Site-directed mutagenesis of a fatty acid elongase ELO-like condensing enzyme

The condensation step of fatty acid elongation is the addition of a C₂ unit from malonyl-CoA to an acyl primer catalyzed by one of two families of enzymes, the 3-ketoacyl-CoA synthases and the ELO-like condensing enzymes. 3-Ketoacyl-CoA synthases use a Claisen-like reaction mechanism while the mechanism of the ELO-catalyzed condensation reaction is unknown. We have used site-directed mutagenesis of Dictyostelium discoideum EloA to identify residues important to catalytic activity and/or structure. Mutation of highly conserved polar residues to alanine resulted in an inactive enzyme strongly suggesting that these residues play a role in the condensation reaction.     

Crystal structure of beta-ketoacyl-acyl carrier protein synthase III. A key condensing enzyme in bacterial fatty acid biosynthesis

Beta-ketoacyl-acyl carrier protein synthase III (FabH), the most divergent member of the family of condensing enzymes, is a key catalyst in bacterial fatty acid biosynthesis and a promising target for novel antibiotics. We report here the crystal structures of FabH determined in the presence and absence of acetyl-CoA. These structures display a fold that is common for condensing enzymes. The observed acetylation of Cys(112) proves its catalytic role and clearly defines the primer binding pocket. Modeling based on a bound CoA molecule suggests catalytic roles for His(244) and Asn(274). The structures provide the molecular basis for FabH substrate specificity and reaction mechanism and are important for structure-based design of novel antibiotics.     

Spectrophotometric assay for the condensing enzyme activity of the microsomal fatty acid chain elongation system

A rapid and simple spectrophotometric method was developed to measure the activity of the condensing enzyme component of the microsomal fatty acid chain elongation system. The intermediate product of the condensation reaction is the beta-ketoacyl CoA which exists in two tautomeric forms, i.e., keto and enol. The addition of bovine serum albumin (BSA) to a cuvette cell containing a beta-ketoacyl CoA derivative resulted in the formation of a 303-nm absorbance peak, characteristic of enolate formation. The beta-ketoacyl CoAs with carbon chain length of 6 to 18 interacted with BSA to produce the 303-nm peak; acetoacetyl CoA was the only beta-keto compound tested which did not interact with BSA to produce the peak. Other compounds which were unaffected by BSA included CoA, free beta-keto acid, beta-hydroxyacyl CoA, acyl CoA, trans-2-enoyl CoA, and malonyl CoA. BSA could not be replaced by ovalbumin; furthermore, denatured (boiling) BSA could not induce the 303-nm peak. The specific activity of the condensing enzyme measured by the spectrophotometric method compares favorably with the activity obtained by the radioactive method. The apparent extinction coefficient (epsilon) for the absorbance peak generated by the beta-keto thioester varied from 5 to 30 mM-1 cm-1 depending on the beta-keto derivative. The spectrophotometric procedure can be used in the determination of the condensing enzyme activity in not only hepatic microsomes but also in kidney and brain microsomes both of which have significantly lower activity. The advantages of the novel method over the radioactive method are that (i) it does not involve the use of radioactive compounds, (ii) it is much less cumbersome and significantly less costly, and (iii) it is rapid and easy to perform.     

A fatty acid condensing enzyme from Physaria fendleri increases hydroxy fatty acid accumulation in transgenic oilseeds of Camelina sativa

Main conclusion

Co-expression of a lesquerella fatty acid elongase and the castor fatty acid hydroxylase in camelina results in higher hydroxy fatty acid containing seeds with normal oil content and viability. Producing hydroxy fatty acids (HFA) in oilseed crops has been a long-standing goal to replace castor oil as a renewable source for numerous industrial applications. A fatty acid hydroxylase, RcFAH, from Ricinus communis, was introduced into Camelina sativa, but yielded only 15 % of HFA in its seed oil, much lower than the 90 % found in castor bean. Furthermore, the transgenic seeds contained decreased oil content and the germination ability was severely affected. Interestingly, HFA accumulation was significantly increased in camelina seed when co-expressing RcFAH with a fatty acid condensing enzyme, LfKCS3, from Physaria fendleri, a native HFA accumulator relative to camelina. The oil content and seed germination of the transgenic seeds also appeared normal compared to non-transgenics. LfKCS3 has been previously characterized to specifically elongate the hydroxylated ricinoleic acid to lesquerolic acid, the 20-carbon HFA found in lesquerella oil. The elongation reaction may facilitate the HFA flux from phosphatidylcholine (PC), the site of HFA formation, into the acyl-CoA pool for more efficient utilization in triacylglycerol (TAG) biosynthesis. This was demonstrated by increased HFA accumulation in TAG concurrent with reduced HFA content in PC during camelina seed development, and increased C20-HFA in HFA-TAG molecules. These effects of LfKCS3 thus may effectively relieve the bottleneck for HFA utilization in TAG biosynthesis and the feedback inhibition to fatty acid synthesis, result in higher HFA accumulation and restore oil content and seed viability.     

Biphenyl-based analogues of thiolactomycin, active against Mycobacterium tuberculosis mtFabH fatty acid condensing enzyme

Analogues of the antibiotic thiolactomycin, with biphenyl-based 5-substituents, were found to have excellent in vitro inhibitory activity against the recombinant Mycobacterium tuberculosis beta-ketoacyl-ACP synthase mtFabH condensing enzyme. In particular, 5-(4'-benzyloxy-biphen-4-ylmethyl)-4-hydroxy-3,5-dimethyl-5H-thiophen-2-one exhibited approximately a 4-fold increased potency against this key condensing enzyme involved in M. tuberculosis mycolic acid biosynthesis, compared to thiolactomycin.     

Acetylene-based analogues of thiolactomycin, active against Mycobacterium tuberculosis mtFabH fatty acid condensing enzyme

Analogues of the natural antibiotic thiolactomycin, with acetylene-based side chains, have the highest recorded in vitro inhibitory activity against the recombinant Mycobacterium tuberculosis beta-ketoacyl-ACP synthase mtFabH condensing enzyme. In particular, 5-[3-(4-acetyl-phenyl)-prop-2-ynyl]-4-hydroxy-3,5-dimethyl-5H-thiophen-2-one exhibited more than an 18-fold increased potency, compared to thiolactomycin, against this key condensing enzyme, involved in M. tuberculosis mycolic acid biosynthesis. Analogues of the antibiotic thiolactomycin, with acetylene-based side chains, have the highest recorded activity against cloned mtFabH condensing enzyme.     

CUT1, an Arabidopsis gene required for cuticular wax biosynthesis and pollen fertility, encodes a very-long-chain fatty acid condensing enzyme.

Land plants secrete a layer of wax onto their aerial surfaces that is essential for survival in a terrestrial environment. This wax is composed of long-chain, aliphatic hydrocarbons derived from very-long-chain fatty acids (VLCFAs). Using the Arabidopsis expressed sequence tag database, we have identified a gene, designated CUT1, that encodes a VLCFA condensing enzyme required for cuticular wax production. Sense suppression of CUT1 in transgenic Arabidopsis plants results in waxless (eceriferum) stems and siliques as well as conditional male sterility. Scanning electron microscopy revealed that this was a severe waxless phenotype, because stems of CUT1-suppressed plants were completely devoid of wax crystals. Furthermore, chemical analyses of waxless plants demonstrated that the stem wax load was reduced to 6 to 7% of wild-type levels. This value is lower than that reported for any of the known eceriferum mutants. The severe waxless phenotype resulted from the downregulation of both the decarbonylation and acyl reduction wax biosynthetic pathways. This result indicates that CUT1 is involved in the production of VLCFA precursors used for the synthesis of all stem wax components in Arabidopsis. In CUT1-suppressed plants, the C24 chain-length wax components predominate, suggesting that CUT1 is required for elongation of C24 VLCFAs. The unique wax composition of CUT1-suppressed plants together with the fact that the location of CUT1 on the genetic map did not coincide with any of the known ECERIFERUM loci suggest that we have identified a novel gene involved in wax biosynthesis. CUT1 is currently the only known gene with a clearly established function in wax production.     

Regulation of cyclopropane fatty acid biosynthesis by variations in enzyme activities

It is proposed that cyclopropane fatty acid biosynthesis in $\text{Lactobacillus plantarum}$ is regulated by $\text{in vivo}$ variations in the activities of two enzymes acting sequentially. S-adenosylhomocysteine hydrolase relieves the end-product inhibition of cyclopropane synthetase by degrading a product (S-adenosyl-homocysteine) of the latter enzyme activity. Both enzymes show an abrupt increase and subsequent decrease in activity at a time during the bacterial growth cycle which corresponds to the period of most rapid synthesis of cyclopropane fatty acid $\text{in vivo}$.     

Modulation of fatty acid biosynthesis by antisense beta-keto reductase expression

Plants with an antisense construct to beta-ketoacyl-acyl carrier protein reductase have reduced seed and leaf fatty acid contents and show distinct phenotypes, including leaf curl, stunted growth and wrinkled seeds. The carbon resource allocation is altered in both of these tissues. In leaves there is an inability to synthesize transient starch and in seeds embryogenesis is foreshortened and storage proteins accumulate prematurely.     

Phosphatidic acid biosynthesis in rat liver mitochondria and microsomal fractions. Regulation of fatty acid positional specificity.

The positional and fatty acid specificity of phosphatidic acid biosynthesis in rat liver mitochondria and microsomal fractions was studied by using acylcarnitines, CoA and an excess of carnitine palmitoyltransferase (EC 2.3.1.21) as the source of acyl-CoA. In the mitochondria, the preference for palmitic acid at the 1-position is increased at high acyl-CoA concentrations, whereas it is decreased in the microsomal fraction. There was no change in the fatty acid specificity at the 2-position with different acyl-CoA concentrations in any of the factions. The preference in mitochondria for linoleic acid at the 2-position is strongly increased at high concentrations of lysophosphatidic acid.     

Fatty acid and sterol specificity in the biosynthesis of steryl esters by enzyme preparations from spinach leaves

Acetone powders prepared from the 20,000g participate fraction of spinach (Spinacia oleracea L.) leaves catalyzed the formation of steryl esters from free sterol and 1,2-diacylglycerol as the acyl donor. There was no sterol specificity when cholesterol, sitosterol, and campesterol were compared. When rates of sterol ester biosynthesis were compared using different 1,2-diacylglycerols it was found that the shorter chain fatty acids and the more unsaturated fatty acids were preferred. When the substrate concentration of diacylglycerol was varied, the maximal velocities obtained with the different substrates were dipalmitoleoyl- >dilinolenoyl- >dioleoyl- >dilinoleoyl-glycerol. It was demonstrated by silver nitrate thin-layer chromatography that the fatty acids of the supplied diacylglycerols were transferred to the sterol. When diacylglycerol mixtures were supplied, it was found that unsaturated diacylglycerols greatly stimulated conversion of saturated diacylglycerols to saturated steryl esters. For an equimolar mixture of dipalmitoyl-, dioleoyl-, dilinoleoyl-, and dilinolenoyl-glycerol, about equal amounts of the four steryl ester species were synthesized.     

Butyryl/Caproyl-CoA:Acetate CoA-transferase: cloning,expression and characterization of the key enzyme involved in medium-chain fatty acid biosynthesis

Coenzyme A transferases (CoATs) are important enzymes involved in carbon chain elongation, contributing to medium-chain fatty acid (MCFA) biosynthesis. For example, butyryl-CoA:acetate CoA transferase (BCoAT) is responsible for the final step of butyrate synthesis from butyryl-CoA. However, little is known about caproyl-CoA:acetate CoA-transferase (CCoAT), which is responsible for the final step of caproate synthesis from caproyl-CoA. In the present study, two CoAT genes from Ruminococcaceae bacterium CPB6 and Clostridium tyrobutyricum BEY8 were identified by gene cloning and expression analysis. Enzyme assays and kinetic studies were carried out using butyryl-CoA or caproyl-CoA as the substrate. CPB6-CoAT can catalyze the conversion of both butyryl-CoA into butyrate and caproyl-CoA into caproate, but its catalytic efficiency with caproyl-CoA as the substrate was 3.8-times higher than that with butyryl-CoA. In contrast, BEY8-CoAT had only BCoAT activity, not CCoAT activity. This demonstrated the existence of a specific CCoAT involved in chain elongation via the reverse β-oxidation pathway. Comparative bioinformatics analysis showed the presence of a highly conserved motif (GGQXDFXXGAXX) in CoATs, which is predicted to be the active center. Single point mutations in the conserved motif of CPB6-CoAT (Asp³⁴⁶ and Ala³⁵¹) led to marked decreases in the activity for butyryl-CoA and caproyl-CoA, indicating that the conserved motif is the active center of CPB6-CoAT and that Asp³⁴⁶ and Ala³⁵¹ have a significant impact on the enzymatic activity. This work provides insight into the function of CCoAT in caproic acid biosynthesis and improves understanding of the chain elongation pathway for MCFA production.     

A phosphopantetheinyl transferase that is essential for mitochondrial fatty acid biosynthesis

In this study we report the molecular genetic characterization of the Arabidopsis mitochondrial phosphopantetheinyl transferase (mtPPT), which catalyzes the phosphopantetheinylation and thus activation of mitochondrial acyl carrier protein (mtACP) of mitochondrial fatty acid synthase (mtFAS). This catalytic capability of the purified mtPPT protein (encoded by AT3G11470) was directly demonstrated in an in vitro assay that phosphopantetheinylated mature Arabidopsis apo‐mtACP isoforms. The mitochondrial localization of the AT3G11470‐encoded proteins was validated by the ability of their N‐terminal 80‐residue leader sequence to guide a chimeric GFP protein to this organelle. A T‐DNA‐tagged null mutant mtppt‐1 allele shows an embryo‐lethal phenotype, illustrating a crucial role of mtPPT for embryogenesis. Arabidopsis RNAi transgenic lines with reduced mtPPT expression display typical phenotypes associated with a deficiency in the mtFAS system, namely miniaturized plant morphology, slow growth, reduced lipoylation of mitochondrial proteins, and the hyperaccumulation of photorespiratory intermediates, glycine and glycolate. These morphological and metabolic alterations are reversed when these plants are grown in a non‐photorespiratory condition (i.e. 1% CO₂ atmosphere), demonstrating that they are a consequence of a deficiency in photorespiration due to the reduced lipoylation of the photorespiratory glycine decarboxylase.     

AtoSC two-component system is involved in cPHB biosynthesis through fatty acid metabolism in E. coli

Background

We have shown previously that AtoSC two-component system regulates the biosynthesis of E. coli cPHB [complexed poly-(R)-3-hydroxybutyrate].

Methods

The AtoSC involvement on fatty acids metabolism, towards cPHB synthesis, was studied using cPHB determination, gene expression, and fatty acid metabolic pathways inhibitors.

Results

Deletion of the atoDAEB operon from the E. coli genome resulted in a consistent reduction of cPHB accumulation. When in ΔatoDAEB cells, the atoDAEB operon and the AtoSC system were introduced extrachromosomally, a significant enhancement of cPHB levels was observed. Moreover, the introduction of a plasmid with atoSC genes regulated positively cPHB biosynthesis. A lesser cPHB enhancement was triggered when plasmids carrying either atoS or atoC were introduced. The intracellular distribution of cPHB was regulated by AtoSC or AtoC according to the inducer (acetoacetate or spermidine). Blockage of β-oxidation by acrylic acid reduced cPHB levels, suggesting the involvement of this pathway in cPHB synthesis; however, the overproduction of AtoSC or its constituents separately resulted in cPHB enhancement. Inhibition of fatty acid biosynthesis by cerulenin resulted to a major cPHB reduction, indicating the contribution of this pathway in cPHB production. Inhibition of both β-oxidation and fatty acid biosynthesis reduced dramatically cPHB, suggesting the contribution of both pathways in cPHB biosynthesis.

Conclusions

Short fatty acid catabolism (atoDAEB operon) and fatty acids metabolic pathways participate in cPHB synthesis through the involvement of AtoSC system.

General significance

The involvement of the AtoSC system in the fatty acids metabolic pathways interplay towards cPHB biosynthesis provides additional perceptions of AtoSC role on E. coli regulatory biochemical processes.