Cryo-EM snapshots of mycobacterial arabinosyltransferase complex EmbB-AcpM

Inhibition of Mycobacterium tuberculosis (Mtb) cell wall assembly is an established strategy for anti-TB chemotherapy. Arabinosyltransferase EmbB, which catalyzes the transfer of arabinose from the donor decaprenyl-phosphate-arabinose (DPA) to its arabinosyl acceptor is an essential enzyme for Mtb cell wall synthesis. Analysis of drug resistance mutations suggests that EmbB is the main target of the front-line anti-TB drug, ethambutol. Herein, we report the cryo-EM structures of Mycobacterium smegmatis EmbB in its "resting state" and DPA-bound "active state". EmbB is a fifteen-transmembrane-spanning protein, assembled as a dimer. Each protomer has an associated acyl-carrier-protein (AcpM) on their cytoplasmic surface. Conformational changes upon DPA binding indicate an asymmetric movement within the EmbB dimer during catalysis. Functional studies have identified critical residues in substrate recognition and catalysis, and demonstrated that ethambutol inhibits transferase activity of EmbB by competing with DPA. The structures represent the first step directed towards a rational approach for anti-TB drug discovery.

Cryo-EM snapshots of mycobacterial arabinosyltransferase complex EmbB-AcpM

Inhibition of Mycobacterium tuberculosis (Mtb) cell wall assembly is an established strategy for anti-TB chemotherapy. Arabinosyltransferase EmbB, which catalyzes the transfer of arabinose from the donor decaprenyl-phosphate-arabinose (DPA) to its arabinosyl acceptor is an essential enzyme for Mtb cell wall synthesis. Analysis of drug resistance mutations suggests that EmbB is the main target of the front-line anti-TB drug, ethambutol. Herein, we report the cryo-EM structures of Mycobacterium smegmatis EmbB in its "resting state" and DPA-bound "active state". EmbB is a fifteen-transmembrane-spanning protein, assembled as a dimer. Each protomer has an associated acyl-carrier-protein (AcpM) on their cytoplasmic surface. Conformational changes upon DPA binding indicate an asymmetric movement within the EmbB dimer during catalysis. Functional studies have identified critical residues in substrate recognition and catalysis, and demonstrated that ethambutol inhibits transferase activity of EmbB by competing with DPA. The structures represent the first step directed towards a rational approach for anti-TB drug discovery.

Identification and functional analysis of soybean stearoyl-ACP Δ⁹ desaturase (GmSAD) gene family / 生物工程学报

Stearoyl-ACP Δ⁹ desaturase (SAD) catalyzes the synthesis of monounsaturated oleic acid or palmitoleic acid in plastids. SAD is the key enzyme to control the ratio of saturated fatty acids to unsaturated fatty acids in plant cells. In order to analyze the regulation mechanism of soybean oleic acid synthesis, soybean (Glycine max) GmSAD family members were genome-wide identified, and their conserved functional domains and physicochemical properties were also analyzed by bioinformatics tools. The spatiotemporal expression profile of each member of GmSADs was detected by qRT-PCR. The expression vectors of GmSAD5 were constructed. The enzyme activity and biological function of GmSAD5 were examined by Agrobacterium-mediated transient expression in Nicotiana tabacum leaves and genetic transformation of oleic acid-deficient yeast (Saccharomyces cerevisiae) mutant BY4389. Results show that the soybean genome contains five GmSAD family members, all encoding an enzyme protein with diiron center and two conservative histidine enrichment motifs (EENRHG and DEKRHE) specific to SAD enzymes. The active enzyme protein was predicted as a homodimer. Phylogenetic analysis indicated that five GmSADs were divided into two subgroups, which were closely related to AtSSI2 and AtSAD6, respectively. The expression profiles of GmSAD members were significantly different in soybean roots, stems, leaves, flowers, and seeds at different developmental stages. Among them, GmSAD5 expressed highly in the middle and late stages of developmental seeds, which coincided with the oil accumulation period. Transient expression of GmSAD5 in tobacco leaves increased the oleic acid and total oil content in leaf tissue by 5.56% and 2.73%, respectively, while stearic acid content was reduced by 2.46%. Functional complementation assay in defective yeast strain BY4389 demonstrated that overexpression of GmSAD5 was able to restore the synthesis of monounsaturated oleic acid, resulting in high oil accumulation. Taken together, soybean GmSAD5 has strong selectivity to stearic acid substrates and can efficiently catalyze the biosynthesis of monounsaturated oleic acid. It lays the foundation for the study of soybean seed oleic acid and total oil accumulation mechanism, providing an excellent target for genetic improvement of oil quality in soybean.

Increasing pinosylvin production in Escherichia coli by reducing the expression level of the gene fabI-encoded enoyl-acyl carrier protein reductase

Background: The plant secondary metabolite pinosylvin is a polyphenol from the stilbene family, which have positive effects on human health. Biotechnological production is an attractive alternative for obtaining this stilbene. In Escherichia coli, malonyl-CoA is the precursor for both stilbene and fatty acid syntheses. In this study, with the aim of increasing pinosylvin production, we evaluated a novel approach that is based on reducing the expression of the gene fabI, which encodes the enzyme enoyl-acyl carrier protein reductase that is involved in fatty acid synthesis. Results: A recombineering method was employed to eliminate the chromosomal -35 promoter sequence and the upstream region of the gene fabI in E. coli strain W3110. Analysis, employing RT-qPCR, showed that such modification caused a 60% reduction in the fabI transcript level in the mutant strain W3110Δ-35fabI::Cm compared to the wild type W3110. Synthetic genes encoding a mutant version of 4-coumaroyl-CoA ligase from Streptomyces coelicolor A3 with improved catalytic activity employing cinnamic acid as substrate and a stilbene synthase from Vitis vinifera were cloned to generate the plasmid pTrc-Sc4CL(M)-VvSTS. The production performance of strains W3110Δ-35fabI::Cm/pTrc-Sc4CL(M)-VvSTS and W3110/pTrc-Sc4CL(M)- VvSTS was determined in shake flask cultures with Luria-Bertani medium supplemented with 10 g/L glycerol and 3 mM cinnamic acid. Under these conditions, the strain W3110Δ-35fabI::Cm/pTrc-Sc4CL(M)-VvSTS produced 52.67 mg/L pinosylvin, a level 1.5-fold higher than that observed with W3110/pTrc-Sc4CL(M)-VvSTS. Conclusion: A reduction in the transcript level of fabI caused by the elimination of the -35 and upstream promoter sequences is a successful strategy to improve pinosylvin production in E. coli.

Docking studies on novel alkaloid tryptanthrin and its analogues against enoyl-acyl carrier protein reductase (InhA) of Mycobacterium tuberculosis.

Isoniazid resistance is a serious threat in the battle against the treatment of multi-drug resistant tuberculosis (MDR-TB) and extremely drug-resistant tuberculosis (XDR-TB). Isoniazid is an inhibitor of enoyl-acyl carrier protein reductase (InhA) of Mycobacterium tuberculosis, which is an important and functional enzyme of the type II fatty acid synthesis system and important therapeutic target. Natural alkaloid tryptanthrin and its analogues have shown anti-tubercular activity against MDR-TB, but their cellular target is unknown. In this work, in silico molecular docking was performed using docking server in order to see the interaction of tryptanthrin and its 15 analogues with InhA of M. tuberculosis. Results showed that among tryptanthrin and its 15 analogues, tryptanthrin and its two analogues exhibited good affinity to the binding site of InhA with free binding energy of -7.94 kcal/mol and inhibition constant (Ki) of 1.50 µm. Active site residues of InhA interacting with tryptanthrin were Ser13, Thr39, Phe41, Leu63, Asp64, Val65, Ile95, Phe97 and Ile122. In binding mode, polar bond were found between O1 (1) with Asp64 of bond length (3.34 Å) and hydrophobic bonds were found between Leu63 with C15 and C12, Val65 with C7, Val65 with C12 and C4, Ile95 with C6 and C7, Ile95 with C10, C12 and C14. Important pi-pi bonds were found between Phe41 with C2, C5, C7, C12, C4, C6, C8, C9, C13 and Phe97 with C9. These interactions indicated stability of tryptanthrin in active residue and suggested it as a potential drug candidate. Thus, good affinity of tryptanthrin to binding site of InhA may lead to synthesis of anti-tubercular drug capable of combating MDR strains of M. tuberculosis

Characterization of ?-Ketoacyl-acyl Carrier Protein Synthase Ⅱ Homologues in Enterococcus faecalis / 生物化学与生物物理进展

FabB(?-ketoacyl-acyl carrier protein synthase Ⅰ)and FabF(?-ketoacyl-acyl carrier protein synthase Ⅱ)are two key enzymes of fatty acid biosynthesis in E.coli.The Gram-positive pathogenic bacterium Enterococcus faecalis has a fatty acid composition very similar to that of E.coli.Bioinformatic analysis reveals that though E.faecalis has two fabF homologues,there is no recognizable fabB homologue in the genome of E.faecalis.Two fabF homologues(fabF1 and fabF2)were amplified by using E.faecalis V583 genomitic DNA as template,and two plasmids,pHW13(fabF1)and pHW14(fabF2),were constructed.The results of experiments in vivo and in vitro have shown that fabF1 gene could complement E.coli fabB mutation and FabF1 possessed ?-ketoacyl-acyl carrier protein synthase Ⅰ(FabB)activity,while fabF2 gene could complement E.coli fabF mutation and FabF2 had ?-ketoacyl-acyl carrier protein synthase Ⅱ(FabF)activity.Meanwhile the data also shown that FabF2 possessed partial function of ?-ketoacyl-acyl carrier protein synthase Ⅰ(FabB),and it could make E.coli fabB mutation synthesized low amount of unsaturated fatty acid.From these data it is clear that FabF species enzymes could have activity of ?-ketoacyl-acyl carrier protein synthase Ⅰ(FabB).

Cloning and Expression of Acyl Carrier Protein Gene from Schizochytrium / 微生物学通报

Acyl carrier protein is an essential component involved in the biosynthesis of DHA(Docosahexaenoic Acid) via PKS(Polyketide synthase) pathway,which takes the growing acyl chain from one enzyme to another.One cDNA clone,with high homology of ACP,was isolated from Schizochytrium sp.FJU-512 cDNA library.The deduced amino acid sequence contained 142 residues with isoelectric point of 5.04 and had the 4'-phosphopantetheine prosthetic(4'-PP) binding site.The target fragment was digested with BamHⅠ/HindⅢand inserted into the expression vector pET-30a resulting in the plasmid pET-30a/acp.The recombinant vector was transformed into E.coli BL21(DE3) and induced by IPTG.SDS-PAGE analysis demonstrated that ACP was effectively expressed.