NAD metabolism and the NAD biosynthetic enzymes nicotinamide nucleotide adenylyltransferases (NMNATs) are thought to play a key neuroprotective role in tauopathies, including Alzheimer ’ s disease. Here, we in- vestigated whether modulating the expression of the NMNAT nuclear isoform NMNAT1, which is important for neuronal maintenance, in ﬂ uences the development of behavioral and neuropathological abnormalities in htau mice, which express non-mutant human tau isoforms and represent a model of tauopathy relevant to Alzheimer ’ s disease. Prior to the development of cognitive symptoms, htau mice exhibit tau hyperphosphorylation associated with a selective de ﬁ cit in food burrowing, a behavior reminiscent to activities of daily living which are impaired early in Alzheimer ’ s disease. We crossed htau mice with Nmnat1 transgenic and knockout mice and tested the resulting oﬀspring until the age of 6 months. We show that overexpression of NMNAT1 ameliorates the early de ﬁ cit in food burrowing characteristic of htau mice. At 6 months of age, htau mice did not show neurode- generative changes in both the cortex and hippocampus, and these were not induced by downregulating NMNAT1 levels. Modulating NMNAT1 levels produced a corresponding eﬀect on NMNAT enzymatic activity but did not alter NAD levels in htau mice. Although changes in local NAD levels and subsequent modulation of NAD- dependent enzymes cannot be ruled out, this suggests that the e ﬀ ects seen on behavior may be due to changes in tau phosphorylation. Our results suggest that increasing NMNAT1 levels can slow the progression of symptoms and neuropathological features of tauopathy, but the underlying mechanisms remain to be established.
we examined several enzyme activities in- volved in lignin biosynthesis, such as phenylalanine ammo- nia-lyase (PAL), cinnamate:4-hydroxylase (C4H), 4CL, and cinnamyl alcohol dehydrogenase (CAD) activity in poplar callus. Substrate utilization of the callus 4CL well supports the findings of previous experiments. 21 The increased en- zyme activity upstream (PAL, C4H, 4CL toward FA) in lignin biosynthesis also explains the increased syringyl lignin biosynthesis in poplar callus by exogenously supplied SA.
One unanswered question is how inhibition of fatty acid biosynthesis attenuates HCMV replication. Our data suggest that fatty acid biosynthesis is required at a relatively late stage during infection (Fig. 7). The simplest explanation is that fatty acid biosynthesis is necessary for the bulk production of fatty acids required for viral envelope phospholipids and that inhi- bition of this pathway limits the production of viral envelope. A related, but slightly different scenario is that the viral enve- lope consists of specific phospholipids whose production is activated and important for production of infectious virions. If this were the case, specific phospholipid-modifying enzymes working downstream of ACC1 would be potential targets for antiviral therapy. To our knowledge, no one has reported a detailed biochemical analysis of the HCMV phospholipid en- velope, which would identify the relevant lipid species and thereby illuminate phospholipid metabolic enzymes of interest. An alternative possibility is that HCMV replication depends on fatty acid biosynthesis for the modification of specific pro- teins. Fatty acid-based protein modifications, for example, my- ristoylation, are often essential for protein localization and function. This could be the case for the HCMV pp28 protein, an essential protein whose myristoylation is necessary for its proper localization and incorporation into virions (24, 51). Whether fatty acid biosynthesis is required for production of phospholipids or fatty acid protein modifications, its require- ment at late times postinfection suggests that HCMV targeting of ACC1 is important for virion assembly and production. It is clear that HCMV infection targets this regulatory enzyme in multiple ways, and future work will elucidate the specific mech- anisms involved and identify the specific fatty acid biosynthetic requirements for HCMV replication.
The first and the rate-limiting enzyme of heme biosynthesis is δ-aminolevulinate synthase (ALAS), which is localized in mitochondria. There are 2 tissue-specific isoforms of ALAS, erythroid-specific (ALAS-E) and nonspecific ALAS (ALAS-N). To identify possible mitochondrial factors that modulate ALAS-E function, we screened a human bone marrow cDNA library, using the mitochondrial form of human ALAS-E as a bait protein in the yeast 2-hybrid system. Our screening led to the isolation of the β subunit of human ATP-specific succinyl CoA synthetase (SCS-βA). Using transient expression and coimmunoprecipitation, we verified that mitochodrially expressed SCS-βA associates specifi- cally with ALAS-E and not with ALAS-N. Furthermore, the ALAS-E mutants R411C and M426V asso- ciated with SCS-βA, but the D190V mutant did not. Because the D190V mutant was identified in a patient with pyridoxine-refractory X-linked sideroblastic anemia, our findings suggest that appro- priate association of SCS-βA and ALAS-E promotes efficient use of succinyl CoA by ALAS-E or helps translocate ALAS-E into mitochondria.
We have found highly predictable patterns of protooncogene expression in cell lines and tumor tissue of neuroblastoma (NB), a tumor of the peripheral nervous system (PNS). These patterns make it possible to recognize two different genetically definable subgroups among histopathologically indistinguishable tumors. Additionally, we have identified a difference in neurotransmitter biosyntheticenzyme activity in these two subgroups of NB. The patterns of protooncogene expression and neurotransmitter biosynthetic enzymes suggests that these tumors arise in different cells of the PNS.
Genomic analyses of histaminergic signaling in daphnids The recent release of the D. pulex genome provides a unique resource in crustacean biology, as thus far it is the only crustacean genome sequenced and available for public use. This resource has been used previously to glean information concerning the neurochemistry of D. pulex. Specifically, the peptides used by D. pulex as locally released neuromodulators and/or circulating neurohormones were deduced via genome mining and bioinformatics (Christie et al., 2011). Here, we have complemented our immunohistochemical mapping of histamine in the daphnid CNS with mining of the D. pulex genome for genes encoding key players in the histaminergic signaling pathway. Specifically, the genome was mined for orthologs of HDC, the rate-limiting biosyntheticenzyme of histamine, as well as for orthologs of two hcls; D. melanogaster sequences were used for this mining. Putative D. pulex genes for each of these proteins were identified. The predicted D. pulex HDC is highly similar in amino acid sequence (85%) to that of D. melanogaster. Similarly, the D. pulex protein orthologs of A- and B-type hcls show high levels of amino acid conservation with their D. melanogaster counterparts (89 and 74%, respectively). Although they are currently predictions, these putative D. pulex histaminergic pathway proteins are, to the best of our knowledge, the first HDC and hcls described from any crustacean. Moreover, the discovery of the genes encoding these molecules now allows for studies of their distribution in daphnids, as well as providing templates for searching the genomes and transcriptomes of other crustacean species for the genes/mRNAs encoding similar proteins (genes nearly identical in nucleotide sequence to those of D. pulex HDC, hclA and hclB are also present in an as of yet unreleased assembly of the D. magna genome (M.D.McC., A.E.C. and J. R. Shaw, unpublished). Likewise, the discovery of these D. pulex genes provide molecular targets for assessing whether specific environmental and or anthropogenic stressors might alter the expression of these proteins and hence influence histaminergic signaling in this important ecotoxicological model species.
of a core peptide of a leaderless substrate. Upon addition of exogenous ‘minimal’ leader or full-length leader peptide, the processing ability of LynD is restored, cyclising both cysteine residues in the leaderless substrate. The efficiency of this trans activation of LynD towards leaderless substrates was significantly increased by fusing the important leader peptide residues to the N-terminus of LynD, creating an ‘activated’ enzyme: AcLynD. AcLynD has been shown to efficiently process multiple leaderless substrates, and at a rate comparable to that of the native enzyme with the full-length PatE 0 substrate. The ability to dispose of the leader peptide is highly advantageous from a biotechnological perspective, as it allows for the synthesis of diverse cyanobactin analogues in vitro, starting from shorter, thus more economic substrates. Furthermore, shorter peptides are more amenable to chemical synthesis, allowing for the incorporation of non-natural amino acids, o↵ering an explosion in diversity. Moreover AcLynD, and homologues thereof are potentially capable of installing heterocycles in substrates, unrelated to cyanobactins, vastly increasing their potential biotechnogical application.
Genome sequencing of secondary-metabolite-producing microorganisms has re- vealed an enormous potential to increase the known chemical space (5), with the promise of new leads in human therapies or for sustainable agriculture. One of the drivers of the renewed interest in NPs was the discovery of so-called cryptic BGCs that are silent under routine laboratory conditions and may therefore specify molecules that had so far been missed during pharmaceutical screening (7–9). An approach that is rapidly gaining momentum is that of expressing cryptic BGCs in a heterologous chassis strain or superhost and exchanging promoter elements within the BGC with those that are expected to result in high levels of expression under laboratory conditions. How- ever, there are several problems associated with this approach. First, it is still difﬁcult to effectively apply it through high-throughput strategies, and it is hard to establish the promise of the activity of a certain BGC on the basis of the DNA sequence alone (10). Second, examples of NPs that are produced from multiple BGCs, or by strains in coculture, are accumulating (11, 12). Alternatively, a single BGC may also be responsible for the production of many (up to over 100) structurally related molecules that differ in terms of their activity (13–15), or BGCs can be associated into “superclusters” that function to produce two or more similar molecules (16, 17). Thus, to optimally exploit the chemical space of NPs, we need to understand the connection between the genomic diversity and the chemical diversity of their biosynthetic pathways.
Many species analyzed in this study that did not produce iri- doids had no detectable GES expression, suggesting that GES is a key gatekeeping step in iridoid biosynthesis. Despite ISY being responsible for the iridoid scaffold formation and catalyzing the first committed step in iridoid biosynthesis, GES is responsible for diverting metabolic flux away from canonical monoterpenes by converting GPP to geraniol. The association of increased expression of genes encoding the upstream en- zymes 1-deoxyxylulose 5-phosphate synthase (DXS; P = 0.0260) and geranylgeranyl pyrophosphate synthase large sub- unit (GGPPS-LSU; P = 0.0011) (Figure 4A and Supplemental Table 9) with iridoid biosynthesis is also notable. DXS is a rate-limiting enzyme in the MEP pathway, which provides pre- cursors for iridoid formation (Estevez et al., 2001). GGPPS- LSU can form GPP when acting with geranyl pyrophosphate synthase small subunit (GPPS-SSU; Rai et al., 2013), although it is surprising that no correlation with GPPS-SSU was observed as this protein causes the enzyme complex to produce GPP as its major product. In Catharanthus roseus, two different en- zymes have been proposed to act as 8-hydroxygeraniol oxido- reductase, 8-hydroxygeraniol oxidoreductase A (8HGOA; KF302069; Miettinen et al., 2014) and 8-hydroxygeraniol oxido- reductase B (8HGOB; AY352047; Krithika et al., 2015). In this study, both enzyme expression patterns correlate significantly with the presence of iridoids, although 8HGOA shows a more significant association (P = 0.0001 versus P = 0.0042) and only 8HGOB shows a significant association with orthogroup occupancy (P = 0.0083); the precise physiological and biochemical roles of 8HGOA and 8HGOB remain to be determined experimentally.
S. scabies 87.22 has become a model organism for studying plant-microbe interactions among Gram-positive plant pathogenic bacteria and the genome sequence for this strain is now published (GenBank Accession no. FN554889). Three out of four of the biosynthetic gene clusters investigated in this work are responsible for the production of the siderophores pyochelin, scabichelin and desferrioxamines, which are iron-chelating molecules. Iron acquisition through the use of siderophores is crucial for the development of infection in the mammalian host by pathogenic bacteria (Miethke and Marahiel, 2007). Studies of iron uptake were mostly conducted in Gram negative bacteria, whereas investigations into iron acquisition by Gram positive plant pathogens remained limited. Analysis of the S. scabies secondary metabolite biosynthetic gene clusters and its metabolic products is important to increase the understanding of its potential role in plant pathogenicity. For example, several genes encoding for the biosynthesis of plant toxins in these S. scabies secondary metabolite biosynthetic gene clusters, were found to be significant virulence factors. Awareness of these virulence factors can be used in the future in the agrochemical development to prevent infection by that pathogen potentially saving important agricultural crops from damage.
Artemisinin synthesis occurs in the glandular tri- chomes (GLTs) present on flowers, floral buds and leaves. Trichome-specific fatty acyl-CoA reductase 1 (TAFR1) is thought to be involved in GLT development and ses- quiterpenoid biosynthesis; which are important for artemisinin production [5, 6]. Other enzymes like amor- phadiene synthase (ADS), cytochrome P450, CYP71AV1 (CYP), double bond reductase 2 (DBR2) and aldehyde dehydrogenase 1 (ALDH1) catalyze different steps of artemisinin biosynthesis [7–11]. Overexpression of these enzymes may lead to increased artemisinin accumulation and also the derivatives. Some studies provide evidence that metabolic engineering of the biosynthetic pathway that leads to artemisinin by the insertion of different genes can increase artemisinin content in planta [12, 13]. In this regard, rol genes of A. rhizogenes are considered to be effective inducers of secondary metabolites produc- tion in plants . Rol A is a DNA binding protein and stimulator of growth, rol B having tyrosine phosphatase activity regulates signal transduction pathway of auxin [15, 16], and rol C has cytokinin glucosidase activity. However, each gene seems to affect plant morphology and stimulate production of different secondary metabo- lites [17–20]. However, there is no report about produc- tion of transgenic A. annua plants with individual rol genes for enhancement of artemisinin and its derivatives. Previous work on transformation of other Artemisia sp. with rol genes showed not only over expression of arte- misinin biosynthetic genes, but also artemisinin in the plant .
RimP affects the translational efficiency and fidelity in E. coli As a ribosome assembly cofactor, RimP may affect transla- tional efficiency. Thus, the translational accuracy was measured using the mutated xylE as a reporter which con- tains a UGA stop codon instead of a UGG tryptophan codon at 47 position. When the stop codon was decoded by a near-cognate tRNA, the full length catechol dioxygenase was expressed and showed enzyme activ- ity. To check the expression of catechol dioxygenase, all recombinant strains (BW25113/pSET152::rrnFp::xylE, rimPDM/pSET152::rrnFp::xylE, BW25113/pSET152::rrnFp:: xylE * and rimPDM/pSET152::rrnFp::xylE * ) were cultured in LB medium at 37°C. Under this condition, the growth rate of BW25113 was a little faster than rimPDM (Figure 4A). Meanwhile, the expression level of wild-type catechol dioxygenase in BW25113 was almost the same as rimPDM (Figure 4B). When introducing a UGA stop codon into the wild-type xylE (named as xylE * ), the catechol dioxygenase activity decreased 3 orders of magnitude. Meanwhile, the activity of XylE * decreased almost 2–4 folds in rimPDM
In the last few years, there has been a resurgence of interest in the discovery of natural products, and this resurgence has been fueled by the explosion in the availability of microbial genomic sequences and the expansion of sampling to previously unstud- ied habitats and environments. However, a very large gap exists between the throughput of sequencing and the rate of discov- ery of novel pathways involved in secondary metabolism, pre- dominantly because of the absence of tools that facilitate this intellectually and computationally difficult task. Additionally, no public resources exist that allow for the global analysis and comparison of putative biosynthetic gene clusters. These anal- yses have been accessible only to laboratories staffed with com- FIG 7 Diversity of architectures of phenazine biosynthetic gene clusters. (A) Architecturally unique BCs containing all core genes of phenazine biosynthesis were sorted based on similarity to the Rhizobium leguminosarum phenazine gene cluster (blue box), which were calculated by using a modification of the average nucleotide identity approach (ANI) (38). Amino acid sequences of proteins encoded in the BC were used to identify bidirectional best hits (BBH) using USEARCH (39). Additionally, amino acid similarity was evaluated instead of identity. For a set of BBH proteins, the average amino acid similarity was calculated in addition to the alignment fraction (AF). The similarity score was calculated as the product of these two values for the R. leguminosarum BC (AAS*AF). Phylum classifications are abbreviated as follow: ACT, Actinobacteria; AP, Alphaproteobacteria; BP, Betaproteobacteria; GP, Gammaproteobacteria. Phenazine clusters that have been previously studied experimentally are underlined. (B) Evidence for the horizontal transfer of the Rhizobium leguminosarum phenazine BC (box). A BLASTp search was performed with each protein in and around the BC as the query against all proteins in the IMG database. Each gene is colored (see legend) based on the phylogenetic class of its top hit (see Table S1 in the supplemental material). Genes marked with an asterisk are transposases.
Abstract: 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 charac- terized 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.
 summarized the current understanding of the pathway of biosynthesis, the function of cloned tri gene and evolution of Tri gene, focusing on Fusarium species. Following the isolation of Tri5 gene encoding the first enzyme trichodine synthase, 10 biosynthetic gene (two regulatory gene, seven pathway gene and one transporter gene were functionally identified in the Tri 5 gene cluster. At least three pathway gene, Tri 101