Computationally Designed Epitope-Mediated Imprinted Polymers versus Conventional Epitope Imprints for the Detection of Human Adenovirus in Water and Human Serum Samples

Detection of pathogenic viruses for point-of-care applications has attracted great attention since the COVID-19 pandemic. Current virus diagnostic tools are laborious and expensive, while requiring medically trained staff. Although user-friendly and cost-effective biosensors are utilized for virus detection, many of them rely on recognition elements that suffer major drawbacks. Herein, computationally designed epitope-imprinted polymers (eIPs) are conjugated with a portable piezoelectric sensing platform to establish a sensitive and robust biosensor for the human pathogenic adenovirus (HAdV). The template epitope is selected from the knob part of the HAdV capsid, ensuring surface accessibility. Computational simulations are performed to evaluate the conformational stability of the selected epitope. Further, molecular dynamics simulations are executed to investigate the interactions between the epitope and the different functional monomers for the smart design of eIPs. The HAdV epitope is imprinted via the solid-phase synthesis method to produce eIPs using in silico-selected ingredients. The synthetic receptors show a remarkable detection sensitivity (LOD: 102 pfu mL–1) and affinity (dissociation constant (Kd): 6.48 × 10–12 M) for HAdV. Moreover, the computational eIPs lead to around twofold improved binding behavior than the eIPs synthesized with a well-established conventional recipe. The proposed computational strategy holds enormous potential for the intelligent design of ultrasensitive imprinted polymer binders

Aromatic <i>C-</i>Methyltransferases with Antipodal Stereoselectivity for Structurally Diverse Phenolic Amino Acids Catalyze the Methylation Step in the Biosynthesis of the Actinomycin Chromophore

The actinomycin biosynthetic gene cluster of Streptomyces chrysomallus harbors two paralogous genes, acmI and acmL, encoding methyltransferases. To unveil their suspected role in the formation of 3-hydroxy-4-methyl-anthranilic acid (4-MHA), the building block of the actinomycin chromophore, each gene was expressed in Escherichia coli. Testing the resulting ∼40 kDa His6-tagged proteins with compounds of biogenetic relevance as substrates and S-adenosyl-l-methionine revealed that each exclusively methylated 3-hydroxykynurenine (3-HK) with formation of 3-hydroxy-4-methylkynurenine (4-MHK) identified by its in vitro conversion to 4-MHA with hydroxykynureninase. AcmI and AcmL methylate also hydroxyphenyl-amino propanoic acids such as p-tyrosine, m-tyrosine, or 3,4-dihydroxy-l-phenylalanine (DOPA) but at a lower rate than 3-HK. The presence of the α-amino group was necessary for substrate recognition. Phenolic acids with shorter chains such as 4-hydoxyphenyl-l-glycine (HPG), 3-hydroxybenzoic acid (3-HB), or 3-hydroxyanthranilic acid (3-HA) gave no product. Both enzymes were stereospecific for the optical configuration at α-C with unprecedented antipodal selectivity for the d-enantiomer of 3-HK and the l-enantiomer of p-tyrosine or m-tyrosine. AcmI and AcmL show sequence similarity to various C- and O-methyltransferases from bacteria. Phylogenetic analysis places them into the clade of C-methyltransferases comprising among others orthologues involved in 4-MHA formation of other biosynthesis systems and methyltransferases putatively involved in the C-methylation of tyrosine. Remarkably, computational remodelling of AcmI and AcmL structures revealed significant similarity with the 3-D structures of type 1 O-methyltransferases from plants such as caffeic acid O-methyltransferase (COMT) and other phenylpropanoid methyltransferases. The relevance of 3-HK or 3-HA methylation in the actinomycin biosynthesis pathways of different actinomycetes is discussed

Cytochrome P450_sky Interacts Directly with the Nonribosomal Peptide Synthetase to Generate Three Amino Acid Precursors in Skyllamycin Biosynthesis

The generation of modified amino acid precursors for incorporation in nonribosomal peptide synthesis (NRPS) plays a crucial, if often understated, role in the generation of peptide natural products. The biosynthesis of the cyclic depsipeptide skyllamycin requires three β-hydroxylated amino acid precursors, with <i>in vivo</i> gene inactivation experiments implicating cytochrome P450_sky (CYP163B3) in the hydroxylation of these amino acids. Here, we demonstrate the <i>in vitro</i> oxidation of l-amino acid substrates bound to peptidyl carrier protein (PCP) domains 5, 7, and 11 of the skyllamycin nonribosomal synthetase by P450_sky. Selectivity for these domains over other PCP domains could be demonstrated, with hydroxylation selective for l-amino acids and stereospecific in nature resulting in the (2<i>S</i>,3<i>S</i>)-configuration. The oxidation of amino acids or small molecule substrate analogues was not supported, demonstrating the necessity of the carrier protein in P450_sky-catalyzed hydroxylation. The binding of aminoacyl-PCP substrates to P450_sky was detected for the catalytically active PCP₇ but not for the catalytically inactive PCP₁₀, indicating carrier protein-mediated selectivity in P450_sky substrate binding. X-ray crystal structures of P450_sky reveal a 3D-structure with a highly open active site, the size of which is dictated by the carrier protein bound nature of the substrate. P450_sky is the first P450 demonstrated to not only interact directly with PCP-bound amino acids within the peptide-forming NRPS but also to do so with three different PCP domains in a specific fashion. This represents an expansion of the complexity and scope of NRPS-mediated peptide synthesis, with the generation of hydroxylated amino acid precursors occurring through the interaction of P450 enzymes following, rather than prior to, the selection of amino acids by NRPS-adenylation domains

Venom Proteomics of Indonesian King Cobra, <i>Ophiophagus hannah</i>: Integrating Top-Down and Bottom-Up Approaches

We report on the first application of top-down mass spectrometry in snake venomics. <i>De novo</i> sequence tags generated by, and ProSight Lite supported analysis of, combined collisional based dissotiations (CID and HCD) recorded in a hybrid LTQ Orbitrap instrument in data-dependent mode identified a number of proteins from different toxin families, namely, 11 three-finger toxins (7–7.9 kDa), a Kunitz-type inhibitor (6.3 kDa), ohanin (11.9 kDa), a novel phospholipase A₂ molecule (13.8 kDa), and the cysteine-rich secretory protein (CRISP) ophanin (25 kDa) from Indonesian king cobra venom. Complementary bottom-up MS/MS analyses contributed to the completion of a locus-resolved venom phenotypic map for Ophiophagus hannah, the world’s longest venomous snake and a species of medical concern across its wide distribution range in forests from India to Southeast Asia. Its venom composition, comprising 32–35 proteins/peptides from 10 protein families, is dominated by α-neurotoxins and convincingly explains the main neurotoxic effects of human envenoming caused by king cobra bite. The integration of efficient chromatographic separation of the venom’s components and locus-resolved toxin identification through top-down and bottom-up MS/MS-based species-specific database searching and <i>de novo</i> sequencing holds promise that the future will be bright for the field of venom research

Deuterium Labeled Peptides Give Insights into the Directionality of Class III Lantibiotic Synthetase LabKC

The biosynthesis of a considerable number of ribosomally synthesized peptide antibiotics involves the modification of Ser and Thr residues of a precursor peptide. This post-translational processing is performed by one or multiple modifying enzymes encoded in the biosynthetic gene cluster. We present a deuterium-label based enzyme assay, utilizing a series of peptide substrates with α-deuterated Ser, for the determination of the dehydration order during the biosynthesis of class III lantibiotic labyrinthopeptin A2. Remarkably, the data show that, in contrast to other modifying enzymes of class I and II lantibiotics, LabKC has a C- to N-terminal processing mode. This surprising finding, which we consider relevant for the biosyntheses of other class III lantibiotics, underlines significant differences of this class of modifying enzymes compared to other investigated systems

Reconstituted Biosynthesis of the Nonribosomal Macrolactone Antibiotic Valinomycin in <i>Escherichia coli</i>

The structural complexity of nonribosomal peptides (NRPs) impeding economic chemical synthesis and poor cultivability of source organisms limits the development of bioprocesses for novel bioactive compounds. Since nonribosomal peptide synthetases (NRPSs) assemble NRPs from simple amino acid building blocks, heterologous expression of NRPSs in a robust and easy to manipulate expression host is an attractive strategy to make pharmaceutically relevant NRPs more accessible and is also a basis for engineering of these enzymes to generate novel synthetic bioactive compounds. Here we show a systematic approach for the heterologous expression of the 654 kDa heterodimeric valinomycin synthetase (VlmSyn) from <i>Streptomyces tsusimaensis</i> in a soluble and active form in <i>Escherichia coli</i>. VlmSyn activity and precursor requirements were determined <i>in vitro</i> and provided evidence for a previously proposed model of valinomycin biosynthesis. <i>In vivo</i> production of recombinant valinomycin, a macrolactone antibiotic with reported antifungal, antibacterial, and antiviral activities, was achieved using an engineered <i>E. coli</i> strain growing in inexpensive media and independent of the supplementation with precursors and further optimization of the cultivation conditions. Tailoring of VlmSyn in <i>E. coli</i> paves the way to the production of novel valinomycin analogues in the future

2-Amino-2-deoxyisochorismate Is a Key Intermediate in <i>Bacillus subtilis p-</i>Aminobenzoic Acid Biosynthesis

2-Amino-2-deoxyisochorismate Is a Key Intermediate in Bacillus subtilis p-Aminobenzoic Acid Biosynthesi

Venom Proteomics of Indonesian King Cobra, <i>Ophiophagus hannah</i>: Integrating Top-Down and Bottom-Up Approaches

We report on the first application of top-down mass spectrometry in snake venomics. <i>De novo</i> sequence tags generated by, and ProSight Lite supported analysis of, combined collisional based dissotiations (CID and HCD) recorded in a hybrid LTQ Orbitrap instrument in data-dependent mode identified a number of proteins from different toxin families, namely, 11 three-finger toxins (7–7.9 kDa), a Kunitz-type inhibitor (6.3 kDa), ohanin (11.9 kDa), a novel phospholipase A₂ molecule (13.8 kDa), and the cysteine-rich secretory protein (CRISP) ophanin (25 kDa) from Indonesian king cobra venom. Complementary bottom-up MS/MS analyses contributed to the completion of a locus-resolved venom phenotypic map for Ophiophagus hannah, the world’s longest venomous snake and a species of medical concern across its wide distribution range in forests from India to Southeast Asia. Its venom composition, comprising 32–35 proteins/peptides from 10 protein families, is dominated by α-neurotoxins and convincingly explains the main neurotoxic effects of human envenoming caused by king cobra bite. The integration of efficient chromatographic separation of the venom’s components and locus-resolved toxin identification through top-down and bottom-up MS/MS-based species-specific database searching and <i>de novo</i> sequencing holds promise that the future will be bright for the field of venom research

Elaiomycins B and C: Alkylhydrazide Antibiotics from <i>Streptomyces</i> sp. BK 190

Two novel alkyhydrazides, elaiomycins B and C, together with the azoxy antibiotic elaiomycin were isolated from Streptomyces sp. BK 190. The structures were established by 1D- and 2D-NMR spectroscopy including 15N NMR studies and high-resolution orbitrap-ESI-mass spectrometry