The crystal structure of PfFabZ, the unique beta-hydroxyacyl-ACP dehydratase involved in fatty acid biosynthesis of Plasmodium falciparum

The unique beta-hydroxyacyl-ACP dehydratase in Plasmodium falciparum, PfFabZ, is involved in fatty acid biosynthesis and catalyzes the dehydration of beta-hydroxy fatty acids linked to acyl carrier protein. The structure was solved by single anomalous dispersion (SAD) phasing using a quick-soaking experiment with potassium iodide and refined to a resolution of 2.1 A. The crystal structure represents the first structure of a Plasmodium beta-hydroxyacyl-ACP dehydratase with broad substrate specificity. The asymmetric unit contains a hexamer that appears as a trimer of dimers. Each dimer shows the known "hot dog" fold that has been observed in only a few other protein structures. Each of the two independent active sites in the dimer is formed by equal contributions from both subunits. The active site is mainly hydrophobic and looks like an L-shaped tunnel. The catalytically important amino acids His 133 and Glu 147' (from the other subunit), together with His98', form the only hydrophilic site in this tunnel. The inner end of the active site tunnel is closed by the phenyl ring of Phe 169, which is located in a flexible, partly visible loop. In order to explain the acceptance of substrates longer than ~C-7, the phenyl ring must move away to open the tunnel. The present structure supports an enzymatic mechanism consisting of an elimination reaction catalyzed by His 133 and Glu147'. 3-decynoyl-N-acetylcysteamine, an inhibitor known to interact with the E. coli dehydratase/isomerase, turned out to interact covalently with PfFabZ. A first model of PfFabZ with this potent inhibitor is presented.     

Evidence for a novel phosphopantetheinyl transferase domain in the polyketide synthase for enediyne biosynthesis

The polyketide synthase associated with the biosynthesis of enediyne-containing calicheamicin contains a putative phosphopantetheinyl transferase (PPTase) domain. By cloning and expressing the C-terminal region of the polyketide synthase and in vitro phosphopantetheinylation assay, we found that the PPTase domain exhibits preferred substrate specificity towards acyl and peptidyl carrier proteins in fatty acid and non-ribosomal peptide synthesis over its cognate partner. We also found evidence suggesting that the PPTase domain adopts a pseudo-trimeric structure, distinct from the pseudo-dimeric structure of type II PPTases. The results revealed a novel type of PPTase with unique structure and substrate specificity, and suggested that the polyketide synthase probably acquired the PPTase domain from a primary metabolic pathway in evolution.     

Negative regulation by Ser/Thr phosphorylation of HadAB and HadBC dehydratases from Mycobacterium tuberculosis type II fatty acid synthase system

The type II fatty acid synthase system of mycobacteria is involved in the biosynthesis of major and essential lipids, mycolic acids, key-factors of Mycobacterium tuberculosis pathogenicity. One reason of the remarkable survival ability of M. tuberculosis in infected hosts is partly related to the presence of cell wall-associated mycolic acids. Despite their importance, the mechanisms that modulate synthesis of these lipids in response to environmental changes are unknown. We demonstrate here that HadAB and HadBC dehydratases of this system are phosphorylated by Ser/Thr protein kinases, which negatively affects their enzymatic activity. The phosphorylation of HadAB/BC is growth phase-dependent, suggesting that it represents a mechanism by which mycobacteria might tightly control mycolic acid biosynthesis under non-replicating condition.     

Inhibitory effects of tannic acid on fatty acid synthase and 3T3-L1 preadipocyte

Tannic acid is a hydrolyzable tannin that exists in many widespread edible plants with a variety of biological activities. In this study, we found that tannic acid potently inhibited the activity of fatty acid synthase (FAS) in a concentration-dependent manner with a half-inhibitory concentration value (IC₅₀) of 0.14 μM. The inhibition kinetic results showed that the inhibition of FAS by tannic acid was mixed competitive and noncompetitive manner with respect to acetyl-CoA and malonyl-CoA, but uncompetitive to NADPH. Tannic acid prevented the differentiation of 3T3-L1 pre-adipocytes, and thus repressed intracellular lipid accumulation. In the meantime, tannic acid decreased the expression of FAS and down-regulated the mRNA level of FAS and PPARγ during adipocyte differentiation. Further studies showed that the inhibitory effect of tannic acid did not relate to FAS non-specific sedimentation. Since FAS was believed to be a therapeutic target of obesity, these findings suggested that tannic acid was considered having potential in the prevention of obesity.     

Cloning and characterization of canadine synthase involved in noscapine biosynthesis in opium poppy

Noscapine biosynthesis in opium poppy is thought to occur via N-methylcanadine, which would be produced through 9-O-methylation of (S)-scoulerine, methylenedioxy bridge formation on (S)-tetrahydrocolumbamine, and N-methylation of (S)-canadine. Only scoulerine 9-O-methyltransferase has been functionally characterized. We report the isolation and characterization of a cytochrome P450 (CYP719A21) from opium poppy that converts (S)-tetrahydrocolumbamine to (S)-canadine. Recombinant CYP719A21 displayed strict substrate specificity and high affinity (K_m = 4.63 ± 0.71 μM) for (S)-tetrahydrocolumbamine. Virus-induced gene silencing of CYP719A21 caused a significant increase in (S)-tetrahydrocolumbamine accumulation and a corresponding decrease in the levels of putative downstream intermediates and noscapine in opium poppy plants.     

Identification and catalytic characterization of a nonribosomal peptide synthetase-like (NRPS-like) enzyme involved in the biosynthesis of echosides from Streptomyces sp. LZ35

Echosides, isolated from Streptomyces sp. LZ35, represent a class of para-terphenyl natural products that display DNA topoisomerase I and IIα inhibitory activities. By analyzing the genome draft of strain LZ35, the ech gene cluster was identified to be responsible for the biosynthesis of echosides, which was further confirmed by gene disruption and HPLC analysis. Meanwhile, the biosynthetic pathway for echosides was proposed. Furthermore, the echA-gene, encoding a tri-domain nonribosomal peptide synthetase (NRPS)-like enzyme, was identified as a polyporic acid synthetase and biochemically characterized in vitro. This is the first study to our knowledge on the biochemical characterization of an Actinobacteria quinone synthetase, which accepts phenylpyruvic acid as a native substrate. Therefore, our results may help investigate the function of other NRPS-like enzymes in Actinobacteria.     

Identification of a novel bifunctional delta12/delta15 fatty acid desaturase from a basidiomycete, Coprinus cinereus TD#822-2

A new gene encoding a delta12 fatty acid desaturase-related protein was cloned from a multicellular basidiomycete Coprinus cinereus TD#822-2. The 1326 bp full-length gene, designated as Cop-odeA, codes for a putative protein of 442 amino acids with a MW of 49224. The Cop-odeA yeast transformant accumulated four new fatty acids identified as 9,12-hexadecadienoic acid, 9,12,15-hexadecatrienoic acid, linoleic acid, and alpha-linolenic acid, which comprised 8.8%, 1.0%, 29.0%, and 0.6% of the total fatty acids, respectively. The Cop-odeA protein was confirmed to be a novel bifunctional fatty acid desaturase with both high delta12 desaturase activity and unusual delta15 desaturase activity.     

Identification of a novel conjugate in human urine: bile acid acyl galactosides

We report a novel conjugate, bile acid acyl galactosides, which exist in the urine of healthy volunteers. To identify the two unknown peaks obtained in urine specimens from healthy subjects, the specimens were subjected to solid phase extraction and then to liquid chromatographic separation. The eluate corresponding to the unknown peaks on the chromatogram was collected. Following alkaline hydrolysis and liquid chromatography (LC)/electrospray ionization (ESI)-mass spectrometric (MS) analysis, cholic acid (CA) and deoxycholic acid (DCA) were identified as liberated bile acids. When a portion of the alkaline hydrolyzate was subjected to a derivatization reaction with 1-phenyl-3-methyl-5-pyrazolone, a derivative of galactose was detected by LC/ESI-MS. Finally, the liquid chromatographic and mass spectrometric properties of these unknown compounds in urine specimens were compared to those of authentic specimens and the structures were confirmed as CA 24-galactoside and DCA 24-galactoside. These results strongly imply that bile acid 24-galactosides, a novel conjugate, were synthesized in the human body.     

The nitrite-oxidizing community in activated sludge from a municipal wastewater treatment plant determined by fatty acid methyl ester-stable isotope probing

Metabolically-active autotrophic nitrite oxidizers from activated sludge were labeled with ¹³C-bicarbonate under exposure to different temperatures and nitrite concentrations. The labeled samples were characterized by FAME-SIP (fatty acid methyl ester-stable isotope probing). The compound cis-11-palmitoleic acid, which is the major lipid of the most abundant nitrite oxidizer in activated sludge, Candidatus Nitrospira defluvii, showed ¹³C-incorporation in all samples exposed to 3 mM nitrite. Subsequently, the lipid cis-7-palmitoleic acid was labeled, and it indicated the activity of a nitrite oxidizer that was different from the known Nitrospira taxa in activated sludge. The highest incorporation of cis-7-palmitoleic acid label was found after incubation with a nitrite concentration of 0.3 mM at 17 and 22 °C. While activity of Nitrobacter populations could not be detected by the FAME-SIP approach, an unknown nitrite oxidizer with the major lipid cis-9 isomer of palmitoleic acid exhibited ¹³C-incorporation at 28 °C with 30 mM nitrite. These results indicated flexibility of nitrite-oxidizing guilds in a complex community responding to different conditions. Labeled lipids so far not described for activated sludge-associated nitrifiers indicated the presence of unknown nitrite oxidizers in this habitat. The FAME-SIP-based information can be used to define appropriate conditions for the enrichment of nitrite-oxidizing guilds from complex samples.     

Accumulation of arachidonic acid-rich triacylglycerols in the microalga Parietochloris incisa (Trebuxiophyceae,Chlorophyta)

The freshwater green microalga Parietochloris incisa is the richest known plant source of the polyunsaturated fatty acid (PUFA), arachidonic acid (20:4omega6, AA). While many microalgae accumulate triacylglycerols (TAG) in the stationary phase or under certain stress conditions, these TAG are generally made of saturated and monounsaturated fatty acids. In contrast, most cellular AA of P. incisa resides in TAG. Using various inhibitors, we have attempted to find out if the induction of the biosynthesis of AA and the accumulation of TAG are codependent. Salicylhydroxamic acid (SHAM) affected a growth reduction that was accompanied with an increase in the content of TAG from 3.0 to 6.2% of dry weight. The proportion of 18:1 increased sharply in all lipids while that of 18:2 and its down stream products, 18:3omega6, 20:3omega6 and AA, decreased, indicating an inhibition of the Delta12 desaturation of 18:1. Treatment with the herbicide SAN 9785 significantly reduced the proportion of TAG. However, the proportion of AA in TAG, as well as in the polar lipids, increased. These findings indicate that while there is a preference for AA as a building block of TAG, the latter can be produced using other fatty acids, when the production of AA is inhibited. On the other hand, inhibiting TAG construction did not affect the production of AA. In order to elucidate the possible role of AA in TAG we have labeled exponential cultures of P. incisa kept at 25 degrees C with [1-14C]arachidonic acid and cultivated the cultures for another 12 h at 25, 12 or 4 degrees C. At the lower temperatures, labeled AA was transferred from TAG to polar lipids, indicating that TAG of P. incisa may have a role as a depot of AA that can be incorporated into the membranes, enabling the organism to quickly respond to low temperature-induced stress.     

Isolation and characterization of genes from the marine microalga Pavlova salina encoding three front-end desaturases involved in docosahexaenoic acid biosynthesis

The marine microalga Pavlova salina produces lipids containing approximately 50% omega-3 long chain polyunsaturated fatty acids (LC-PUFA) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). Three cDNA sequences, designated PsD4Des, PsD5Des, PsD8Des, were isolated from P. salina and shown to encode three front-end desaturases with Delta4, Delta5 and Delta8 specificity, respectively. Southern analysis indicated that the P. salina genome contained single copies of all three front-end fatty acid desaturase genes. When grown at three different temperatures, analysis of fatty acid profiles indicated P. salina desaturation conversions occurred with greater than 95% efficiency. Real-Time PCR revealed that expression of PsD8Des was higher than for the other two genes under normal growth conditions, while PsD5Des had the lowest expression level. The deduced amino acid sequences from all three genes contained three conserved histidine boxes and a cytochrome b(5) domain. Sequence alignment showed that the three genes were homologous to corresponding desaturases from other microalgae and fungi. The predicted activities of these three front-end desaturases leading to the synthesis of LC-PUFA were also confirmed in yeast and in higher plants.     

Identification of a novel missense mutation in Brazilian patient with a severe form of mucopolysaccharidosis type IVA

Mucopolysaccharidosis type IVA (MPS IVA) or Morquio syndrome type A is an autosomal recessive disease caused by deficiency of the lysosomal enzyme N-acetylgalactosamine-6-sulfatase (GALNS). We report molecular characterization of a patient who presents the new missense mutation p.C165Y in homozygosis. Bioinformatics analysis predicted this mutation as being probably pathogenic. To evaluate the possibility that this alteration was a polymorphism we tested 100 alleles and all the results were negative. These findings together with the observation that this alteration is not present in controls, suggest that it is a disease-causing mutation, which was correlated with the severe phenotype observed in our patient. We conclude that molecular analysis of the GALNS gene, in addition to enzyme assays, is important for diagnosis and contributes to the better understanding of the relationship between genotype and phenotype, which is important as enzyme replacement therapy (ERT) will soon become available and treatment decisions will have to be take in such cases.     

Use of 3-hydroxy fatty acid concentrations in a murine air pouch infection model as a surrogate marker for LPS activity: a feasibility study using environmental Burkholderia cenocepacia isolates

Using a murine hypodermic air pouch infection model designed to mimic the release of bacterial products at physiological levels, 3-hydroxy fatty acid (3-OH FA) and endotoxin unit levels from Burkholderia cenocepacia isolates were assessed. The B. cenocepacia environmental isolates (n = 35) survived in the hypodermic air pouch but did not invade across the peritoneal epithelial layer during a 72-h infection. For all 35 strains, when the molar ratio of C_14:0 3-OH FA to C_16:0 3-OH FA in the air pouch fluid wash samples was between 1.4 and 2.5, the concentrations of C_14:0 3-OH FA were correlated with the endotoxin unit levels. However, both surrogate markers exhibited different correlations to the inflammatory response. The linear regression coefficient was 0.4234 for C_14:0 3-OH FA concentrations vs. NO productions, 0.223 for endotoxin unit levels vs. NO productions, 0.5008 for C_14:0 3-OH FA concentrations vs. TNF-alpha productions and 0.2869 for endotoxin unit levels vs. TNF-alpha productions. Therefore, C_14:0 3-OH FA concentrations, rather than endotoxin unit levels, acted as an immunostimulatory indicator for LPS in the B. cenocepacia isolates.     

Identification and functional analysis of a novel mutation in the SOX10 gene associated with Waardenburg syndrome type IV

Waardenburg syndrome type IV (WS4) is a rare genetic disorder, characterized by auditory–pigmentary abnormalities and Hirschsprung disease. Mutations of the EDNRB gene, EDN3 gene, or SOX10 gene are responsible for WS4. In the present study, we reported a case of a Chinese patient with clinical features of WS4. In addition, the three genes mentioned above were sequenced in order to identify whether mutations are responsible for the case. We revealed a novel nonsense mutation, c.1063C>T (p.Q355*), in the last coding exon of SOX10. The same mutation was not found in three unaffected family members or 100 unrelated controls. Then, the function and mechanism of the mutation were investigated in vitro. We found both wild-type (WT) and mutant SOX10 p.Q355* were detected at the expected size and their expression levels are equivalent. The mutant protein also localized in the nucleus and retained the DNA-binding activity as WT counterpart; however, it lost its transactivation capability on the MITF promoter and acted as a dominant-negative repressor impairing function of the WT SOX10.     

Identification of tenuazonic acid as a novel type of natural photosystem II inhibitor binding in QB-site of Chlamydomonas reinhardtii

Tenuazonic acid (TeA) is a natural phytotoxin produced by Alternaria alternata, the causal agent of brown leaf spot disease of Eupatorium adenophorum. Results from chlorophyll fluorescence revealed TeA can block electron flow from Q_A to Q_B at photosystem II acceptor side. Based on studies with D1-mutants of Chlamydomonas reinhardtii, the No. 256 amino acid plays a key role in TeA binding to the Q_B-niche. The results of competitive replacement with [¹⁴C]atrazine combined with JIP-test and D1-mutant showed that TeA should be considered as a new type of photosystem II inhibitor because it has a different binding behavior within Q_B-niche from other known photosystem II inhibitors. Bioassay of TeA and its analogues indicated 3-acyl-5-alkyltetramic and even tetramic acid compounds may represent a new structural framework for photosynthetic inhibitors.     

Biotechnological approaches for the production of polyhydroxyalkanoates in microorganisms and plants - a review

The increasing effect of non-degradable plastic wastes is a growing concern. Polyhydroxyalkanoates (PHAs), macromolecule-polyesters naturally produced by many species of microorganisms, are being considered as a replacement for conventional plastics. Unlike petroleum-derived plastics that take several decades to degrade, PHAs can be completely bio-degraded within a year by a variety of microorganisms. This biodegradation results in carbon dioxide and water, which return to the environment. Attempts based on various methods have been undertaken for mass production of PHAs. Promising strategies involve genetic engineering of microorganisms and plants to introduce production pathways. This challenge requires the expression of several genes along with optimization of PHA synthesis in the host. Although excellent progress has been made in recombinant hosts, the barriers to obtaining high quantities of PHA at low cost still remain to be solved. The commercially viable production of PHA in crops, however, appears to be a realistic goal for the future.