Enzyme In Biosynthesis Pathway Research Articles

Transcription Factor Engineering for High-Throughput Strain Evolution and Organic Acid Bioproduction: A Review.

Metabolic regulation of gene expression for the microbial production of fine chemicals, such as organic acids, is an important research topic in post-genomic metabolic engineering. In particular, the ability of transcription factors (TFs) to respond precisely in time and space to various small molecules, signals and stimuli from the internal and external environment is essential for metabolic pathway engineering and strain development. As a key component, TFs are used to construct many biosensors in vivo using synthetic biology methods, which can be used to monitor the concentration of intracellular metabolites in organic acid production that would otherwise remain “invisible” within the intracellular environment. TF-based biosensors also provide a high-throughput screening method for rapid strain evolution. Furthermore, TFs are important global regulators that control the expression levels of key enzymes in organic acid biosynthesis pathways, therefore determining the outcome of metabolic networks. Here we review recent advances in TF identification, engineering, and applications for metabolic engineering, with an emphasis on metabolite monitoring and high-throughput strain evolution for the organic acid bioproduction.

Two Distinct Mechanisms of Inhibition of LpxA Acyltransferase Essential for Lipopolysaccharide Biosynthesis.

The lipopolysaccharide biosynthesis pathway is considered an attractive drug target against the rising threat of multi-drug-resistant Gram-negative bacteria. Here, we report two novel small-molecule inhibitors (compounds 1 and 2) of the acyltransferase LpxA, the first enzyme in the lipopolysaccharide biosynthesis pathway. We show genetically that the antibacterial activities of the compounds against efflux-deficient Escherichia coli are mediated by LpxA inhibition. Consistently, the compounds inhibited the LpxA enzymatic reaction in vitro. Intriguingly, using biochemical, biophysical, and structural characterization, we reveal two distinct mechanisms of LpxA inhibition; compound 1 is a substrate-competitive inhibitor targeting apo LpxA, and compound 2 is an uncompetitive inhibitor targeting the LpxA/product complex. Compound 2 exhibited more favorable biological and physicochemical properties than compound 1 and was optimized using structural information to achieve improved antibacterial activity against wild-type E. coli. These results show that LpxA is a promising antibacterial target and imply the advantages of targeting enzyme/product complexes in drug discovery.

A phosphorylation-based switch controls TAA1-mediated auxin biosynthesis in plants

Auxin determines the developmental fate of plant tissues, and local auxin concentration is precisely controlled. The role of auxin transport in modulating local auxin concentration has been widely studied but the regulation of local auxin biosynthesis is less well understood. Here, we show that TRYPTOPHAN AMINOTRANSFERASE OF ARABIDOPSIS (TAA1), a key enzyme in the auxin biosynthesis pathway in Arabidopsis thaliana is phosphorylated at Threonine 101 (T101). T101 phosphorylation status can act as an on/off switch to control TAA1-dependent auxin biosynthesis and is required for proper regulation of root meristem size and root hair development. This phosphosite is evolutionarily conserved suggesting post-translational regulation of auxin biosynthesis may be a general phenomenon. In addition, we show that auxin itself, in part via TRANS-MEMBRANE KINASE 4 (TMK4), can induce T101 phosphorylation of TAA1 suggesting a self-regulatory loop whereby local auxin signalling can suppress biosynthesis. We conclude that phosphorylation-dependent control of TAA1 enzymatic activity may contribute to regulation of auxin concentration in response to endogenous and/or external cues.

Transcription of lncRNA ACoS-AS1 is essential to trans-splicing between SlPsy1 and ACoS-AS1 that causes yellow fruit in tomato

ABSTRACTPhytoene synthase (PSY) has been considered as an important regulatory enzyme in carotenoids biosynthesis pathway. Previous study finds that the yellow fruit in Solanum lycopersicum var. cerasiforme accession PI 114490 is caused by loss-of-function of SlPSY1 due to trans-splicing between SlPsy1 and an unknown gene transcribed from neighbour opposite strand DNA of SlPsy1. The genomic DNA sequences of SlPsy1 between red and yellow-fruited tomato lines have one single-nucleotide polymorphism (SNP) in the fourth intron and one SSR in the intergenic region. In the current study, the cause of trans-splicing event was further investigated. The data showed that the previously defined unknown gene was a putative long non-coding RNA ACoS-AS1 with three variants in many yellow-fruited tomato lines. The intronic SNP and intergenic SSR were tightly associated with trans-splicing event SlPsy1-ACoS-AS1. However, transgenic tomato lines carrying the genomic DNA of SlPsy1 from PI 114490 did not generate transcripts of ACoS-AS1and SlPsy1-ACoS-AS1 suggesting that only the intronic SNP could not cause the trans-splicing event. Over-expression of SlPsy1-ACoS-AS1 in red-fruited tomato line M82 did not have any phenotype change while over-expression of wild type SlPsy1 resulted in altered leaf colour. Sub-cellular localization analysis showed that SlPSY1-ACoS-AS1 could not enter plastids where SlPSY1 has its enzyme activity. Mutation of ACoS-AS1 in PI 114490 generated by CRISPR/Cas9 techniques resulted in red fruits implying that ACoS-AS1 was essential to trans-splicing event SlPsy1-ACoS-AS1. The results obtained here will extend knowledge to understand the mechanism of trans-splicing event SlPsy1-ACoS-AS1 and provide additional information for the regulation of carotenoids biosynthesis.

Functional and structural analysis of trehalose-6-phosphate phosphatase from Burkholderia pseudomallei: Insights into the catalytic mechanism

We report the functional and structural characterization of trehalose-6-phosphate phosphatase (TPP), from the Gram-negative bacterium B. pseudomallei that causes melioidosis, a severe infectious disease endemic in Southeast Asia and Northern Australia. TPP is a key enzyme in the trehalose biosynthesis pathway, which plays an important role in bacterial stress responses. Due to the absence of this biosynthetic pathway in mammals, TPP has drawn attention as a potential drug target, to combat antibiotic resistance.In this context, we present a detailed biochemical analysis of purified recombinant TPP, reporting its specific high catalytic activity toward the trehalose-6-phosphate substrate, and an absolute requirement for its Mg2+ cofactor. Furthermore, we present the crystal structure of TPP solved at 1.74 Å, revealing the canonical haloacid dehalogenase (HAD) superfamily fold and conserved substrate binding pocket, from which insights into the catalytic mechanism may be deduced. Our data represent a starting point for the rational design of antibacterial drugs.

Statins Limit Coenzyme Q Synthesis and Metabolically Synergize with MEK Inhibition in Pancreatic Tumors.

Tumors frequently increase expression of enzymes in the mevalonate biosynthesis pathway. Statins inhibit flux through this pathway, but if and how such treatments elicit a therapeutic benefit in cancer remains unclear. In this issue of Cancer Research, McGregor and colleagues perform in vivo metabolic tracing to demonstrate that mouse pancreatic ductal adenocarcinoma (PDAC) tumors and human PDAC cell lines require this pathway for coenzyme Q (CoQ) synthesis and redox homeostasis. Simvastatin treatment reduces CoQ synthesis and promotes oxidative stress and apoptosis in tumors when administered in combination with a MEK inhibitor, providing a new mechanism through which statin treatment may impact PDAC growth.See related article by McGregor et al., p. 175.

Unraveling the importance of molecules of natural origin in antifungal drug development through targeting ergosterol biosynthesis pathway

Over the past decades, the incidence of life-threatening fungal infections has increased dramatically in particular among patients with hampered immune function. Fungal infections cause around 1.5 million deaths annually, superior to malaria and tuberculosis. With respect to high toxicity, narrow spectrum of activity and drug resistance to current antifungals, there is an urgent need to discover novel leads from molecules of natural origin especially those derived from plants and microorganisms for antifungal drug discovery. Among antifungal drugs introduced into the clinic, those affecting ergosterol biosynthesis are still superior to other classes and the vital role of ergosterol in fungal growth and development. This review highlights current knowledge about available antifungal agents and further issues on antifungal drug discovery from compounds of natural origin which affect ergosterol biosynthesis. Special attention is made to the fungal sterol C24-methyltransferase (SMT), a crucial enzyme in ergosterol biosynthesis pathway as a novel target for rational drug design.

In silico identification and structure function analysis of a putative coclaurine N-methyltransferase from Aristolochia fimbriata

In this study we isolated and performed in silico analysis of a putative coclaurine N-methyltransferase (CNMT) from the basal angiosperm Aristolochia fimbriata. The Aristolochiaceae plant family produces alkaloids similar to the Papavaraceae family, and CNMTs are central enzymes in biosynthesis pathways producing compounds of ethnopharmacological interest. We used bioinformatics and computational tools to predict a three-dimensional homology model and to investigate the putative function of the protein and its mechanism for methylation. The putative CNMT is a unique (S)-adenosyl-L-methionine (SAM)-dependent N-methyltransferase, catalyzing transfer of a methyl group from SAM to the amino group of coclaurine. The model revealed a mixed α/β structure comprising seven twisted β-strands surrounded by twelve α-helices. Sequence comparisons and the model indicate an N-terminal catalytic Core domain and a C-terminal domain, of which the latter forms a pocket for coclaurine. An additional binding pocket for SAM is connected to the coclaurine binding pocket by a small opening. CNMT activity is proposed to follow an SN2-type mechanism as observed for a similarly conformed enzyme. Residues predicted for the methyl transfer reaction are Tyr79 and Glu96, which are conserved in the sequence from A. fimbriata and in homologous N-methyltransferases. The isolated CNMT is the first to be investigated from any basal angiosperm.

Overexpression of the GbF3'H1 Gene Enhanced the Epigallocatechin, Gallocatechin, and Catechin Contents in Transgenic Populus.

Ginkgo biloba L. leaves are a flavonoid resource for the pharmaceutical industry. The flavonoid 3'-hydroxylase (F3'H) is a key enzyme in the flavonoid biosynthesis pathway. However, the role of F3'H in flavonoid biosynthesis and metabolism is unclear. In this study, we characterized and functionally analyzed the ginkgo F3'H gene GbF3'H1 that encodes a protein of 520 amino acids. Expression profiling showed that GbF3'H1 was highly expressed in the leaves of ginkgo in September. Subcellular localization showed that GbF3'H1 occurred predominately in the cytoplasm. Transgenic poplars overexpressing GbF3'H1 had more red pigmentation in leaves than did wild-type (WT) plants. Furthermore, the concentrations of epigallocatechin, gallocatechin, and catechin in the downstream products synthesized by flavonoids were significantly higher in the transgenic plants than in the WT plants. These results indicate that the overexpression of GbF3'H1 enhances flavonoid production in transgenic plants and provides new insights into flavonoid biosynthesis and metabolism.

Fatty Acids Regulate Porcine Reproductive and Respiratory Syndrome Virus Infection via the AMPK-ACC1 Signaling Pathway.

Lipids play a crucial role in the replication of porcine reproductive and respiratory syndrome virus (PRRSV), a porcine virus that is endemic throughout the world. However, little is known about the effect of fatty acids (FAs), a type of vital lipid, on PRRSV infection. In this study, we found that treatment with a FA biosynthetic inhibitor significantly inhibited PRRSV propagation, indicating the necessity of FAs for optimal replication of PRRSV. Further study revealed that 5′-adenosine monophosphate (AMP)-activated protein kinase (AMPK), a key kinase antagonizing FA biosynthesis, was strongly activated by PRRSV and the pharmacological activator of AMPK exhibited anti-PRRSV activity. Additionally, we found that acetyl-CoA carboxylase 1 (ACC1), the first rate-limiting enzyme in the FA biosynthesis pathway, was phosphorylated (inactive form) by PRRSV-activated AMPK, and active ACC1 was required for PRRSV proliferation, suggesting that the PRRSV infection induced the activation of the AMPK–ACC1 pathway, which was not conducive to PRRSV replication. This work provides new evidence about the mechanisms involved in host lipid metabolism during PRRSV infection and identifies novel potential antiviral targets for PRRSV.

Integrative transcriptome analysis discloses the molecular basis of a heterogeneous fungal phytopathogen complex, Rhizoctonia solani AG-1 subgroups

Rhizoctonia solani is a fungal species complex that causes necrotrophic crop diseases. It comprises several anastomosis groups, some of which include intra-subgroups, such as AG-1 IA and AG-1 IB, exhibiting varying pathogenicity. Owing to its heterozygous and multinucleate features, genomic analyses of R. solani are still challenging, and understanding of its genetic diversity and genic components is limited. In this study, in order to elucidate the molecular basis of this phytopathogen complex, an integrated transcriptome analysis was undertaken for three subgroups of AG-1, i.e. AG-1 IA, AG-1 IB, and AG-1 IC. Sequence variations suggested substantial evolutionary distances within AG-1. Transcript simple sequence repeats showed comparable characteristics among AG-1, but contained polymorphic sites. Intra-subgroup polymorphisms suggested varying genic heterozygosity within AG-1, suggesting their independent evolutionary trajectory. Sequences of pathogenic factors, phytotoxin biosynthesis pathway enzymes, secreted lignocellulosic enzymes, secreted reactive oxygen species detoxification enzymes, apoplastic/cytoplasmic effector candidates, were conserved among those subgroups. dN/dS ratios of a secretome subset suggested core secreted proteins in AG-1 and distinct evolution of Cys-rich small secreted proteins after differentiation of AG-1 subgroups. Identification of likely pathogenic factors including allergen protein homologues, oxidative phosphorylation and ethylene biosynthesis pathways, and diversification of polysaccharide monooxygenases provides molecular insight into key genomic components that play a role in R. solani pathogenesis.

Sp1 Mediates the Constitutive Expression and Repression of the PDSS2 Gene in Lung Cancer Cells.

Prenyl diphosphate synthase subunit 2 (PDSS2) is the first key enzyme in the CoQ10 biosynthesis pathway, and contributes to various metabolic and nephritic diseases. It has been reported that PDSS2 is downregulated in several types of tumors and acts as a potential tumor suppressor gene to inhibit the proliferation and migration of cancer cells. However, the regulatory mechanism of PDSS2 expression remains elusive. In the present study, we first identified and characterized the PDSS2 promoter region. We established four different luciferase reporter constructs which mainly cover the 2 kb region upstream of the PDSS2 gene transcription initiation site. Series luciferase reporter assay demonstrated that all four constructs have prominent promoter activity, and the core promoter of PDSS2 is mainly located within the 202 bp region near its transcription initiation site. Transcription factor binding site analysis revealed that the PDSS2 promoter contains binding sites for canonical transcription factors such as Sp1 and GATA-1. Overexpression of Sp1 significantly inhibited PDSS2 promoter activity, as well as its endogenous expression, at both mRNA and protein levels in lung cancer cells. Site-directed mutagenesis assay further confirmed that the Sp1 binding sites are essential for proximal prompter activity of PDSS2. Consistently, a selective Sp1 inhibitor, mithramycin A, treatment repressed the PDSS2 promoter activity, as well as its endogenous expression. Chromatin immunoprecipitation (ChIP) assay revealed that Sp1 binds to the PDSS2 promoter in vivo. Of note, the expression of Sp1 and PDSS2 are negatively correlated, and higher Sp1 expression with low PDSS2 expression is significantly associated with poor prognosis in lung cancer. Taken together, our results strongly suggest the essential role of Sp1 in maintaining the basic constitutive expression of PDSS2, and the pathogenic implication of Sp1-mediated PDSS2 transcriptional repression in lung cancer cells.

Targeting DHODH with AG-636 Induces Apoptosis and Differentiation and Inhibits Mitochondrial Function in AML, Translating into Anti-Tumor Efficacy in Vitro and in Vivo

Targeting DHODH with AG-636 Induces Apoptosis and Differentiation and Inhibits Mitochondrial Function in AML, Translating into Anti-Tumor Efficacy in Vitro and in Vivo

Structure-based design of guanosine analogue inhibitors targeting GTP cyclohydrolase IB towards a new class of antibiotics

GTP cyclohydrolase (GCYH-I) is an enzyme in the folate biosynthesis pathway that has not been previously exploited as an antibiotic target, although several pathogens including N. gonorrhoeae use a form of the enzyme GCYH-IB that is structurally distinct from the human homologue GCYH-IA. A comparison of the crystal structures of GCYH-IA and -IB with the nM inhibitor 8-oxo-GTP bound shows that the active site of GCYH-IB is larger and differently shaped. Based on this structural information, we designed and synthesized a small set of 8-oxo-G derivatives with ether linkages at O6 and O8 expected to displace water molecules from the expanded active site of GCYH-IB. The most potent of these compounds, G3, is selective for GCYH-IB, supporting the premise that potent and selective inhibitors of GCYH-IB could constitute a new class of small molecule antibiotics.

Differential effects of simvastatin on membrane organization and dynamics in varying phases

Simvastatin belongs to the statin family of cholesterol lowering drugs which act as competitive inhibitors of HMG-CoA reductase, the rate-determining enzyme in cholesterol biosynthesis pathway. Simvastatin is a semi-synthetic, highly lipophilic statin, and has several side effects. Since HMG-CoA reductase is localized in the endoplasmic reticulum, orally administered simvastatin needs to cross the cellular plasma membrane to be able to act on HMG-CoA reductase. With an overall goal of exploring the interaction of simvastatin with membranes, we examined the effect of simvastatin on the organization and dynamics in membranes of varying phase, in a depth-dependent manner. For this, we employed DPH and TMA-DPH, which represent fluorescent membrane probes localized at two different locations (depths) in the membrane. Analysis of fluorescence anisotropy and lifetime data of these depth-specific probes in membranes of varying phase (gel/fluid/liquid-ordered) showed that the maximum membrane disordering was observed in gel phase, while moderate effects were observed in liquid-ordered phase, with no significant change in membrane order in fluid phase membranes. We conclude that simvastatin induces change in membrane order in a depth-dependent and phase-specific manner. These results provide novel insight in the membrane interaction of simvastatin and could be crucial for understanding its pharmacological effect.

A distinct lineage of giant viruses brings a rhodopsin photosystem to unicellular marine predators

Giant viruses are remarkable for their large genomes, often rivaling those of small bacteria, and for having genes thought exclusive to cellular life. Most isolated to date infect nonmarine protists, leaving their strategies and prevalence in marine environments largely unknown. Using eukaryotic single-cell metagenomics in the Pacific, we discovered a Mimiviridae lineage of giant viruses, which infects choanoflagellates, widespread protistan predators related to metazoans. The ChoanoVirus genomes are the largest yet from pelagic ecosystems, with 442 of 862 predicted proteins lacking known homologs. They are enriched in enzymes for modifying organic compounds, including degradation of chitin, an abundant polysaccharide in oceans, and they encode 3 divergent type-1 rhodopsins (VirR) with distinct evolutionary histories from those that capture sunlight in cellular organisms. One (VirRDTS) is similar to the only other putative rhodopsin from a virus (PgV) with a known host (a marine alga). Unlike the algal virus, ChoanoViruses encode the entire pigment biosynthesis pathway and cleavage enzyme for producing the required chromophore, retinal. We demonstrate that the rhodopsin shared by ChoanoViruses and PgV binds retinal and pumps protons. Moreover, our 1.65-Å resolved VirRDTS crystal structure and mutational analyses exposed differences from previously characterized type-1 rhodopsins, all of which come from cellular organisms. Multiple VirR types are present in metagenomes from across surface oceans, where they are correlated with and nearly as abundant as a canonical marker gene from Mimiviridae Our findings indicate that light-dependent energy transfer systems are likely common components of giant viruses of photosynthetic and phagotrophic unicellular marine eukaryotes.

The essential role of the transporter ABCG2 in the pathophysiology of erythropoietic protoporphyria.

Erythropoietic protoporphyria (EPP) is an inherited disease caused by loss-of-function mutations of ferrochelatase, an enzyme in the heme biosynthesis pathway that converts protoporphyrin IX (PPIX) into heme. PPIX accumulation in patients with EPP leads to phototoxicity and hepatotoxicity, and there is no cure. Here, we demonstrated that the PPIX efflux transporter ABCG2 (also called BCRP) determines EPP-associated phototoxicity and hepatotoxicity. We found that ABCG2 deficiency decreases PPIX distribution to the skin and therefore prevents EPP-associated phototoxicity. We also found that ABCG2 deficiency protects against EPP-associated hepatotoxicity by modulating PPIX distribution, metabolism, and excretion. In summary, our work has uncovered an essential role of ABCG2 in the pathophysiology of EPP, which suggests the potential for novel strategies in the development of therapy for EPP.

High levels of expression of multiple enzymes in the Smirnoff-Wheeler pathway are important for high accumulation of ascorbic acid in acerola fruits.

Acerola fruits contain abundant ascorbic acid (AsA). The gene expression levels of three upstream enzymes in the primary AsA biosynthesis pathway were correlated with AsA contents in the fruits of two acerola cultivars. Multiple overexpression of the enzymes increased AsA contents, suggesting their high expression is important for high AsA accumulation in acerola fruits and the breeding of AsA-rich plants. Abbreviations: AsA: ascorbic acid; PMI: phosphomannose isomerase; PMM: phosphomannomutase; GMP: GDP-d-mannose pyrophosphorylase; GME: GDP-d-mannose 3',5'-epimerase; GGP: GDP-l-galactose phosphorylase; GPP: l-galactose-1-phosphate phosphatase; GDH: l-galactose dehydrogenase; GLDH: l-galactono-1,4-lactone dehydrogenase.

δ-Aminolevulinate induces fetal hemoglobin expression by enhancing cellular heme biosynthesis.

Sickle cell disease (SCD) and β-thalassemia are inherited blood disorders caused by genetic defects in the β-globin gene on chromosome 11, producing severe disease in people worldwide. Induction of fetal hemoglobin consisting of two α-globin and two γ-globin chains ameliorates the clinical symptoms of both disorders. In the present study, we investigated the ability of δ-aminolevulinate (ALA), the heme precursor, to activate γ-globin gene expression as well as its effects on cellular functions in erythroid cell systems. We demonstrated that ALA induced γ-globin expression at both the transcriptional and protein levels in the KU812 erythroid cell line. Using inhibitors targeting two enzymes in the heme biosynthesis pathway, we showed that cellular heme biosynthesis was involved in ALA-mediated γ-globin activation. Moreover, the transcription factor NRF2 (nuclear factor [erythroid-derived 2]-like 2), a critical regulator of the cellular antioxidant response, was activated by ALA and contributed to mechanisms of γ-globin activation; ALA did not affect cell proliferation and was not toxic to cells. Subsequent studies demonstrated ALA-induced γ-globin activation in erythroid progenitors generated from normal human CD34+ stem cells. These data support future study to explore the potential of stimulating intracellular heme biosynthesis by ALA or similar compounds as a novel therapeutic strategy for treating SCD and β-thalassemia. Impact statement Inherited mutations in the β-globin-like genes result in the most common forms of genetic blood disease including sickle cell disease (SCD) and β-thalassemia worldwide. Therefore, effective inexpensive therapies that can be distributed widely are highly desirable. Currently, drug-mediated fetal hemoglobin (HbF) induction can ameliorate clinical symptoms of SCD and β-thalassemia and is the most effective strategy for developing new therapeutic options. In the current study, we confirmed that δ-Aminolevulinate (ALA), the precursor of heme, induces γ-globin expression at both the transcriptional and translational levels in primary human erythroid progenitors. Moreover, the results indicate activation of the transcription factor NRF2 (nuclear factor (erythroid-derived 2)-like 2) by ALA to enhance HbF expression. These data support future study to explore the potential of stimulating intracellular heme biosynthesis by ALA or similar compounds as a novel therapeutic strategy for treating SCD and β-thalassemia.

Cloning,expression and characterization of chalcone isomerase from medicinal plant Chinese sumac (Rhus chinensis)

Flavonoids are a group of secondary metabolites found in plants. They have many pharmacological functions and play an important role in Chinese sumac( Rhus chinensis),which is a well-known traditional Chinese medicinal plant. Chalcone isomerase( CHI,EC 5. 5. 1. 6) is one of the key enzymes in the flavonoids biosynthesis pathway. In this paper,the full-length c DNA sequence encoding the chalcone isomerase from R. chinensis( designated as Rc CHI) was cloned by RT-PCR and rapid-amplification of c DNA Ends( RACE). The Rc CHI c DNA sequence was 1 058 bp and the open reading frame( ORF) was 738 bp. The ORF predicted to encode a 245-amino acid polypeptide. Rc CHI gene contained an intron and two exons. The sequence alignments revealed Rc CHI shared47. 1%-71. 6% identity with the homologues in other plants. Real-time PCR analysis showed that the total flavonoid levels were positively correlated with tissue-specific expressions of Rc CHI mRNA in different tissues. The recombinant protein was successfully expressed in an Escherichia coli strain with the p GEX-6 P-1 vector. In this paper,the CHI gene was cloned and characterized in the family of Anacardiaceae and will help us to obtain better knowledge of the flavonoids biosynthesis of the flavonoid compounds in R. chinensis.