acyl-CoA Reductase Activities Research Articles

Wax ester synthesis is required for Mycobacterium tuberculosis to enter in vitro dormancy.

Mycobacterium tuberculosis (Mtb) is known to produce wax esters (WE) when subjected to stress. However, nothing is known about the enzymes involved in biosynthesis of WE and their role in mycobacterial dormancy. We report that two putative Mtb fatty acyl-CoA reductase genes (fcr) expressed in E. coli display catalytic reduction of fatty acyl-CoA to fatty aldehyde and fatty alcohol. Both enzymes (FCR1/Rv3391) and FCR2/Rv1543) showed a requirement for NADPH as the reductant, a preference for oleoyl-CoA over saturated fatty acyl-CoA and were inhibited by thiol-directed reagents. We generated Mtb gene-knockout mutants for each reductase. Metabolic incorporation of 14C-oleate into fatty alcohols and WE was severely diminished in the mutants under dormancy-inducing stress conditions that are thought to be encountered by the pathogen in the host. The fatty acyl-CoA reductase activity in cell lysates of the mutants under nitric oxide stress was significantly reduced when compared with the wild type. Complementation restored the lost activity completely in the Δfcr1 mutant and partially in the Δfcr2 mutant. WE synthesis was inhibited in both Δfcr mutants. The Δfcr mutants exhibited faster growth rates, an increased uptake of 14C-glycerol suggesting increased permeability of the cell wall, increased metabolic activity levels and impaired phenotypic antibiotic tolerance under dormancy-inducing combined multiple stress conditions. Complementation of the mutants did not restore the development of antibiotic tolerance to wild-type levels. Transcript analysis of Δfcr mutants showed upregulation of genes involved in energy generation and transcription, indicating the inability of the mutants to become dormant. Our results indicate that the fcr1 and fcr2 gene products are involved in WE synthesis under in vitro dormancy-inducing conditions and that WE play a critical role in reaching a dormant state. Drugs targeted against the Mtb reductases may inhibit its ability to go into dormancy and therefore increase susceptibility of Mtb to currently used antibiotics thereby enhancing clearance of the pathogen from patients.

A histidine residue in the catalytic mechanism distinguishes Vibrio harveyi aldehyde dehydrogenase from other members of the aldehyde dehydrogenase superfamily.

Aldehyde dehydrogenases (ALDHs) catalyze the transfer to NAD(P) of a hydride ion from a thiohemiacetal derivative of the aldehyde coupled with a cysteine residue in the active site. In Vibrio harveyi aldehyde dehydrogenase (Vh-ALDH), a histidine residue (H450) is in proximity (3.8 A) to the cysteine nucleophile (C289) and is thus capable of increasing its reactivity in sharp contrast to other ALDHs in which more distantly located glutamic acid residues are proposed to act as the general base. Mutation of H450 in Vh-ALDH to Gln and Asn resulted in loss of dehydrogenase, (thio)esterase, and acyl-CoA reductase activities; the residual activity of H450Q was higher than that of the H450N mutant in agreement with the capability of Gln but not Asn to partially replace the epsilon-imino group of H450. Coupled with a change in the rate-limiting step, these results indicate that H450 increases the reactivity of C289. Moreover, for the first time, the acylated enzyme intermediate could be directly monitored after reaction with [(3)H]tetradecanoyl-CoA showing that the H450Q mutant was acylated more rapidly than the H450N mutant. Inactivation of the wild-type enzyme with N-ethylmaleimide was much more rapid than the H450Q mutant which in turn was faster than the H450N mutant, demonstrating directly that the nucleophilicity of C289 was affected by H450. As the glutamic acid residue implicated as the general base in promoting cysteine nucleophilicity in other ALDHs is conserved in Vh-ALDH, elucidation of why a histidine residue has evolved to assist in this function in Vh-ALDH will be important to understand the mechanism of ALDHs in general, as well as help delineate the specific roles of the active site glutamic acid residues.

Cysteine-286 as the site of acylation of the Lux-specific fatty acyl-CoA reductase

The channelling of fatty acids into the fatty aldehyde substrate for the bacterial bioluminescence reaction is catalyzed by a fatty acid reductase multienzyme complex, which channels fatty acids through the thioesterase (LuxD), synthetase (LuxE) and reductase (LuxC) components. Although all three components can be readily acylated in extracts of different luminescent bacteria, this complex has been successfully purified only from Photobacterium phosphoreum and the sites of acylation identified on LuxD and LuxE. To identify the acylation site on LuxC, the nucleotide sequence of P. phosphoreum luxC has been determined and the gene expressed in a mutant Escherichia coli strain. Even in crude extracts, the acylated reductase intermediate as well as acyl-CoA reductase activity could be readily detected, providing the basis for analysis of mutant reductases. Comparison of the amino-acid sequences of LuxC from P. phosphoreum, P. leiognathi and other luminescent bacteria, showed that only three cysteine residues (C 171, C 279, and C 286) were conserved. As a cysteine residue on LuxC has been implicated in fatty acyl transfer, each of the conserved cysteine residues of the P. phosphoreum and P. leiognathi reductases was converted to a serine residue, and the properties of the mutant proteins examined. Only mutation of C 286-blocked reductase activity and prevented formation of the acylated reductase intermediate, showing that C 286 is the site of acylation on LuxC.

Resolution and Purification of an Aldehyde-Generating and an Alcohol-Generating Fatty Acyl-CoA Reductase from Pea Leaves (Pisum sativumL.).

Higher plant tissues produce both wax esters generated from fatty alcohols and hydrocarbons generated from fatty aldehydes. If two different reductases are responsible for the synthesis of aldehydes and alcohols, both types of reductases may be present in such tissues. To test for this possibility, pea leaves, known to produce both types of wax components, were examined. Subcellular fractionation showed that acyl-CoA reductase activities were localized mainly in the microsomal fraction. Fatty aldehyde formation was rectilinear for 30 min and subsequently decreased, whereas fatty alcohol formation remained linear for 2 h. The two activities in the microsomes were differently affected by pH; alcohol formation was optimal between pH 5 and pH 6, whereas aldehyde formation was optimal at around pH 7.5. With solubilized microsomes, protein concentration dependence of alcohol formation showed a sigmoidal pattern, possibly suggesting inhibition by hexadecanoyl-CoA at low protein concentrations. Bovine serum albumin (BSA) enhanced alcohol formation. In contrast, the aldehyde generation showed a typical protein concentration dependence, and BSA severely inhibited aldehyde generation. Phosphatidylcholine showed over twofold stimulation for alcohol formation, whereas aldehyde formation was only slightly stimulated. All of this biochemical evidence suggested the presence of two different reductases. Confirming this hypothesis, an aldehyde-generating and an alcohol-generating reductase were resolved from the solubilized microsomal proteins using Blue A agarose, gel filtration, and hexadecanoyl-CoA affinity chromatography. SDS–PAGE of the purified proteins showed that the alcohol-generating enzyme was a 58-kDa protein and the aldehyde-forming one was a 28-kDa protein. It is proposed that two different elongating systems are functionally coupled to the alcohol-generating and aldehyde-generating reductases, which in turn are coupled to the transacylase to produce wax esters and to the decarbonylase to produce hydrocarbons, respectively.

Vibrio harveyi aldehyde dehydrogenase. Partial reversal of aldehyde oxidation and its possible role in the reduction of fatty acids for the bioluminescence reaction.

Vibrio harveyi aldehyde dehydrogenase, which catalyzes the oxidation of long chain aliphatic aldehydes to acids, has been discovered to have both acyl-CoA reductase and thioesterase activities. Tetradecanoyl-CoA was reduced to tetradecanal in the presence of NAD(P)H, as monitored by the stimulation of luciferase activity by the aldehyde product (acyl-CoA reductase). In the absence of NADPH, [3H]tetradecanoyl-CoA was hydrolyzed to the hexane-soluble fatty acid (thioesterase). Inhibition data with N-ethylmaleimide suggest that a single active site on aldehyde dehydrogenase is responsible for all three enzymatic activities. The acyl-CoA reductase activity was maximal at low NADPH concentration (about 1 microM), whereas much higher concentrations of NADH (greater than 100-fold) were required for optimal activity. Further increases in NADPH or NADH concentrations inhibited both the acyl-CoA reductase and thioesterase reactions. On the basis of the specificity of aldehyde dehydrogenase for NADP(H), an improved purification procedure employing affinity chromatography on 2', 5'-ADP-Sepharose is described. Although fatty acid reductase activity could not be reconstituted, aldehyde dehydrogenase specifically stimulated the rate of acylation of the acyl protein synthetase component from the Photobacterium phosphoreum fatty acid reductase system. This observation, combined with the partial reversal of aldehyde oxidation described above, suggests a possible role for aldehyde dehydrogenase in aldehyde biosynthesis for the luminescent reaction in V. harveyi.

Differential acylation in vitro with tetradecanoyl coenzyme A and tetradecanoic acid (+ATP) of three polypeptides shown to have induced synthesis in Photobacterium phosphoreum.

Acylation of extracts of Photobacterium phosphoreum at different stages of growth with [3H]tetradecanoic acid (+ATP) has shown that two polypeptides found in the fatty acid reductase complex, the fatty acid activating enzyme (50K) and the 34K polypeptide, were specifically labeled during induction of the luminescent system. An alternate method for in vitro acylation of polypeptides in the luminescent system was developed using tetradecanoyl-CoA. Both the 34K polypeptide and, to a lesser extent, the acyl-CoA reductase component (58K) in the complex, were acylated with [3H]tetradecanoyl-CoA. In contrast, the fatty acid activating enzyme (50K) was not labeled. Labeling of both the 34K and 58K polypeptides with [3H]tetradecanoyl-CoA as well as the acyl-CoA reductase activity in extracts paralleled the induction of luciferase during growth. Differential labeling of P. phosphoreum cells with [35S]methionine before luminescence induction and with [3H]methionine after the onset of luminescence followed by purification of luciferase and the polypeptides in the fatty acid reductase complex demonstrated that the alpha and beta subunits of luciferase and the 34K, 50K, and 58K polypeptides of the complex had 3H/35S ratios at least 7-fold higher than the constitutive proteins. These results give evidence that the synthesis of the component polypeptides of the fatty acid reductase are induced during the development of bioluminescence and may be under the same control as luciferase. The experiments also showed that P. phosphoreum may have the highest content of luciferase of any luminescent bacterium, constituting approximately 20% of the total soluble protein in extracts.

Resolution of the fatty acid reductase from Photobacterium phosphoreum into acyl protein synthetase and acyl-CoA reductase activities. Evidence for an enzyme complex.

Resolution of the fatty acid reductase from Photobacterium phosphoreum into acyl protein synthetase and acyl-CoA reductase activities. Evidence for an enzyme complex.

Regulation of acyl coenzyme A reductase by a heat-stable cytosolic protein during preputial gland development

A low-molecular-weight protein located in the cytosol of mouse preputial glands has been shown to stimulate the activity of a microsomal acyl coenzyme A (CoA) reductase in the gland. This cytoplasmic protein was stable to heating and lyophilization, but was destroyed by trypsin digestion. It was able to bind palmitoyl-CoA and gel elution behavior indicated it had a molecular weight of 10,000–12,000. The level of this stimulatory cytosolic protein and the activity of acyl-CoA reductase were shown to correlate with differentiation of the preputial gland during development of puberty in male mice; the acyl-CoA reductase activity first appeared at 4 weeks of age and increased dramatically up to 6 weeks of age. By 8 weeks, when sexual maturity was attained, the reductase activity decreased to that level found in mature male mice. The cytosol from the preputial glands of the youngest mice (3 weeks) contained sufficient heat-stable acyl-CoA binding protein to stimulate acyl-CoA reduction; however, the 3-week-old preputial gland microsomes had little or no acyl-CoA reductase activity. As the animal matured, the stimulatory capacity in the heat-treated cytosol increased, reaching a maximum at 6 weeks; by 8 weeks, the stimulatory capacity of the soluble fraction had decreased to that found in mature male mouse. Results of this study suggest that the concentration of acyl-CoA, cytoplasmic acyl-CoA binding protein, and acyl-CoA reductase activity regulate the level of fatty alcohols in vivo and that the reductase activity and binding protein have similar patterns of development during puberty.

Properties of microsomal acyl coenzyme A reductase in mouse preputial glands

Synthesis of long-chain fatty alcohols in preputial glands of mice is catalyzed by an NADPH-dependent acyl coenzyme A (CoA) reductase located in microsomal membranes; sensitivity to trypsin digestion indicates that the reductase is on the cytoplasmic side of the membrane. Results with pyrazole and phenobarbital demonstrate the reaction is not catalyzed by a nonspecific alcohol dehydrogenase or an aldehyde reductase. Acyl-CoA reductase activity is sensitive to sulfhydryl and serine reagent modification, is stimulated by bovine serum albumin, and produces an aldehyde intermediate. The activity is extremely detergent sensitive and cannot be restored even after removal of the detergents. Phospholipase C or asolectin treatment does not release the acyl-CoA reductase from microsomal membranes, but causes a significant decrease in the activity recovered in the membrane pellet. Glycerol does not solubilize the reductase activity, nor does 3.0 m NaCl; however, the combination of glycerol and 3.0 m NaCl did release about 50% of the acyl-CoA reductase from the microsomal pellet. Substrate concentration curves obtained in the presence or absence of bovine serum albumin show significant differences in enzyme activities. The reductase is sensitive to the concentration of palmitoyl-CoA and is progressively inhibited at levels beyond the critical micellar concentration of the substrate. The apparent K m for acyl-CoA reductase is 14 μ m; however, the maximum velocity varies with the concentration of albumin used. Expression of enzyme activity in delipidated microsomes requires specific phospholipids, which suggests that in vivo regulation of acyl-CoA reductase activity could be achieved through modifications in membrane lipid composition.

Solubilization of fatty acid synthetase, acyl-CoA reductase, and fatty acyl-CoA alcohol transacylase from the microsomes of Euglena gracilis

The enzymes involved in the de novo synthesis of wax esters were solubilized from the microsomes of dark-grown Euglena gracilis by the use of deoxycholate or high concentration of sodium chloride. Solubilization with deoxycholate provided a preparation which catalyzed only the synthesis of fatty acids and this activity was stimulated by addition of phospholipids while the fatty acid synthetase from the soluble cytoplasm was not affected by the addition of phospholipids. Detergent Solubilization did not provide active soluble acyl-CoA reductase. Solubilization of the microsomal fatty acid synthetase together with acyl-CoA reductase and acyl-CoA hexadecanol transacylase was achieved upon treatment of the membranes with 3 m sodium chloride. Removal of sodium chloride by dialysis resulted in turbidity and the insoluble material recovered from this turbid solution by centrifugation at 105,000 g contained the bulk of the acyl-CoA reductase activity while the supernatant contained the major portion of the fatty acid synthetase. This aggregation of the reductase and its high detergent sensitivity suggest that this enzyme is highly hydrophobic and probably buried in the membrane matrix. Sucrose density gradient centrifugation of the NaCl solubilized preparation in the presence of 3 m sodium chloride resulted in the resolution of fatty acid synthetase from the acyl-CoA reductase and the transacylase.

A tissue culture system for studies of the regulation of ether-linked and ester-linked aliphatic moieties in glycerolipids

The incorporation of [1- 14 C]hexadecanol and [1- 14 C]palmitic acid into ether- and ester-linked lipids was investigated using suspension and monolayer cultures of L-M fibroblasts. Cells maintained in suspension cultures incorporated relatively high amounts of [1- 14 C]hexadecanol into alkyldiacylglycerols, whereas the same cells grown as monolayers did not. The suspension cultures also synthesized small amounts of ethanolamine plasmalogens when the cells were incubated for 6 hr; none of the other lipid classes contained 14 C in the O -alk-l-enyl moieties. In the monolayer cultures most of the label from the hexadecanol was incorporated into the acyl moieties, indicating that the enzymes responsible for oxidation of the alcohol is extremely active under these conditions. Of the total activity incorporated into the lipids of the suspension cultures, 2–5% of the 14 C was present in the alkyldiacylglycerols when palmitic acid was the precursor and 5–15% when hexadecanol was the precursor. Approximately 1% of the incorporated 14 C was in the alkyldiacylglycerol fraction from the monolayer cultures. Analysis of the alkyldiacylglycerols isolated from the suspension cultures incubated with hexadecanol demonstrated that approximately 60% of the 14 C was in the alkyl moieties, whereas the alkyldiacylglycerols isolated from the samples incubated with palmitic acid contained about 35% of the 14 C in the alkyl moieties. The incorporation of label from the palmitic acid into O -alkyl moieties shows that acyl-CoA reductase activities are present at a much higher level in the L-M fibroblasts maintained in suspension cultures, than in the monolayer cultures. In some experiments, the O -alkyl moieties of cells maintained in suspension cultures were prelabeled with [1- 14 C]hexadecanol so that the turnover of the O -alkyl moieties could be traced when the cells were transferred back to monolayer cultures. After these cells grew overnight as monolayers, they contained essentially no labeled alkyldiacylglycerols although substantial amounts of labled acyl groups were still present. The incorporation of [1- 14 C]hexadecanol into the alkyl moieties of the glycerolipids in the cells maintained in suspension culture could also be altered by pH of the growth media. At a pH of 6.2, the 14 C associated with the alkyldiacyl-glycerols was 40–50% lower than that observed at pH 8.0. These results demonstrate that the metabolism of ether and ester groupings of glycerolipids in L-M fibroblasts can be manipulated and that the tissue culture systems described should be useful for exploring regulatory mechanisms involving glycerolipids that contain these groupings.