 367 mg/L 6-MSA production. Additional genomic integration of the genes had a strongly promoting effect and 6-MSA titers reached more than 2 g/L. Simultaneous expression of 6-MSA decarboxylase patG from A. clavatus led to the complete conversion of 6-MSA and production of up to 589 mg/L m-cresol. As addition of 450–750 mg/L m-cresol to yeast cultures nearly completely inhibited growth our data suggest that the toxicity of m-cresol might be the limiting factor for higher production titers.

Modular type I polyketide synthase acyl carrier protein domains share a common N-terminally extended fold

Acyl carrier protein (ACP) domains act as interaction hubs within modular polyketide synthase (PKS) systems, employing specific protein-protein interactions to present acyl substrates to a series of enzyme active sites. Many domains from the multimodular PKS that generates the toxin mycolactone display an unusually high degree of sequence similarity, implying that the few sites which vary may do so for functional reasons. When domain boundaries based on prior studies were used to prepare two isolated ACP segments from this system for studies of their interaction properties, one fragment adopted the expected tertiary structure, but the other failed to fold, despite sharing a sequence identity of 49%. Secondary structure prediction uncovered a previously undetected helical region (H0) that precedes the canonical helix-bundle ACP topology in both cases. This article reports the NMR solution structures of two N-terminally extended mycolactone mACP constructs, mH0ACPa and mH0ACPb, both of which possess an additional α-helix that behaves like a rigid component of the domain. The interactions of these species with a phosphopantetheinyl transferase and a ketoreductase domain are unaffected by the presence of H0, but a shorter construct that lacks the H0 region is shown to be substantially less thermostable than mH0ACPb. Bioinformatics analysis suggests that the extended H0-ACP motif is present in 98% of type I cis-acyltransferase PKS chain-extension modules. The polypeptide linker that connects an H0-ACP motif to the preceding domain must therefore be ~12 residues shorter than previously thought, imposing strict limits on ACP-mediated substrate delivery within and between PKS modules.

Opposing reactions in coenzyme A metabolism sensitize Mycobacterium tuberculosis to enzyme inhibition.

Mycobacterium tuberculosis (Mtb) is the leading infectious cause of death in humans. Synthesis of lipids critical for Mtb's cell wall and virulence depends on phosphopantetheinyl transferase (PptT), an enzyme that transfers 4'-phosphopantetheine (Ppt) from coenzyme A (CoA) to diverse acyl carrier proteins. We identified a compound that kills Mtb by binding and partially inhibiting PptT. Killing of Mtb by the compound is potentiated by another enzyme encoded in the same operon, Ppt hydrolase (PptH), that undoes the PptT reaction. Thus, loss-of-function mutants of PptH displayed antimicrobial resistance. Our PptT-inhibitor cocrystal structure may aid further development of antimycobacterial agents against this long-sought target. The opposing reactions of PptT and PptH uncover a regulatory pathway in CoA physiology.

A wrench in the gears of life

Tuberculosis Tuberculosis is a global health crisis that threatens to become worse as resistance to existing drugs emerges. Identifying ideal targets for drug development requires knowledge of weak points in biochemical pathways that are specific for the pathogen but are absent in hosts. Ballinger et al. identified a small molecule that inhibits the enzyme phosphopantetheinyl transferase (PptT), which is crucial for biosynthesis of mycobacterial structural and virulence lipids (see the Perspective by Mizrahi and Warner). Treatment resulted in selective killing of the bacteria in vitro and in a mouse model. The target pathways were made sensitive to PptT inhibition by a second enzyme, phosphopantetheine hydrolase, whose activity opposes that of the transferase. Science , this issue p. [eaau8959][1]; see also p. [457][2] [1]: /lookup/doi/10.1126/science.aau8959 [2]: /lookup/doi/10.1126/science.aaw5224

Biosynthesis in vitro of bacillamide intermediate-heterocyclic AlaCysthiazole by heterologous expression of nonribosomal peptide synthetase (NRPS)

Bacillamide C, a potential natural antialgae active compound, is produced by Bacillus atrophaeus C89 derived from marine sponge Dysidea avara. A nonribosomal peptide synthetase (NRPS) cluster is hypothesized to be involved in the biosynthesis of bacillamide C. The NRPS with a domain string of A1-PCP1-Cy-A2-PCP2-C can be divided into three functional modules. After heterologous expression and purification of module A1-PCP1 and module Cy-A2-PCP2, their catalytic activities were biochemically proven in vitro by the reaction with the apo-PCP domain transformed to the holo-PCP domain through a phosphopantetheinyl transferase, ATP, and substrate amino acids. Five– membered heterocyclic AlaCysthiazole with molecular weight of 172.0389 was detected. This proved the formation of the heterocyclic dipeptide AlaCysthiazole, which is considered to be a building block for the biosynthesis of bacillamide. This study provides a basis for further biosynthesis of bacillamides.

Site-Specific Antibody Labeling Using Phosphopantetheinyl Transferase-Catalyzed Ligation.

4'-Phosphopantetheinyl transferases (PPTases) have been employed by researchers as versatile biocatalysts for the site-specific modification of numerous protein targets with structurally diverse molecules. Here we describe the use of these enzymes for the production of homogeneous antibody-drug conjugates (ADCs), which have garnered much attention as innovative anticancer drugs. The exceptionally broad substrate tolerance of PPTases allows for one-step and two-step conjugation strategies for site-specific ADC synthesis. While one-step conjugation involves direct coupling of a drug molecule to an antibody, two-step conjugation provides increased flexibility and efficiency of the conjugation process by first attaching a bioorthogonal chemical handle that is then used for drug molecule attachment in a second step. The aim of this chapter is to outline detailed protocols for both labeling procedures, as well as to provide guidance on enzyme and substrate preparation.