scholarly journals Differences in Substrate Specificities of Five Bacterial Wax Ester Synthases

2012 ◽  
Vol 78 (16) ◽  
pp. 5734-5745 ◽  
Author(s):  
Brett M. Barney ◽  
Bradley D. Wahlen ◽  
EmmaLee Garner ◽  
Jiashi Wei ◽  
Lance C. Seefeldt
Keyword(s):  
Kinetic Studies ◽  
Fatty Acyl ◽  
Biofuel Production ◽  
Wax Ester ◽  
Acinetobacter Baylyi ◽  
Content Type ◽  
Rapid Purification ◽  
Range Characterization ◽  
Acyl Coenzyme A

ABSTRACTWax esters are produced in certain bacteria as a potential carbon and energy storage compound. The final enzyme in the biosynthetic pathway responsible for wax ester production is the bifunctional wax ester synthase/acyl-coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT), which utilizes a range of fatty alcohols and fatty acyl-CoAs to synthesize the corresponding wax ester. We report here the isolation and substrate range characterization for five WS/DGAT enzymes from four different bacteria:Marinobacter aquaeoleiVT8,Acinetobacter baylyi,Rhodococcus jostiiRHA1, andPsychrobacter cryohalolentisK5. The results from kinetic studies of isolated enzymes reveal a differential activity based on the order of substrate addition and reveal subtle differences between the substrate selectivity of the different enzymes. Thesein vitroresults are compared to the wax ester and triacylglyceride product profiles obtained from each organism grown under neutral lipid accumulating conditions, providing potential insights into the role that the WS/DGAT enzyme plays in determining the final wax ester products that are produced under conditions of nutrient stress in each of these bacteria. Further, the analysis revealed that one enzyme in particular fromM. aquaeoleiVT8 showed the greatest potential for future study based on rapid purification and significantly higher activity than was found for the other isolated WS/DGAT enzymes. The results provide a framework to test prospective differences between these enzymes for potential biotechnological applications such as high-value petrochemicals and biofuel production.

scholarly journals Identification of a Residue Affecting Fatty Alcohol Selectivity in Wax Ester Synthase

2012 ◽  
Vol 79 (1) ◽  
pp. 396-399 ◽  
Author(s):  
Brett M. Barney ◽  
Rachel L. Mann ◽  
Janet M. Ohlert
Keyword(s):  
Kinetic Parameters ◽  
Fatty Alcohol ◽  
Biosynthetic Pathway ◽  
Fatty Acyl ◽  
Wax Ester ◽  
Acinetobacter Baylyi ◽  
Content Type ◽  
Marinobacter Aquaeolei ◽  
Acyl Coenzyme A ◽  
Wax Ester Synthase

ABSTRACT The terminal enzyme in the bacterial wax ester biosynthetic pathway is the bifunctional wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT), which utilizes a fatty alcohol and a fatty acyl-coenzyme A (CoA) to synthesize the corresponding wax ester. In this report, we identify a specific residue in WS/DGAT enzymes obtained from Marinobacter aquaeolei VT8 and Acinetobacter baylyi that alters fatty alcohol selectivity and kinetic parameters when modified to alternative residues.

scholarly journals Fatty Alcohols for Wax Esters in Marinobacter aquaeolei VT8: Two Optional Routes in the Wax Biosynthesis Pathway

2013 ◽  
Vol 79 (22) ◽  
pp. 7055-7062 ◽  
Author(s):  
Eric M. Lenneman ◽  
Janet M. Ohlert ◽  
Nagendra P. Palani ◽  
Brett M. Barney
Keyword(s):  
Fatty Alcohol ◽  
Fatty Acyl ◽  
Wax Esters ◽  
Transcriptional Analysis ◽  
Wax Ester ◽  
Fatty Aldehyde ◽  
Marinobacter Aquaeolei

ABSTRACTThe biosynthesis of wax esters in bacteria is accomplished by a unique pathway that combines a fatty alcohol and a fatty acyl coenzyme A substrate. Previousin vitroenzymatic studies indicated that two different enzymes could be involved in the synthesis of the required fatty alcohol inMarinobacter aquaeoleiVT8. In this study, we demonstrate through a series of gene deletions and transcriptional analysis that either enzyme is capable of fulfilling the role of providing the fatty alcohol required for wax ester biosynthesisin vivo, but evolution has clearly selected one of these, a previously characterized fatty aldehyde reductase, as the preferred enzyme to perform this reaction under typical wax ester-accumulating conditions. These results complement previousin vitrostudies and provide the first glimpse into the role of each enzymein vivoin the native organism.

scholarly journals A Fatty Acyl Coenzyme A Reductase Promotes Wax Ester Accumulation in Rhodococcus jostii RHA1

2017 ◽  
Vol 83 (20) ◽  
Author(s):  
James Round ◽  
Raphael Roccor ◽  
Shu-Nan Li ◽  
Lindsay D. Eltis
Keyword(s):  
Specific Activity ◽  
Dry Weight ◽  
Fatty Acyl ◽  
Wax Esters ◽  
Wax Ester ◽  
Wild Type ◽  
Considerable Potential ◽  
Commodity Chemicals ◽  
Acyl Coenzyme A ◽  
Rhodococcus Jostii

ABSTRACT Many rhodococci are oleaginous and, as such, have considerable potential for the sustainable production of lipid-based commodity chemicals. Herein, we demonstrated that Rhodococcus jostii RHA1, a soil bacterium that catabolizes a wide range of organic compounds, produced wax esters (WEs) up to 0.0002% of its cellular dry weight during exponential growth on glucose. These WEs were fully saturated and contained primarily 31 to 34 carbon atoms. Moreover, they were present at higher levels during exponential growth than under lipid-accumulating conditions. Bioinformatics analyses revealed that RHA1 contains a gene encoding a putative fatty acyl coenzyme A (acyl-CoA) reductase (FcrA). The purified enzyme catalyzed the NADPH-dependent transformation of stearoyl-CoA to stearyl alcohol with a specific activity of 45 ± 3 nmol/mg · min and dodecanal to dodecanol with a specific activity of 5,300 ± 300 nmol/mg · min. Deletion of fcrA did not affect WE accumulation when grown in either carbon- or nitrogen-limited medium. However, the ΔfcrA mutant accumulated less than 20% of the amount of WEs as the wild-type strain under conditions of nitric oxide stress. A strain of RHA1 overproducing FcrA accumulated WEs to ∼13% cellular dry weight under lipid-accumulating conditions, and their acyl moieties had longer average chain lengths than those in wild-type cells (C17 versus C16). The results provide insight into the biosynthesis of WEs in rhodococci and facilitate the development of this genus for the production of high-value neutral lipids. IMPORTANCE Among the best-studied oleaginous bacteria, rhodococci have considerable potential for the sustainable production of lipid-based commodity chemicals, such as wax esters. However, many aspects of lipid synthesis in these bacteria are poorly understood. The current study identifies a key enzyme in wax ester synthesis in rhodococci and exploits it to significantly improve the yield of wax esters in bacteria. In so doing, this work contributes to the development of novel bioprocesses for an important class of oleochemicals that may ultimately allow us to phase out their unsustainable production from sources such as petroleum and palm oil.

scholarly journals The enzymology of short-chain fatty acyl-coenzyme A synthetase from seeds of Pinus radiata. Kinetic studies and a proposed reaction mechanism

1974 ◽  
Vol 137 (3) ◽  
pp. 435-442 ◽  
Author(s):  
Owen A. Young ◽  
John W. Anderson
Keyword(s):  
Fatty Acid ◽  
Pinus Radiata ◽  
Kinetic Studies ◽  
Single Species ◽  
Competitive Inhibitor ◽  
Fatty Acyl ◽  
Short Chain ◽  
Low Concentrations ◽  
High Concentrations ◽  
Acyl Coenzyme A

1. Short-chain fatty acyl-CoA synthetase from seeds of Pinus radiata was examined by acetate- and propionate-dependent PPi–ATP exchange. Reaction mixtures came to equilibrium almost instantly as judged by rates of exchange and analysis of an incubation mixture. 2. The activity of the enzyme was correlated with the concentration of MgP2O72- but not with the concentration of Mg2+, as judged by PPi–ATP exchange and fatty acyl AMP-dependent synthesis of ATP in the presence of PPi. In PPi–ATP exchange assays, no clear relationship between activity and any single species of ATP was apparent. 3. High concentrations of fatty acid inhibited PPi–ATP exchange. PPi–dATP exchange was less than PPi–ATP exchange at low concentrations of fatty acid, but at higher concentrations PPi–dATP exchange exceeded PPi–ATP exchange. The rate of synthesis of fatty acyl-CoA in the presence of dATP was less than with ATP. 4. ATP and propionate inhibited the synthesis of ATP from propionyl-AMP and PPi. The inhibition by ATP was competitive with respect to propionyl-AMP and non-competitive with respect to PPi. The inhibition by propionate was non-competitive with respect to propionyl-AMP and PPi. 5. AMP was a competitive inhibitor of propionyl-AMP-dependent synthesis of ATP and competitively inhibited propionate-dependent PPi–ATP exchange when ATP was the variable substrate. 6. It was concluded that the first partial reaction catalysed by the enzyme is ordered; ATP is the first substrate to react with the enzyme and PPi is probably the only product released.

scholarly journals Active Multienzyme Assemblies for Long-Chain Olefinic Hydrocarbon Biosynthesis

2017 ◽  
Vol 199 (9) ◽  
Author(s):  
James K. Christenson ◽  
Matthew R. Jensen ◽  
Brandon R. Goblirsch ◽  
Fatuma Mohamed ◽  
Wei Zhang ◽  
...  
Keyword(s):  
Active Sites ◽  
Xanthomonas Campestris ◽  
Fatty Acyl ◽  
Biosynthetic Activity ◽  
Content Type ◽  
Large Structures ◽  
Negative Stain ◽  
Acyl Coenzyme A ◽  
Hydrocarbon Biosynthesis ◽  
Long Chain

ABSTRACT Bacteria from different phyla produce long-chain olefinic hydrocarbons derived from an OleA-catalyzed Claisen condensation of two fatty acyl coenzyme A (acyl-CoA) substrates, followed by reduction and oxygen elimination reactions catalyzed by the proteins OleB, OleC, and OleD. In this report, OleA, OleB, OleC, and OleD were individually purified as soluble proteins, and all were found to be essential for reconstituting hydrocarbon biosynthesis. Recombinant coexpression of tagged OleABCD proteins from Xanthomonas campestris in Escherichia coli and purification over His6 and FLAG columns resulted in OleA separating, while OleBCD purified together, irrespective of which of the four Ole proteins were tagged. Hydrocarbon biosynthetic activity of copurified OleBCD assemblies could be reconstituted by adding separately purified OleA. Immunoblots of nondenaturing gels using anti-OleC reacted with X. campestris crude protein lysate indicated the presence of a large protein assembly containing OleC in the native host. Negative-stain electron microscopy of recombinant OleBCD revealed distinct large structures with diameters primarily between 24 and 40 nm. Assembling OleB, OleC, and OleD into a complex may be important to maintain stereochemical integrity of intermediates, facilitate the movement of hydrophobic metabolites between enzyme active sites, and protect the cell against the highly reactive β-lactone intermediate produced by the OleC-catalyzed reaction. IMPORTANCE Bacteria biosynthesize hydrophobic molecules to maintain a membrane, store carbon, and for antibiotics that help them survive in their niche. The hydrophobic compounds are often synthesized by a multidomain protein or by large multienzyme assemblies. The present study reports on the discovery that long-chain olefinic hydrocarbons made by bacteria from different phyla are produced by multienzyme assemblies in X. campestris. The OleBCD multienzyme assemblies are thought to compartmentalize and sequester olefin biosynthesis from the rest of the cell. This system provides additional insights into how bacteria control specific biosynthetic pathways.

scholarly journals Assessment of Mycobacterium tuberculosis Pantothenate Kinase Vulnerability through Target Knockdown and Mechanistically Diverse Inhibitors

2014 ◽  
Vol 58 (6) ◽  
pp. 3312-3326 ◽  
Author(s):  
B. K. Kishore Reddy ◽  
Sudhir Landge ◽  
Sudha Ravishankar ◽  
Vikas Patil ◽  
Vikas Shinde ◽  
...  
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Present Report ◽  
Kinetic Studies ◽  
Antimycobacterial Activity ◽  
Type I ◽  
Pantothenate Kinase ◽  
Content Type ◽  
Rate Limiting Step

ABSTRACTPantothenate kinase (PanK) catalyzes the phosphorylation of pantothenate, the first committed and rate-limiting step toward coenzyme A (CoA) biosynthesis. In our earlier reports, we had established that the type I isoform encoded by thecoaAgene is an essential pantothenate kinase inMycobacterium tuberculosis, and this vital information was then exploited to screen large libraries for identification of mechanistically different classes of PanK inhibitors. The present report summarizes the synthesis and expansion efforts to understand the structure-activity relationships leading to the optimization of enzyme inhibition along with antimycobacterial activity. Additionally, we report the progression of two distinct classes of inhibitors, the triazoles, which are ATP competitors, and the biaryl acetic acids, with a mixed mode of inhibition. Cocrystallization studies provided evidence of these inhibitors binding to the enzyme. This was further substantiated with the biaryl acids having MIC against the wild-typeM. tuberculosisstrain and the subsequent establishment of a target link with an upshift in MIC in a strain overexpressing PanK. On the other hand, the ATP competitors had cellular activity only in aM. tuberculosisknockdown strain with reduced PanK expression levels. Additionally,in vitroandin vivosurvival kinetic studies performed with aM. tuberculosisPanK (MtPanK) knockdown strain indicated that the target levels have to be significantly reduced to bring in growth inhibition. The dual approaches employed here thus established the poor vulnerability of PanK inM. tuberculosis.

scholarly journals Multiple Pathways for Triacylglycerol Biosynthesis in Streptomyces coelicolor

2008 ◽  
Vol 74 (9) ◽  
pp. 2573-2582 ◽  
Author(s):  
Ana Arabolaza ◽  
Eduardo Rodriguez ◽  
Silvia Altabe ◽  
Hector Alvarez ◽  
Hugo Gramajo
Keyword(s):  
Mutant Strain ◽  
De Novo ◽  
Streptomyces Coelicolor ◽  
Lipid Production ◽  
Wax Ester ◽  
Acinetobacter Baylyi ◽  
Triple Mutant ◽  
Storage Lipids ◽  
Tag Biosynthesis

ABSTRACT The terminal reaction in triacylglyceride (TAG) biosynthesis is the esterification of diacylglycerol (DAG) with a fatty acid molecule. To study this reaction in Streptomyces coelicolor, we analyzed three candidate genes (sco0958, sco1280, and sco0123) whose products significantly resemble the recently identified wax ester synthase/acyl-coenzyme A (CoA):DAG acyltransferase (DGAT) from Acinetobacter baylyi. The deletion of either sco0123 or sco1280 resulted in no detectable decrease in TAG accumulation. In contrast, the deletion of sco0958 produced a dramatic reduction in neutral lipid production, whereas the overexpression of this gene yielded a significant increase in de novo TAG biosynthesis. In vitro activity assays showed that Sco0958 mediates the esterification of DAG using long-chain acyl-CoAs (C14 to C18) as acyl donors. The Km and V max values of this enzyme for myristoyl-CoA were 45 μM and 822 nmol mg−1 min−1, respectively. Significantly, the triple mutant strain was not completely devoid of storage lipids, indicating the existence of alternative TAG-biosynthetic routes. We present strong evidence demonstrating that the residual production of TAG in this mutant strain is mediated, at least in part, by an acyl-CoA-dependent pathway, since the triple mutant still exhibited DGAT activity. More importantly, there was substantial phospholipid:DGAT (PDAT) activity in the wild type and in the triple mutant. This is the first time that a PDAT activity has been reported for bacteria, highlighting the extreme metabolic diversity of this industrially important soil microorganism.

scholarly journals Amelioration of Cryptosporidium parvum Infection In Vitro and In Vivo by Targeting Parasite Fatty Acyl-Coenzyme A Synthetases

2013 ◽  
Vol 209 (8) ◽  
pp. 1279-1287 ◽  
Author(s):  
F. Guo ◽  
H. Zhang ◽  
J. M. Fritzler ◽  
S. D. Rider ◽  
L. Xiang ◽  
...  
Keyword(s):  
Cryptosporidium Parvum ◽  
Coenzyme A ◽  
Fatty Acyl ◽  
Acyl Coenzyme A

scholarly journals Toxin-Mediated Paracellular Transport of Antitoxin Antibodies Facilitates Protection against Clostridium difficile Infection

2014 ◽  
Vol 83 (1) ◽  
pp. 405-416 ◽  
Author(s):  
Z. Zhang ◽  
X. Chen ◽  
L. D. Hernandez ◽  
P. Lipari ◽  
A. Flattery ◽  
...  
Keyword(s):  
Clostridium Difficile ◽  
Humoral Response ◽  
Kinetic Studies ◽  
General Mechanism ◽  
Similar Data ◽  
Protective Effects ◽  
Content Type ◽  
Genetic Ablation ◽  
Apical Side

The exotoxins TcdA and TcdB are the major virulence factors ofClostridium difficile. Circulating neutralizing antitoxin antibodies are protective inC. difficileinfection (CDI), as demonstrated, in part, by the protective effects of actoxumab and bezlotoxumab, which bind to and neutralize TcdA and TcdB, respectively. The question of how systemic IgG antibodies neutralize toxins in the gut lumen remains unresolved, although it has been suggested that the Fc receptor FcRn may be involved in active antibody transport across the gut epithelium. In this study, we demonstrated that genetic ablation of FcRn and excess irrelevant human IgG have no impact on actoxumab-bezlotoxumab-mediated protection in murine and hamster models of CDI, suggesting that Fc-dependent transport of antibodies across the gut wall is not required for efficacy. Tissue distribution studies in hamsters suggest, rather, that the transport of antibodies depends on toxin-induced damage to the gut lining. In anin vitrotwo-dimensional culture system that mimics the architecture of the intestinal mucosal epithelium, toxins on the apical side of epithelial cell monolayers are neutralized by basolateral antibodies, and antibody transport across the cell layer is dramatically increased upon addition of toxin to the apical side. Similar data were obtained with F(ab′)2fragments, which lack an Fc domain, consistent with FcRn-independent paracellular, rather than transcellular, transport of antibodies. Kinetic studies show that initial damage caused by apical toxin is required for efficient neutralization by basolateral antibodies. These data may represent a general mechanism of humoral response-mediated protection against enteric pathogens.

scholarly journals Long-Chain Fatty Acyl Coenzyme A Ligase FadD2 Mediates Intrinsic Pyrazinamide Resistance in Mycobacterium tuberculosis

2016 ◽  
Vol 61 (2) ◽  
Author(s):  
Brandon C. Rosen ◽  
Nicholas A. Dillon ◽  
Nicholas D. Peterson ◽  
Yusuke Minato ◽  
Anthony D. Baughn
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Coenzyme A ◽  
Fatty Acyl ◽  
Wild Type ◽  
Intrinsic Resistance ◽  
First Line ◽  
Minimum Concentration ◽  
Content Type ◽  
Acyl Coenzyme A ◽  
Long Chain

ABSTRACT Pyrazinamide (PZA) is a first-line tuberculosis (TB) drug that has been in clinical use for 60 years yet still has an unresolved mechanism of action. Based upon the observation that the minimum concentration of PZA required to inhibit the growth of Mycobacterium tuberculosis is approximately 1,000-fold higher than that of other first-line drugs, we hypothesized that M. tuberculosis expresses factors that mediate intrinsic resistance to PZA. To identify genes associated with intrinsic PZA resistance, a library of transposon-mutagenized Mycobacterium bovis BCG strains was screened for strains showing hypersusceptibility to the active form of PZA, pyrazinoic acid (POA). Disruption of the long-chain fatty acyl coenzyme A (CoA) ligase FadD2 enhanced POA susceptibility by 16-fold on agar medium, and the wild-type level of susceptibility was restored upon expression of fadD2 from an integrating mycobacterial vector. Consistent with the recent observation that POA perturbs mycobacterial CoA metabolism, the fadD2 mutant strain was more vulnerable to POA-mediated CoA depletion than the wild-type strain. Ectopic expression of the M. tuberculosis pyrazinamidase PncA, necessary for conversion of PZA to POA, in the fadD2 transposon insertion mutant conferred at least a 16-fold increase in PZA susceptibility under active growth conditions in liquid culture at neutral pH. Importantly, deletion of fadD2 in M. tuberculosis strain H37Rv also resulted in enhanced susceptibility to POA. These results indicate that FadD2 is associated with intrinsic PZA and POA resistance and provide a proof of concept for the target-based potentiation of PZA activity in M. tuberculosis.

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