Delineating the biosynthesis of gentamicin x2, the common precursor of the gentamicin C antibiotic complex.

Gentamicin C complex is a mixture of aminoglycoside antibiotics used worldwide to treat severe Gram-negative bacterial infections. Despite its clinical importance, the enzymology of its biosynthetic pathway has remained obscure. We report here insights into the four enzyme-catalyzed steps that lead from the first-formed pseudotrisaccharide gentamicin A2 to gentamicin X2, the last common intermediate for all components of the C complex. We have used both targeted mutations of individual genes and reconstitution of portions of the pathway in vitro to show that the secondary alcohol function at C-3″ of A2 is first converted to an amine, catalyzed by the tandem operation of oxidoreductase GenD2 and transaminase GenS2. The amine is then specifically methylated by the S-adenosyl-l-methionine (SAM)-dependent N-methyltransferase GenN to form gentamicin A. Finally, C-methylation at C-4″ to form gentamicin X2 is catalyzed by the radical SAM-dependent and cobalamin-dependent enzyme GenD1.This work was supported by a project grant from the Medical Research Council, UK (G1001687) to P.F.L.; and by the 973 and 863 programs from the Ministry of Science and Technology of China, National Science Foundation of China, and the Translational Medical Research Fund of Wuhan University School of Medicine to Y.S.; E.M. thanks the Gates Cambridge Trust for a scholarship. We also gratefully acknowledge Dr. Xinzhou Yang, SouthCentral University for Nationalities, for his assistance in separation of gentamicin A2. We thank Dr. Andrew Truman (John Innes Institute) for helpful discussions.This is the final published version. It was originally published in Chemistry and Biology, Volume 22, Issue 2, 19 February 2015, Pages 251–261, doi:10.1016/j.chembiol.2014.12.01

Biosynthetic Gene Cluster of the Glycopeptide Antibiotic Teicoplanin Characterization of Two Glycosyltransferases and the Key Acyltransferase

AbstractThe gene cluster encoding biosynthesis of the clinically important glycopeptide antibiotic teicoplanin has been cloned from Actinoplanes teichomyceticus. Forty-nine putative open reading frames (ORFs) were identified within an 89 kbp genetic locus and assigned roles in teicoplanin biosynthesis, export, resistance, and regulation. Two ORFs, designated orfs 1 and 10*, showed significant homology to known glycosyltransferases. When heterologously expressed in Escherichia coli, these glycosyltransferases were shown to catalyze the transfer of UDP-(N-acetyl)-glucosamine onto, respectively, 3-chloro-β-hydroxytyrosine-6 (3-Cl6βHty) and 4-hydroxyphenylglycine-4 (4Hpg) of the teicoplanin heptapeptide aglycone. The product of another ORF, orf11*, was demonstrated in vitro to transfer n-acetyl-, n-butyryl-, and n-octanoyl-groups from acyl-CoA donors either to a free UDP-aminosugar or to an aminosugar moiety in the teicoplanin pseudoaglycone, thus identifying Orf11* as the key acyltransferase in teicoplanin maturation. These findings should accelerate the combinatorial engineering of new and improved glycopeptide drugs

The Gene Cluster for Fluorometabolite Biosynthesis in Streptomyces cattleya: A Thioesterase Confers Resistance to Fluoroacetyl-Coenzyme A

SummaryA genomic library of Streptomyces cattleya was screened to isolate a gene cluster encoding enzymes responsible for the production of fluorine-containing metabolites. In addition to the previously described fluorinase FlA which catalyzes the formation of 5′-fluoro-5′-deoxyadenosine from S-adenosylmethionine and fluoride, 11 other putative open reading frames have been identified. Three of the proteins encoded by these genes have been characterized. FlB was determined to be the second enzyme in the pathway, catalyzing the phosphorolytic cleavage of 5′-fluoro-5′-deoxyadenosine to produce 5-fluoro-5-deoxy-D-ribose-1-phosphate. The enzyme FlI was found to be an S-adenosylhomocysteine hydrolase, which may act to relieve S-adenosylhomocysteine inhibition of the fluorinase. Finally, flK encodes a thioesterase which catalyzes the selective breakdown of fluoroacetyl-CoA but not acetyl-CoA, suggesting that it provides the producing strain with a mechanism for resistance to fluoroacetate

Methyltransferases of gentamicin biosynthesis

Gentamicin C complex from Micromonospora echinospora remains a globally important antibiotic, and there is revived interest in the semisynthesis of analogs that might show improved therapeutic properties. The complex consists of five components differing in their methylation pattern at one or more sites in the molecule. We show here, using specific gene deletion and chemical complementation, that the gentamicin pathway up to the branch point is defined by the selectivity of the methyltransferases GenN, GenD1, and GenK. Unexpectedly, they comprise a methylation network in which early intermediates are ectopically modified. Using whole-genome sequence, we have also discovered the terminal 6'-N-methyltransfer required to produce gentamicin C2b from C1a or gentamicin C1 from C2, an example of an essential biosynthetic enzyme being located not in the biosynthetic gene cluster but far removed on the chromosome. These findings fully account for the methylation pattern in gentamicins and open the way to production of individual gentamicins by fermentation, as starting materials for semisynthesis.This work was supported by National Natural Science Foundation of China Grant 31470186; by the 973 Program Grant 2012CB721005 from the Ministry of Science and Technology of China; by Open Project Grant MMLKF15-12 from the State Key Laboratory of Microbial Metabolism, Shanghai Jiao Tong University (to Y.S.); by Medical Research Council (MRC) Grants G1001687 and MR/M019020/1 (to P.F.L.); and by an MRC postgraduate studentship (1343325) (to A.R.)

Structural basis for the activity and substrate specificity of fluoroacetyl-CoA thioesterase FlK.

The thioesterase FlK from the fluoroacetate-producing Streptomyces cattleya catalyzes the hydrolysis of fluoroacetyl-coenzyme A. This provides an effective self-defense mechanism, preventing any fluoroacetyl-coenzyme A formed from being further metabolized to 4-hydroxy-trans-aconitate, a lethal inhibitor of the tricarboxylic acid cycle. Remarkably, FlK does not accept acetyl-coenzyme A as a substrate. Crystal structure analysis shows that FlK forms a dimer, in which each subunit adopts a hot dog fold as observed for type II thioesterases. Unlike other type II thioesterases, which invariably utilize either an aspartate or a glutamate as catalytic base, we show by site-directed mutagenesis and crystallography that FlK employs a catalytic triad composed of Thr(42), His(76), and a water molecule, analogous to the Ser/Cys-His-acid triad of type I thioesterases. Structural comparison of FlK complexed with various substrate analogues suggests that the interaction between the fluorine of the substrate and the side chain of Arg(120) located opposite to the catalytic triad is essential for correct coordination of the substrate at the active site and therefore accounts for the substrate specificity

TAGAP expression influences CD4+ T cell differentiation, immune infiltration, and cytotoxicity in LUAD through the STAT pathway: implications for immunotherapy

BackgroundT-cell Activation GTPase Activating Protein (TAGAP) plays a role in immune cell regulation. This study aimed to investigate TAGAP’s expression and its potential impact on CD4+ T cell function and prognosis in lung adenocarcinoma (LUAD).MethodsWe analyzed TAGAP expression and its correlation with immune infiltration and clinical data in LUAD patients using multiple datasets, including The Cancer Genome Atlas (TCGA-LUAD), Gene Expression Omnibus (GEO), and scRNA-seq datasets. In vitro and in vivo experiments were conducted to explore the role of TAGAP in CD4+ T cell function, chemotaxis, and cytotoxicity.ResultsTAGAP expression was significantly lower in LUAD tissues compared to normal tissues, and high TAGAP expression correlated with better prognosis in LUAD patients. TAGAP was positively correlated with immune/stromal/ESTIMATE scores and immune cell infiltration in LUAD. Single-cell RNA sequencing revealed that TAGAP was primarily distributed in CD4+/CD8+ T cells. In vitro experiments showed that TAGAP overexpression enhanced CD4+ T cell cytotoxicity, proliferation, and chemotaxis. Gene Set Enrichment Analysis (GSEA) indicated that TAGAP was enriched in the JAK-STAT signaling pathway. In vivo experiments in a xenograft tumor model demonstrated that TAGAP overexpression suppressed tumor growth and promoted CD4+ T cell cytotoxicity.ConclusionsTAGAP influences CD4+ T cell differentiation and function in LUAD through the STAT pathway, promoting immune infiltration and cytotoxicity. This study provides a scientific basis for developing novel LUAD immunotherapy strategies and exploring new therapeutic targets

Insights into 6-methylsalicylic acid bio-assembly by using chemical probes

Chemical probes capable of reacting with KS (ketosynthase)-bound biosynthetic intermediates were utilized for the investigation of the model type I iterative polyketide synthase 6-methylsalicylic acid synthase (6-MSAS) in vivo and in vitro. From the fermentation of fungal and bacterial 6-MSAS hosts in the presence of chain termination probes, a full range of biosynthetic intermediates was isolated and characterized for the first time. Meanwhile, in vitro studies of recombinant 6-MSA synthases with both nonhydrolyzable and hydrolyzable substrate mimics have provided additional insights into substrate recognition, providing the basis for further exploration of the enzyme catalytic activities

Chimeric Glycosyltransferases for the Generation of Hybrid Glycopeptides

SummaryGlycodiversification, an invaluable tool for generating biochemical diversity, can be catalyzed by glycosyltransferases, which attach activated sugar “donors” onto “acceptor” molecules. However, many glycosyltransferases can tolerate only minor modifications to their native substrates, thus making them unsuitable tools for current glycodiversification strategies. Here we report the production of functional chimeric glycosyltransferases by mixing and matching the N- and C-terminal domains of glycopeptide glycosyltransferases. Using this method we have generated hybrid glycopeptides and have demonstrated that domain swapping can result in a predictable switch of substrate specificity, illustrating that N- and C-terminal domains predominantly dictate acceptor and donor specificity, respectively. The determination of the structure of a chimera in complex with a sugar donor analog shows that almost all sugar-glycosyltransferase binding interactions occur in the C-terminal domain

Canonical Wnt Signaling Pathway on Polarity Formation of Utricle Hair Cells

As part of the inner ear, the vestibular system is responsible for sense of balance, which consists of three semicircular canals, the utricle, and the saccule. Increasing evidence has indicated that the noncanonical Wnt/PCP signaling pathway plays a significant role in the development of the polarity of the inner ear. However, the role of canonical Wnt signaling in the polarity of the vestibule is still not completely clear. In this study, we found that canonical Wnt pathway-related genes are expressed in the early stage of development of the utricle and change dynamically. We conditionally knocked out β-catenin, a canonical Wnt signaling core protein, and found that the cilia orientation of hair cells was disordered with reduced number of hair cells in the utricle. Moreover, regulating the canonical Wnt pathway (Licl and IWP2) in vitro also affected hair cell polarity and indicated that Axin2 may be important in this process. In conclusion, our results not only confirm that the regulation of canonical Wnt signaling affects the number of hair cells in the utricle but also provide evidence for its role in polarity development

Construction of 11 metabolic-related lncRNAs to predict the prognosis in lung adenocarcinoma

Abstract Objective To explore the metabolism-related lncRNAs in the tumorigenesis of lung adenocarcinoma. Methods The transcriptome data and clinical information about lung adenocarcinoma patients were acquired in TCGA (The Cancer Genome Atlas). Metabolism-related genes were from the GSEA (Gene Set Enrichment Analysis) database. Through differential expression analysis and Pearson correlation analysis, lncRNAs about lung adenocarcinoma metabolism were identified. The samples were separated into the training and validation sets in the proportion of 2:1. The prognostic lncRNAs were determined by univariate Cox regression analysis and LASSO (Least absolute shrinkage and selection operator) regression. A risk model was built using Multivariate Cox regression analysis, evaluated by the internal validation data. The model prediction ability was assessed by subgroup analysis. The Nomogram was constructed by combining clinical indicators with independent prognostic significance and risk scores. C-index, calibration curve, DCA (Decision Curve Analysis) clinical decision and ROC (Receiver Operating Characteristic Curve) curves were obtained to assess the prediction ability of the model. Based on the CIBERSORT analysis, the correlation between lncRNAs and tumor infiltrating lymphocytes was obtained. Results From 497 lung adenocarcinoma and 54 paracancerous samples, 233 metabolic-related and 11 prognostic-related lncRNAs were further screened. According to the findings of the survival study, the low-risk group had a greater OS (Overall survival) than the high-risk group. ROC analysis indicated AUC (Area Under Curve) value was 0.726. Then, a nomogram with T, N stage and risk ratings was developed according to COX regression analysis. The C-index was 0.743, and the AUC values of 3- and 5-year survival were 0.741 and 0.775, respectively. The above results suggested the nomogram had a good prediction ability. The results based on the CIBERSORT algorithm demonstrated the lncRNAs used to construct the model had a strong correlation with the polarization of immune cells. Conclusions The study identified 11 metabolic-related lncRNAs for lung adenocarcinoma prognosis, on which basis a prognostic risk scoring model was created. This model may have a good predictive potential for lung adenocarcinoma