scholarly journals Biochemical and Structural Studies of NADH-Dependent FabG Used To Increase the Bacterial Production of Fatty Acids under Anaerobic Conditions

2013 ◽  
Vol 80 (2) ◽  
pp. 497-505 ◽  
Author(s):  
Pouya Javidpour ◽  
Jose H. Pereira ◽  
Ee-Been Goh ◽  
Ryan P. McAndrew ◽  
Suzanne M. Ma ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Methyl Ketone ◽  
Fatty Acid Biosynthesis ◽  
Cell Physiology ◽  
Crystallographic Structure ◽  
Anaerobic Conditions ◽  
Content Type ◽  
Atp Production ◽  
Starting Point

ABSTRACTMajor efforts in bioenergy research have focused on producing fuels that can directly replace petroleum-derived gasoline and diesel fuel through metabolic engineering of microbial fatty acid biosynthetic pathways. Typically, growth and pathway induction are conducted under aerobic conditions, but for operational efficiency in an industrial context, anaerobic culture conditions would be preferred to obviate the need to maintain specific dissolved oxygen concentrations and to maximize the proportion of reducing equivalents directed to biofuel biosynthesis rather than ATP production. A major concern with fermentative growth conditions is elevated NADH levels, which can adversely affect cell physiology. The purpose of this study was to identify homologs ofEscherichia coliFabG, an essential reductase involved in fatty acid biosynthesis, that display a higher preference for NADH than for NADPH as a cofactor. Four potential NADH-dependent FabG variants were identified through bioinformatic analyses supported by crystallographic structure determination (1.3- to 2.0-Å resolution).In vitroassays of cofactor (NADH/NADPH) preference in the four variants showed up to ∼35-fold preference for NADH, which was observed with theCupriavidus taiwanensisFabG variant. In addition, FabG homologs were overexpressed in fatty acid- and methyl ketone-overproducingE. colihost strains under anaerobic conditions, and theC. taiwanensisvariant led to a 60% higher free fatty acid titer and 75% higher methyl ketone titer relative to the titers of the control strains. With further engineering, this work could serve as a starting point for establishing a microbial host strain for production of fatty acid-derived biofuels (e.g., methyl ketones) under anaerobic conditions.

scholarly journals A Caleosin-Like Protein with Peroxygenase Activity Mediates Aspergillus flavus Development, Aflatoxin Accumulation, and Seed Infection

2015 ◽  
Vol 81 (18) ◽  
pp. 6129-6144 ◽  
Author(s):  
Abdulsamie Hanano ◽  
Ibrahem Almousally ◽  
Mouhnad Shaban ◽  
Elizabeth Blee
Keyword(s):  
Fatty Acid ◽  
Aspergillus Flavus ◽  
Polyunsaturated Fatty Acid ◽  
Calcium Binding ◽  
Antioxidant Activities ◽  
Fungal Growth ◽  
Oxidation Reactions ◽  
Content Type ◽  
Fatty Acid Hydroperoxides

ABSTRACTCaleosins are a small family of calcium-binding proteins endowed with peroxygenase activity in plants. Caleosin-like genes are present in fungi; however, their functions have not been reported yet. In this work, we identify a plant caleosin-like protein inAspergillus flavusthat is highly expressed during the early stages of spore germination. A recombinant purified 32-kDa caleosin-like protein supported peroxygenase activities, including co-oxidation reactions and reduction of polyunsaturated fatty acid hydroperoxides. Deletion of the caleosin gene prevented fungal development. Alternatively, silencing of the gene led to the increased accumulation of endogenous polyunsaturated fatty acid hydroperoxides and antioxidant activities but to a reduction of fungal growth and conidium formation. Two key genes of the aflatoxin biosynthesis pathway,aflRandaflD, were downregulated in the strains in whichA. flavusPXG(AfPXG) was silenced, leading to reduced aflatoxin B1 productionin vitro. Application of caleosin/peroxygenase-derived oxylipins restored the wild-type phenotype in the strains in whichAfPXGwas silenced.PXG-deficientA. flavusstrains were severely compromised in their capacity to infect maize seeds and to produce aflatoxin. Our results uncover a new branch of the fungal oxylipin pathway and may lead to the development of novel targets for controlling fungal disease.

scholarly journals Evidence for Inhibition of Topoisomerase 1A by Gold(III) Macrocycles and Chelates TargetingMycobacterium tuberculosisandMycobacterium abscessus

2018 ◽  
Vol 62 (5) ◽  
Author(s):  
Rashmi Gupta ◽  
Carolina Rodrigues Felix ◽  
Matthew P. Akerman ◽  
Kate J. Akerman ◽  
Cathryn A. Slabber ◽  
...  
Keyword(s):  
Mycobacterium Abscessus ◽  
Cross Resistance ◽  
Human Pathogens ◽  
Intrinsic Resistance ◽  
Content Type ◽  
Starting Point ◽  
Scalable Synthesis ◽  
High Level ◽  
Novel Drug

ABSTRACTMycobacterium tuberculosisand the fast-growing speciesMycobacterium abscessusare two important human pathogens causing persistent pulmonary infections that are difficult to cure and require long treatment times. The emergence of drug-resistantM. tuberculosisstrains and the high level of intrinsic resistance ofM. abscessuscall for novel drug scaffolds that effectively target both pathogens. In this study, we evaluated the activity of bis(pyrrolide-imine) gold(III) macrocycles and chelates, originally designed as DNA intercalators capable of targeting human topoisomerase types I and II (Topo1 and Topo2), againstM. abscessusandM. tuberculosis. We identified a total of 5 noncytotoxic compounds active against both mycobacterial pathogens under replicatingin vitroconditions. We chose one of these hits, compound 14, for detailed analysis due to its potent bactericidal mode of inhibition and scalable synthesis. The clinical relevance of this compound was demonstrated by its ability to inhibit a panel of diverseM. tuberculosisandM. abscessusclinical isolates. Prompted by previous data suggesting that compound 14 may target topoisomerase/gyrase enzymes, we demonstrated that it lacked cross-resistance with fluoroquinolones, which target theM. tuberculosisgyrase.In vitroenzyme assays confirmed the potent activity of compound 14 against bacterial topoisomerase 1A (Topo1) enzymes but not gyrase. Novel scaffolds like compound 14 with potent, selective bactericidal activity againstM. tuberculosisandM. abscessusthat act on validated but underexploited targets like Topo1 represent a promising starting point for the development of novel therapeutics for infections by pathogenic mycobacteria.

scholarly journals A FASII Inhibitor Prevents Staphylococcal Evasion of Daptomycin by Inhibiting Phospholipid Decoy Production

2019 ◽  
Vol 63 (4) ◽  
Author(s):  
Carmen J. E. Pee ◽  
Vera Pader ◽  
Elizabeth V. K. Ledger ◽  
Andrew M. Edwards
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Therapeutic Interventions ◽  
Bacterial Killing ◽  
Content Type ◽  
Serious Infections ◽  
Fatty Acid Biosynthetic Pathway ◽  
Emergence Of Resistance ◽  
Host Tissues

ABSTRACT Daptomycin is a treatment of last resort for serious infections caused by drug-resistant Gram-positive pathogens, such as methicillin-resistant Staphylococcus aureus. We have shown recently that S. aureus can evade daptomycin by releasing phospholipid decoys that sequester and inactivate the antibiotic, leading to treatment failure. Since phospholipid release occurs via an active process, we hypothesized that it could be inhibited, thereby increasing daptomycin efficacy. To identify opportunities for therapeutic interventions that block phospholipid release, we first determined how the host environment influences the release of phospholipids and the inactivation of daptomycin by S. aureus. The addition of certain host-associated fatty acids to the growth medium enhanced phospholipid release. However, in serum, the sequestration of fatty acids by albumin restricted their availability to S. aureus sufficiently to prevent their use in the generation of released phospholipids. This finding implies that in host tissues S. aureus may be completely dependent upon endogenous phospholipid biosynthesis to generate lipids for release, providing a target for therapeutic intervention. To test this, we exposed S. aureus to AFN-1252, an inhibitor of the staphylococcal FASII fatty acid biosynthetic pathway, together with daptomycin. AFN-1252 efficiently blocked daptomycin-induced phospholipid decoy production, even in the case of isolates resistant to AFN-1252, which prevented the inactivation of daptomycin and resulted in sustained bacterial killing. In turn, daptomycin prevented the fatty acid-dependent emergence of AFN-1252-resistant isolates in vitro. In summary, AFN-1252 significantly enhances daptomycin activity against S. aureus in vitro by blocking the production of phospholipid decoys, while daptomycin blocks the emergence of resistance to AFN-1252.

scholarly journals A kinetic rationale for functional redundancy in fatty acid biosynthesis

2020 ◽  
Vol 117 (38) ◽  
pp. 23557-23564
Author(s):  
Alex Ruppe ◽  
Kathryn Mains ◽  
Jerome M. Fox
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Fatty Acid Synthase ◽  
Unsaturated Fatty Acids ◽  
Fatty Acid Biosynthesis ◽  
Average Length ◽  
Functional Redundancy ◽  
Experimental Investigations ◽  
Total Production

Cells build fatty acids with biocatalytic assembly lines in which a subset of enzymes often exhibit overlapping activities (e.g., two enzymes catalyze one or more identical reactions). Although the discrete enzymes that make up fatty acid pathways are well characterized, the importance of catalytic overlap between them is poorly understood. We developed a detailed kinetic model of the fatty acid synthase (FAS) ofEscherichia coliand paired that model with a fully reconstituted in vitro system to examine the capabilities afforded by functional redundancy in fatty acid synthesis. The model captures—and helps explain—the effects of experimental perturbations to FAS systems and provides a powerful tool for guiding experimental investigations of fatty acid assembly. Compositional analyses carried out in silico and in vitro indicate that FASs with multiple partially redundant enzymes enable tighter (i.e., more independent and/or broader range) control of distinct biochemical objectives—the total production, unsaturated fraction, and average length of fatty acids—than FASs with only a single multifunctional version of each enzyme (i.e., one enzyme with the catalytic capabilities of two partially redundant enzymes). Maximal production of unsaturated fatty acids, for example, requires a second dehydratase that is not essential for their synthesis. This work provides a kinetic, control-theoretic rationale for the inclusion of partially redundant enzymes in fatty acid pathways and supplies a valuable framework for carrying out detailed studies of FAS kinetics.

SYNTHESIS OF FATTY ACIDS BY TESTICULAR TISSUE IN VITRO

1963 ◽  
Vol 41 (1) ◽  
pp. 1267-1274
Author(s):  
Peter F. Hall ◽  
Edward E. Nishizawa ◽  
Kristen B. Eik-Nes
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Fatty Acid Biosynthesis ◽  
De Novo ◽  
Fatty Acid Fraction ◽  
Testicular Tissue ◽  
Acid Biosynthesis ◽  
Adrenal Tissue ◽  
Substrate Fatty Acid

The fatty acids palmitic, palmitoleic, stearic, and oleic have been isolated from rabbit testis and evidence for the synthesis of palmitic and stearic acids de novo from acetate-1-C14is presented. ICSH did not produce demonstrable stimulation of the synthesis of these acids in vitro although the hormone stimulated the production of testosterone-C14by the same tissue. Adrenal tissue was shown to contain palmitic, stearic, and oleic acids, and ACTH did not increase the incorporation of acetate-1-C14into a fatty acid fraction extracted following incubation of adrenal tissue in the presence of this substrate. Fatty acid biosynthesis, therefore, is probably not influenced by the mechanisms by which tropic hormones increase steroid formation.

scholarly journals Novel Rhizosphere Soil Alleles for the Enzyme 1-Aminocyclopropane-1-Carboxylate Deaminase Queried for Function with anIn VivoCompetition Assay

2015 ◽  
Vol 82 (4) ◽  
pp. 1050-1059 ◽  
Author(s):  
Zhao Jin ◽  
Sara C. Di Rienzi ◽  
Anders Janzon ◽  
Jeff J. Werner ◽  
Largus T. Angenent ◽  
...  
Keyword(s):  
Protein Function ◽  
Gene Diversity ◽  
Functional Characterization ◽  
Acc Deaminase ◽  
Active Site Residues ◽  
Protein Coding ◽  
Content Type ◽  
Soil Microbial ◽  
Starting Point

ABSTRACTMetagenomes derived from environmental microbiota encode a vast diversity of protein homologs. How this diversity impacts protein function can be explored through selection assays aimed to optimize function. While artificially generated gene sequence pools are typically used in selection assays, their usage may be limited because of technical or ethical reasons. Here, we investigate an alternative strategy, the use of soil microbial DNA as a starting point. We demonstrate this approach by optimizing the function of a widely occurring soil bacterial enzyme, 1-aminocyclopropane-1-carboxylate (ACC) deaminase. We identified a specific ACC deaminase domain region (ACCD-DR) that, when PCR amplified from the soil, produced a variant pool that we could swap into functional plasmids carrying ACC deaminase-encoding genes. Functional clones of ACC deaminase were selected for in a competition assay based on their capacity to provide nitrogen toEscherichia coliin vitro. The most successful ACCD-DR variants were identified after multiple rounds of selection by sequence analysis. We observed that previously identified essential active-site residues were fixed in the original unselected library and that additional residues went to fixation after selection. We identified a divergent essential residue whose presence hints at the possible use of alternative substrates and a cluster of neutral residues that did not influence ACCD performance. Using an artificial ACCD-DR variant library generated by DNA oligomer synthesis, we validated the same fixation patterns. Our study demonstrates that soil metagenomes are useful starting pools of protein-coding-gene diversity that can be utilized for protein optimization and functional characterization when synthetic libraries are not appropriate.

scholarly journals In Vitro Inhibition of the Mycobacterium tuberculosis β-Ketoacyl-Acyl Carrier Protein Reductase MabA by Isoniazid

2004 ◽  
Vol 48 (1) ◽  
pp. 242-249 ◽  
Author(s):  
Stéphanie Ducasse-Cabanot ◽  
Martin Cohen-Gonsaud ◽  
Hedia Marrakchi ◽  
Michel Nguyen ◽  
Didier Zerbib ◽  
...  
Keyword(s):  
Fatty Acid ◽  
Fatty Acid Biosynthesis ◽  
Dynamic Equilibrium ◽  
Acyl Carrier Protein ◽  
Multidrug Resistant ◽  
Carrier Protein ◽  
Ligand Complex ◽  
Fas Ii

ABSTRACT The first-line specific antituberculous drug isoniazid inhibits the fatty acid elongation system (FAS) FAS-II involved in the biosynthesis of mycolic acids, which are major lipids of the mycobacterial envelope. The MabA protein that catalyzes the second step of the FAS-II elongation cycle is structurally and functionally related to the in vivo target of isoniazid, InhA, an NADH-dependent enoyl-acyl carrier protein reductase. The present work shows that the NADPH-dependent β-ketoacyl reduction activity of MabA is efficiently inhibited by isoniazid in vitro by a mechanism similar to that by which isoniazid inhibits InhA activity. It involves the formation of a covalent adduct between MnIII-activated isoniazid and the MabA cofactor. Liquid chromatography-mass spectrometry analyses revealed that the isonicotinoyl-NADP adduct has multiple chemical forms in dynamic equilibrium. Both kinetic experiments with isolated forms and purification of the enzyme-ligand complex strongly suggested that the molecules active against MabA activity are the oxidized derivative and a major cyclic form. Spectrofluorimetry showed that the adduct binds to the MabA active site. Modeling of the MabA-adduct complex predicted an interaction between the isonicotinoyl moiety of the inhibitor and Tyr185. This hypothesis was supported by the fact that a higher 50% inhibitory concentration of the adduct was measured for MabA Y185L than for the wild-type enzyme, while both proteins presented similar affinities for NADP+. The crystal structure of MabA Y185L that was solved showed that the substitution of Tyr185 induced no significant conformational change. The description of the first inhibitor of the β-ketoacyl reduction step of fatty acid biosynthesis should help in the design of new antituberculous drugs efficient against multidrug-resistant tubercle bacilli.

scholarly journals A High-Throughput Fatty Acid Profiling Screen Reveals Novel Variations in Fatty Acid Biosynthesis in Chlamydomonas reinhardtii and Related Algae

2014 ◽  
Vol 13 (11) ◽  
pp. 1431-1438 ◽  
Author(s):  
Erin L. Pflaster ◽  
Michael J. Schwabe ◽  
Joyanne Becker ◽  
Melissa S. Wilkinson ◽  
Ashley Parmer ◽  
...  
Keyword(s):  
Fatty Acids ◽  
Fatty Acid ◽  
Fatty Acid Biosynthesis ◽  
Methyl Esters ◽  
Screening Method ◽  
Laboratory Strain ◽  
Gene Encoding ◽  
Preparation Methods ◽  
Content Type ◽  
Species Specific

ABSTRACTAnalysis of fatty acid methyl esters (FAMEs) by gas chromatography (GC) is a common technique for the quantitative and qualitative analysis of acyl lipids. Methods for FAME preparation are typically time-consuming and labor-intensive and require multiple transfers of reagents and products between reaction tubes and autosampler vials. In order to increase throughput and lower the time and materials costs required for FAME preparation prior to GC analysis, we have developed a method in which 10-to-20-mg samples of microbial biomass are transferred to standard GC autosampler vials, transesterified using an emulsion of methanolic trimethylsulfonium hydroxide and hexane, and analyzed directly by GC without further sample handling. This method gives results that are essentially identical to those obtained by the more labor- and material-intensive FAME preparation methods, such as transmethylation with methanolic HCl. We applied this method to the screening of laboratory and environmental isolates of the green algaChlamydomonasfor variations in fatty acid composition. This screening method facilitated two novel discoveries. First, we identified a common laboratory strain ofC. reinhardtii, CC-620, completely lacking all ω-3 fatty acids normally found in this organism and showed that this strain contains an inactivating mutation in the CrFAD7 gene, encoding the sole ω-3 desaturase activity in this organism. Second, we showed that some species ofChlamydomonasmake Δ6-unsaturated polyunsaturated fatty acids (PUFA) rather than the Δ5 species normally made by the previously characterized laboratory strains ofChlamydomonas, suggesting that there is species-specific variation in the regiospecificity and substrate selectivity of front-end desaturases in this algal genus.

scholarly journals Glutamate Racemase Is the Primary Target of β-Chloro-d-Alanine in Mycobacterium tuberculosis

2016 ◽  
Vol 60 (10) ◽  
pp. 6091-6099 ◽  
Author(s):  
Gareth A. Prosser ◽  
Anne Rodenburg ◽  
Hania Khoury ◽  
Cesira de Chiara ◽  
Steve Howell ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Early Stage ◽  
Human Pathogens ◽  
Intact Cells ◽  
Content Type ◽  
Peptidoglycan Biosynthesis ◽  
Glutamate Racemase ◽  
Starting Point ◽  
Effective Drugs

ABSTRACTThe increasing global prevalence of drug resistance among many leading human pathogens necessitates both the development of antibiotics with novel mechanisms of action and a better understanding of the physiological activities of preexisting clinically effective drugs. Inhibition of peptidoglycan (PG) biosynthesis and cross-linking has traditionally enjoyed immense success as an antibiotic target in multiple bacterial pathogens, except inMycobacterium tuberculosis, where it has so far been underexploited.d-Cycloserine, a clinically approved antituberculosis therapeutic, inhibits enzymes within thed-alanine subbranch of the PG-biosynthetic pathway and has been a focus in our laboratory for understanding peptidoglycan biosynthesis inhibition and for drug development in studies ofM. tuberculosis. During our studies on alternative inhibitors of thed-alanine pathway, we discovered that the canonical alanine racemase (Alr) inhibitor β-chloro–d-alanine (BCDA) is a very poor inhibitor of recombinantM. tuberculosisAlr, despite having potent antituberculosis activity. Through a combination of enzymology, microbiology, metabolomics, and proteomics, we show here that BCDA does not inhibit thed-alanine pathway in intact cells, consistent with its poorin vitroactivity, and that it is instead a mechanism-based inactivator of glutamate racemase (MurI), an upstream enzyme in the same early stage of PG biosynthesis. This is the first report to our knowledge of inhibition of MurI inM. tuberculosisand thus provides a valuable tool for studying this essential and enigmatic enzyme and a starting point for future MurI-targeted antibacterial development.

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