Amide N-glycosylation by Asm25, an N-glycosyltransferase of ansamitocins

Ansamitocins are potent antitumor maytansinoids produced by Actinosynnema pretiosum. Their biosynthesis involves the initial assembly of a macrolactam polyketide, followed by a series of postpolyketide synthase (PKS) modifications. Three ansamitocin glycosides were isolated from A. pretiosum and fully characterized structurally as novel ansamitocin derivatives, carrying a beta-D-glucosyl group attached to the macrolactam amide nitrogen in place of the N-methyl group. By gene inactivation and complementation, asm25 was identified as the N-glycosyltransferase gene responsible for the macrolactam amide N-glycosylation of ansamitocins. Soluble, enzymatically active Asm25 protein was obtained from asm25-expressing E. coli by solubilization from inclusion bodies. Its optimal reaction conditions, including temperature, pH, metal ion requirement, and Km/Kcat, were determined. Asm25 also showed broad substrate specificity toward other ansamycins and synthetic indolin-2-ones. To the best of our knowledge, this represents the first in vitro characterization of a purified antibiotic N-glycosyltransferase.     

Analysis of the ambruticin and jerangolid gene clusters of Sorangium cellulosum reveals unusual mechanisms of polyketide biosynthesis

Ambruticins and jerangolids are structurally related antifungal polyketides produced by Sorangium cellulosum strains. Comparative analysis of the gene clusters and characterization of compounds produced by gene knockout strains suggested hypothetical schemes for biosynthesis of these compounds. Polyketide synthase (PKS) architecture suggests that the pyran ring structure common to ambruticins and jerangolids forms by an intramolecular reaction on a PKS-bound intermediate. Disrupting ambM, encoding a discrete enzyme homologous to PKS C-methyltransferase domains, gave 15-desmethylambruticins. Thus, AmbM is required for C-methylation, but not pyran ring formation. Several steps in the post-PKS modification of ambruticin involve new enzymology. Remarkably, the methylcyclopropane ring and putative carbon atom excision during ambruticin biosynthesis apparently occur on the PKS assembly line. The mechanism probably involves a Favorskii rearrangement, but further work is required to elucidate these complex events.     

A squalene epoxidase is involved in biosynthesis of both the antitumor compound clavaric acid and sterols in the basidiomycete H. sublateritium

The basidiomycete Hypholoma sublateritium produces the triterpenoid antitumor clavaric acid, an inhibitor of the human Ras-farnesyl transferase. The H. sublateritium squalene epoxidase gene (erg1) has been cloned and shown to encode a flavoprotein monooxygenase that requires FAD, NADPH, and P450 cofactors. Silencing of the erg1 gene in H. sublateritium using constructions expressed from the gdh promoter of Agaricus bisporus showed that the squalene epoxidase is involved in clavaric acid formation and in ergosterol biosynthesis; silenced expression of erg1 resulted in an ergosterol-dependent phenotype for full growth. Overexpression of erg1 gene resulted in up to 32% to 97% increment of clavaric acid production confirming its involvement in the biosynthesis of this antitumor product. Oxidosqualene (or dioxidosqualene) appears to be the branching point for primary metabolism (sterols) and secondary metabolites in basidiomycetes.     

Synthesis of polyester by means of genetic code reprogramming

Here we report the ribosomal polymerization of alpha-hydroxy acids by means of genetic code reprogramming. The flexizyme system, a ribozyme-based tRNA acylation tool, was used to re-assign individual codons to seven types of alpha-hydroxy acids, and then polyesters were synthesized under controls of the reprogrammed genetic code using a reconstituted cell-free translation system. The sequence and length of the polyester segments were specified by the mRNA template, indicating that high-fidelity ribosome expression of polyesters was possible. This work opens a door for the mRNA-directed synthesis of backbone-altered biopolymers.     

DKxanthene biosynthesis--understanding the basis for diversity-oriented synthesis in myxobacterial secondary metabolism

The DKxanthenes are a family of yellow pigments which play a critical role in myxobacterial development. Thirteen unique structures from Myxococcus xanthus DK1622 differ in the length of their characteristic polyene functionality, as well as the extent of methyl branching. We aimed to understand the mechanistic basis for this "molecular promiscuity" by analyzing the gene cluster in DK1622, and comparing it to the DKxanthene biosynthetic locus in a second myxobacterium, Stigmatella aurantiaca DW4/3-1, which produces a more limited range of compounds. While the core biosynthetic machinery is highly conserved, M. xanthus contains a putative asparagine hydroxylase function which is not present in S. aurantiaca. This observation accounts, in part, for the significantly larger metabolite family in M. xanthus. Detailed analysis of the encoded hybrid polyketide synthase (PKS)-nonribosomal peptide synthetase (NRPS) assembly line provides direct evidence for the mechanism underlying the variable polyene length and the observed pattern of methyl functionalities.     

Loop grafting of Bacillus subtilis lipase A: inversion of enantioselectivity

Lipases are successfully applied in enantioselective biocatalysis. Most lipases contain a lid domain controlling access to the active site, but Bacillus subtilis Lipase A (LipA) is a notable exception: its active site is solvent exposed. To improve the enantioselectivity of LipA in the kinetic resolution of 1,2-O-isopropylidene-sn-glycerol (IPG) esters, we replaced a loop near the active-site entrance by longer loops originating from Fusarium solani cutinase and Penicillium purpurogenum acetylxylan esterase, thereby aiming to increase the interaction surface for the substrate. The resulting loop hybrids showed enantioselectivities inverted toward the desired enantiomer of IPG. The acetylxylan esterase-derived variant showed an inversion in enantiomeric excess (ee) from -12.9% to +6.0%, whereas the cutinase-derived variant was improved to an ee of +26.5%. The enantioselectivity of the cutinase-derived variant was further improved by directed evolution to an ee of +57.4%.     

Gain-of-function mutational activation of human tRNA synthetase procytokine

Disease-causing mutations occur in genes for aminoacyl tRNA synthetases. That some mutations are dominant suggests a gain of function. Native tRNA synthetases, such as tyrosyl-tRNA synthetase (TyrRS) and tryptophanyl-tRNA synthetase, catalyze aminoacylation and are also procytokines that are activated by natural fragmentation. In principle, however, gain-of-function phenotypes could arise from mutational activation of synthetase procytokines. From crystal structure analysis, we hypothesized that a steric block of a critical Glu-Leu-Arg (ELR) motif in full-length TyrRS suppresses the cytokine activity of a natural fragment. To test this hypothesis, we attempted to uncover ELR in the procytokine by mutating a conserved tyrosine (Y341) that tethers ELR. Site-specific proteolytic cleavage and small-angle X-ray scattering established subtle opening of the structure by the mutation. Strikingly, four different assays demonstrated mutational activation of cytokine functions. The results prove the possibilities for constitutive gain-of-function mutations in tRNA synthetases.     

Tryptophan aminopeptidase activity of several indole prenyltransferases from Aspergillus fumigatus

Recently, five indole prenyltransferases from Aspergillus fumigatus have been proven biochemically to be responsible for prenylations of diverse substrates. In this study, we show peptidase activities of 7-DMATS, FgaPT1, CdpNPT, and FtmPT1, with preference for linear peptides containing a tryptophanyl moiety at the N terminus. Testing of 31 peptides revealed that these enzymes shared similar substrate specificity and accepted H-L-Trp-L-Ala-OH and H-L-Trp-Gly-OH as best substrates for aminopeptidase activity. By using H-L-Trp-Gly-OH as substrate, Km values at 350, 380, 300, and 420 microM and enzymatic rate constants kcat/Km at 0.51, 0.24, 0.53, and 0.14 mM(-1)s(-1) were determined for 7-DMATS, FgaPT1, CdpNPT, and FtmPT1, respectively. In contrast to prenyltransferase activities, the aminopeptidase activities were strongly or completely inhibited by EDTA. Mn2+ increased the aminopeptidase activities of FtmPT1 and CdpNPT up to 4- and 6-fold, respectively. To the best of our knowledge, this is the first report on the catalytic promiscuity of prenyltransferases.     

Catalytic residues are shared between two pseudosubunits of the dehydratase domain of the animal fatty acid synthase

Expression, characterization, and mutagenesis of a series of N-terminal fragments of an animal fatty acid synthase, containing the beta-ketoacyl synthase, acyl transferase, and dehydratase domains, demonstrate that the dehydratase domain consists of two pseudosubunits, derived from contiguous regions of the same polypeptide, in which a single active site is formed by the cooperation of the catalytic histidine 878 residue of the first pseudosubunit with aspartate 1032 of the second pseudosubunit. Mutagenesis and modeling studies revealed an essential role for glutamine 1036 in anchoring the position of the catalytic aspartate. These findings establish that sequence elements previously assigned to a central structural core region of the type I fatty acid synthases and some modular polyketide synthase counterparts play an essential catalytic role as part of the dehydratase domain.     

Potent and selective peptidyl boronic acid inhibitors of the serine protease prostate-specific antigen

Prostate cancer cells produce high (microgram to milligram/milliliter) levels of the serine protease Prostate-Specific Antigen (PSA). PSA is enzymatically active in the extracellular fluid surrounding prostate cancers but is found at 1,000- to 10,000-fold lower concentrations in the circulation, where it is inactivated due to binding to abundant serum protease inhibitors. The exclusive presence of high levels of active PSA within prostate cancer sites makes PSA an attractive candidate for targeted imaging and therapeutics. A synthetic approach based on a peptide substrate identified first peptide aldehyde and then boronic acid inhibitors of PSA. The best of these had the sequence Cbz-Ser-Ser-Lys-Leu-(boro)Leu, with a K_i for PSA of 65 nM. The inhibitor had a 60-fold higher K_i for chymotrypsin. A validated model of PSA's catalytic site confirmed the critical interactions between the inhibitor and residues within the PSA enzyme.     

Arabidopsis P-glycoprotein19 participates in the inhibition of gravitropism by gravacin

ATP-binding cassette (ABC) transporters have been implicated in a multitude of biological pathways. In plants, some ABC transporters are involved in the polar transport of the plant hormone auxin and the gravitropic response. We previously identified Gravacin as a potent inhibitor of gravitropism in Arabidopsis thaliana. We demonstrate that P-glycoprotein19 (PGP19) is a target for Gravacin and participates in its inhibition of gravitropism. Gravacin inhibited the auxin transport activity of PGP19 and PGP19-PIN complexes. Furthermore, we identified E1174 as an important residue for PGP19 activity and its ability to form active transport complexes with PIN1. Gravacin is an auxin transport inhibitor that inhibits PGPs, particularly PGP19, which can be used to further dissect the role of PGP19 without the inhibition of other auxin transporters, namely PIN proteins.     

Nanomolar CFTR inhibition by pore-occluding divalent polyethylene glycol-malonic acid hydrazides

Inhibitors of the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel have potential application as antisecretory therapy in cholera. We synthesized mono- and divalent CFTR inhibitors consisting of a malonic acid hydrazide (MalH) coupled via a disulfonic stilbene linker to polyethylene glycols (PEGs; 0.2-100 kDa). IC50 values for CFTR inhibition were 10-15 microM for the monovalent MalH-PEGs, but substantially lower for divalent MalH-PEG-MalH compounds, decreasing from 1.5 to 0.3 microM with increasing PEG size and showing positive cooperativity. Whole-cell patch-clamp showed voltage-dependent CFTR block with inward rectification. Outside-out patch-clamp showed shortened single-channel openings, indicating CFTR pore block from the extracellular side. Luminally added MalH-PEG-MalH blocked by >90% cholera toxin-induced fluid secretion in mouse intestinal loops (IC50 approximately 10 pmol/loop), and greatly reduced mortality in a suckling mouse cholera model. These conjugates may provide safe, inexpensive antisecretory therapy.     

Glycosidase-substrate interactions analysis by STD-NMR spectroscopy: study of alpha-L-fucosidase

Saturation transfer difference-nuclear magnetic resonance (STD-NMR) is a recently developed method used to study the interaction between large proteins and small ligands. It has been successfully employed for various interactions including those between oligosaccharides or glycomimetics, and binding proteins such as lectins and antibodies. We have demonstrated that this method can be used to directly study the interaction between glycosidases and their substrates. We chose to study alpha-l-fucosidases, which, despite their wide distribution among living organisms and their biological significance, have not been studied with regard to their reaction mechanism. We were able for the first time to show direct evidences for the minimum structural requirements for a compound to interact with these enzymes. These findings will be useful for the design of new inhibitors and to optimize the synthetic properties (transglycosylation) of alpha-l-fucosidases.     

Determination of macrolide antibiotics by liquid chromatography

The liquid chromatographic separation of seven macrolides used in food producing animals in the European Union has been studied. Separation was performed by using an end-capped high-purity silica-based C₁₈ column and mobile phases consisting of phosphate buffer (pH 2.5)–acetonitrile mixtures. The effect of pH and acetonitrile percentage on the separation was examined. Two UV-based detection systems, wavelength programming and diode array, were assayed. Detection limits were in the range 6–33 μg l⁻¹ for spiramycin, tilmicosin, tylosin, kitasamicin and josamicin and about 400 μg l⁻¹ for erythromycin and oleandomycin. The suitability of the method for multiresidue determination of the five macrolides is demonstrated by the analysis of spiked samples of chicken muscle.     

Covalent inhibitors of human monoacylglycerol lipase: ligand-assisted characterization of the catalytic site by mass spectrometry and mutational analysis

The active site of recombinant hexa-histidine-tagged human monoacylglycerol lipase (hMGL) is characterized by mass spectrometry using the inhibitors 5-((biphenyl-4-yl)methyl)-N,N-dimethyl-2H-tetrazole-2-carboxamide (AM6701), and N-arachidonylmaleimide (NAM) as probes. Carbamylation of Ser(129) by AM6701 in the putative hMGL catalytic triad demonstrates this residue's essential role in catalysis. Partial NAM alkylation of hMGL cysteine residues 215 and/or 249 was sufficient to achieve approximately 80% enzyme inhibition. Although Cys(215) and/or Cys(249) mutations to alanine(s) did not affect hMGL hydrolytic activity as compared with nonmutated hMGL, the C215A displayed heightened NAM sensitivity, whereas the C249A evidenced reduced NAM sensitivity. These data conclusively demonstrate a sulfhydryl-based mechanism for NAM inhibition of hMGL in which Cys(249) is of paramount importance. Identification of amino acids critical to the catalytic activity and pharmacological modulation of hMGL informs the design of selective MGL inhibitors as potential drugs.     

HPLC-MS/MS检测猪肉中六种大环内酯类抗生素

建立了以高效液相色谱-质谱(HPLC-MS/MS)检测猪肉中替米考星(TILM)、红霉素(ERY)、泰乐菌素(TYL)、吉他霉素(KIT)、罗红菌素(ROX)及交沙霉素(JOS)等6种大环内酯类抗生素的多残留分析方法.样品用乙腈提取后,经正己烷脱脂,过C18 柱净化,4%氨甲醇洗脱后经氮气吹干,采用多反应监测,对6种大环内酯类药物作定性和定量分析.在10～1000μg/L的范围内,6种药物线性良好(R>0.998).在50、100、200μg/Kg添加水平下,6种药物的回收率在62.2%～102%,相对标准偏差为2.7%～16%.替米考星和吉他霉素的检出限(S/N=3)为0.1μg/Kg,其它药物为0.05μg/kg.     

Investigation of the binding of a carbohydrate-mimetic peptide to its complementary anticarbohydrate antibody by STD-NMR spectroscopy and molecular-dynamics simulations

Saturation transfer difference (STD)‐NMR spectroscopy was used to probe experimentally the bioactive solution conformation of the carbohydrate mimic MDWNMHAA 1 of the O‐polysaccharide of Shigella flexneri Y when bound to its complementary antibody, mAb SYA/J6. Molecular dynamics simulations using the ZymeCAD? Molecular Dynamics platform were also undertaken to give a more accurate picture of the conformational flexibility and the possibilities for bound ligand conformations. The ligand topology, or the dynamic epitope, was mapped with the CORCEMA‐ST (COmplete Relaxation and Conformational Exchange Matrix Analysis of Saturation Transfer) program that calculates a total matrix analysis of relaxation and exchange effects to generate predicted STD‐NMR intensities from simulation. The comparison of these predicted STD enhancements with experimental data was used to select a representative binding mode. A protocol that employed theoretical STD effects calculated at snapshots during the entire course of a molecular dynamics (MD) trajectory of the peptide bound to the Fv portion of the antibody, and not the averaged atomic positions of receptor–ligand complexes, was also examined. In addition, the R factor was calculated on the basis of STD (fit) to avoid T1 bias, and an effective R factor, R_eff, was defined such that if the calculated STD (fit) for proton k was within error of the experimental STD (fit) for proton k, then that calculated STD (fit) for proton k was not included in the calculation of the R factor. This protocol was effective in deriving the antibody‐bound solution conformation of the peptide which also differed from the bound conformation determined by X‐ray crystallography; however, several discrepancies between experimental and calculated STD (fit) values were observed. The bound conformation was therefore further refined with a simulated annealing refinement protocol known as STD‐NMR intensity‐restrained CORCEMA optimization (SICO) to give a more accurate representation of the bound peptide epitope. Further optimization was required in this case, but a satisfactory correlation between experimental and calculated STD values was obtained. Attempts were also made to obtain STD enhancements with a synthetic pentasaccharide hapten, corresponding to the O‐polysaccharide, while bound to the antibody. However, unfavorable kinetics of binding in this system prevented sufficient STD build‐up, which, in turn, hindered a rigorous analysis via full STD build‐up curves.     

Parametrization of macrolide antibiotics using the force field toolkit

Macrolides are an important class of antibiotics that target the bacterial ribosome. Computer simulations of macrolides are limited as specific force field parameters have not been previously developed for them. Here, we determine CHARMM‐compatible force field parameters for erythromycin, azithromycin, and telithromycin, using the force field toolkit (ffTK) plugin in VMD. Because of their large size, novel approaches for parametrizing them had to be developed. Two methods for determining partial atomic charges, from interactions with TIP3P water and from the electrostatic potential, as well as several approaches for fitting the dihedral parameters were tested. The performance of the different parameter sets was evaluated by molecular dynamics simulations of the macrolides in ribosome, with a distinct improvement in maintenance of key interactions observed after refinement of the initial parameters. Based on the results of the macrolide tests, recommended procedures for parametrizing very large molecules using ffTK are given. © 2015 Wiley Periodicals, Inc.