The 2.1 Å crystal structure of an acyl‐CoA synthetase from Methanosarcina acetivorans reveals an alternate acyl‐binding pocket for small branched acyl substrates

The acyl‐AMP forming family of adenylating enzymes catalyze two‐step reactions to activate a carboxylate with the chemical energy derived from ATP hydrolysis. X‐ray crystal structures have been determined for multiple members of this family and, together with biochemical studies, provide insights into the active site and catalytic mechanisms used by these enzymes. These studies have shown that the enzymes use a domain rotation of 140° to reconfigure a single active site to catalyze the two partial reactions. We present here the crystal structure of a new medium chain acyl‐CoA synthetase from Methanosarcina acetivorans. The binding pocket for the three substrates is analyzed, with many conserved residues present in the AMP binding pocket. The CoA binding pocket is compared to the pockets of both acetyl‐CoA synthetase and 4‐chlorobenzoate:CoA ligase. Most interestingly, the acyl‐binding pocket of the new structure is compared with other acyl‐ and aryl‐CoA synthetases. A comparison of the acyl‐binding pocket of the acyl‐CoA synthetase from M. acetivorans with other structures identifies a shallow pocket that is used to bind the medium chain carboxylates. These insights emphasize the high sequence and structural diversity among this family in the area of the acyl‐binding pocket. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Metabolic reconstructions identify plant 3‐methylglutaconyl‐CoA hydratase that is crucial for branched‐chain amino acid catabolism in mitochondria

The proteinogenic branched‐chain amino acids (BCAAs) leucine, isoleucine and valine are essential nutrients for mammals. In plants, BCAAs double as alternative energy sources when carbohydrates become limiting, the catabolism of BCAAs providing electrons to the respiratory chain and intermediates to the tricarboxylic acid cycle. Yet, the actual architecture of the degradation pathways of BCAAs is not well understood. In this study, gene network modeling in Arabidopsis and rice, and plant‐prokaryote comparative genomics detected candidates for 3‐methylglutaconyl‐CoA hydratase (4.2.1.18), one of the missing plant enzymes of leucine catabolism. Alignments of these protein candidates sampled from various spermatophytes revealed non‐homologous N‐terminal extensions that are lacking in their bacterial counterparts, and green fluorescent protein‐fusion experiments demonstrated that the Arabidopsis protein, product of gene At4g16800, is targeted to mitochondria. Recombinant At4g16800 catalyzed the dehydration of 3‐hydroxymethylglutaryl‐CoA into 3‐methylglutaconyl‐CoA, and displayed kinetic features similar to those of its prokaryotic homolog. When at4g16800 knockout plants were subjected to dark‐induced carbon starvation, their rosette leaves displayed accelerated senescence as compared with control plants, and this phenotype was paralleled by a marked increase in the accumulation of free and total leucine, isoleucine and valine. The seeds of the at4g16800 mutant showed a similar accumulation of free BCAAs. These data suggest that 3‐methylglutaconyl‐CoA hydratase is not solely involved in the degradation of leucine, but is also a significant contributor to that of isoleucine and valine. Furthermore, evidence is shown that unlike the situation observed in Trypanosomatidae, leucine catabolism does not contribute to the formation of the terpenoid precursor mevalonate.     

Kinetic improvement of an algal diacylglycerol acyltransferase 1 via fusion with an acyl‐CoA binding protein

Microalgal oils in the form of triacylglycerols (TAGs) are broadly used as nutritional supplements and biofuels. Diacylglycerol acyltransferase (DGAT) catalyzes the final step of acyl‐CoA‐dependent biosynthesis of TAG, and is considered a key target for manipulating oil production. Although a growing number of DGAT1s have been identified and over‐expressed in some algal species, the detailed structure?function relationship, as well as the improvement of DGAT1 performance via protein engineering, remain largely untapped. Here, we explored the structure?function features of the hydrophilic N‐terminal domain of DGAT1 from the green microalga Chromochloris zofingiensis (CzDGAT1). The results indicated that the N‐terminal domain of CzDGAT1 was less disordered than those of the higher eukaryotic enzymes and its partial truncation or complete removal could substantially decrease enzyme activity, suggesting its possible role in maintaining enzyme performance. Although the N‐terminal domains of animal and plant DGAT1s were previously found to bind acyl‐CoAs, replacement of CzDGAT1 N‐terminus by an acyl‐CoA binding protein (ACBP) could not restore enzyme activity. Interestingly, the fusion of ACBP to the N‐terminus of the full‐length CzDGAT1 could enhance the enzyme affinity for acyl‐CoAs and augment protein accumulation levels, which ultimately drove oil accumulation in yeast cells and tobacco leaves to higher levels than the full‐length CzDGAT1. Overall, our findings unravel the distinct features of the N‐terminus of algal DGAT1 and provide a strategy to engineer enhanced performance in DGAT1 via protein fusion, which may open a vista in generating improved membrane‐bound acyl‐CoA‐dependent enzymes and boosting oil biosynthesis in plants and oleaginous microorganisms.     

Chlamydomonas carries out fatty acid β‐oxidation in ancestral peroxisomes using a bona fide acyl‐CoA oxidase

Peroxisomes are thought to have played a key role in the evolution of metabolic networks of photosynthetic organisms by connecting oxidative and biosynthetic routes operating in different compartments. While the various oxidative pathways operating in the peroxisomes of higher plants are fairly well characterized, the reactions present in the primitive peroxisomes (microbodies) of algae are poorly understood. Screening of a Chlamydomonas insertional mutant library identified a strain strongly impaired in oil remobilization and defective in Cre05.g232002 (CrACX2), a gene encoding a member of the acyl‐CoA oxidase/dehydrogenase superfamily. The purified recombinant CrACX2 expressed in Escherichia coli catalyzed the oxidation of fatty acyl‐CoAs into trans‐2‐enoyl‐CoA and produced H₂O₂. This result demonstrated that CrACX2 is a genuine acyl‐CoA oxidase, which is responsible for the first step of the peroxisomal fatty acid (FA) β‐oxidation spiral. A fluorescent protein‐tagging study pointed to a peroxisomal location of CrACX2. The importance of peroxisomal FA β‐oxidation in algal physiology was shown by the impact of the mutation on FA turnover during day/night cycles. Moreover, under nitrogen depletion the mutant accumulated 20% more oil than the wild type, illustrating the potential of β‐oxidation mutants for algal biotechnology. This study provides experimental evidence that a plant‐type FA β‐oxidation involving H₂O₂‐producing acyl‐CoA oxidation activity has already evolved in the microbodies of the unicellular green alga Chlamydomonas reinhardtii.     

In Streptomyces lividans,acetyl‐CoA synthetase activity is controlled by O‐serine and Nɛ‐lysine acetylation

Protein acetylation is a rapid mechanism for control of protein function. Acetyl‐CoA synthetase (AMP‐forming, Acs) is the paradigm for the control of metabolic enzymes by lysine acetylation. In many bacteria, type I or II protein acetyltransferases acetylate Acs, however, in actinomycetes type III protein acetyltransferases control the activity of Acs. We measured changes in the activity of the Streptomyces lividans Acs (SlAcs) enzyme upon acetylation by PatB using in vitro and in vivo analyses. In addition to the acetylation of residue K610, residue S608 within the acetylation motif of SlAcs was also acetylated (PKTRSGK⁶¹⁰). S608 acetylation rendered SlAcs inactive and non‐acetylatable by PatB. It is unclear whether acetylation of S608 is enzymatic, but it was clear that this modification occurred in vivo in Streptomyces. In S. lividans, an NAD⁺‐dependent sirtuin deacetylase from Streptomyces, SrtA (a homologue of the human SIRT4 protein) was needed to maintain SlAcs function in vivo. We have characterized a sirtuin‐dependent reversible lysine acetylation system in Streptomyces lividans that targets and controls the Acs enzyme of this bacterium. These studies raise questions about acetyltransferase specificity, and describe the first Acs enzyme in any organism whose activity is modulated by O‐Ser and N^?‐Lys acetylation.     

Molecular characterization of a rabbit long-chain fatty acyl CoA synthetase that is highly expressed in the vascular endothelium

The formation of coenzyme A thioesters from long-chain fatty acids represents a metabolic branch point. We have isolated, cloned and sequenced a long-chain fatty acyl CoA synthetase (LCFACoAS) that is localized to the endothelium of rabbit heart and aorta. Immunofluoresence and in situ hybridization studies show intense staining of the intimal layer of the aorta and coronary vessels. The microvessels, including the capillaries, of the coronary circulation also show intense immunofluoresence. The enzyme shares only about 30% to 70% homology with the primary amino acid sequence of the other known LCFACoAS. There is a region of 44 amino acids at the carboxy terminus, which is unique to the vascular enzyme. This domain contains the most hydrophobic region of the molecule, indicating that it may function as a membrane anchoring site. These results suggest that this LCFACoAS represents a novel isoform, whose functional significance remains to be determined.     

Differential expression patterns of two delta‐9‐acyl‐CoA desaturases in Thitarodes pui (Lepidoptera: Hepialidae) during different seasons and cold exposure

Thitarodes pui larvae have a limited distribution in the Tibetan Plateau and are the host of a parasitic fungus, Ophiocordyceps sinensis. Low temperature is a main environmental stress. However, understanding of T. pui cold adaptation mechanisms is insufficient. Delta‐9‐acyl‐CoA desaturase (D9D) is closely correlated with cold adaptation for many organisms. To further understand the cold adaptation processes in T. pui larvae, two D9Ds, TpdesatA and TpdesatB were sequenced, and expression patterns were investigated during different seasons and cold exposure (under 0°C) in the laboratory. The full lengths of two cDNAs are 1,290 bp and 1,603 bp, and the ORFs encode a polypeptide of 348 and 359 amino acids, respectively. Four transmembrane domains, three conserved histidine residues and five hydrophobic regions exist in these two sequences. The expression level of TpdesatA is up‐regulated in the long‐term cold exposure and negatively correlated with temperature in seasonal patterns. TpdesatB responds to cold temperature in short‐term cold exposure and positively corresponds temporarily in seasonal expression. Two D9Ds may have different substrate specificities, TpdesatA tends to use C16:0 and C18:0 as substrate while TpdesatB prefers C18:0. In conclusion, TpdesatA may play a very important role in T. pui cold tolerance and TpdesatB regulates function in short‐term cold exposure and content change of fatty acids in the body.     

Characterization and functional assay of a fatty acyl‐CoA reductase gene in the scale insect,Ericerus pela Chavannes (Hemiptera: Coccoidae)

Ericerus pela Chavannes (Hemiptera: Coccoidae) is an economically important scale insect because the second instar males secrete a harvestable wax‐like substance. In this study, we report the molecular cloning of a fatty acyl‐CoA reductase gene (EpFAR) of E. pela. We predicted a 520‐aa protein with the FAR family features from the deduced amino acid sequence. The EpFAR mRNA was expressed in five tested tissues, testis, alimentary canal, fat body, Malpighian tubules, and mostly in cuticle. The EpFAR protein was localized by immunofluorescence only in the wax glands and testis. EpFAR expression in High Five insect cells documented the recombinant EpFAR reduced 26‐0:(S) CoA and to its corresponding alcohol. The data illuminate the molecular mechanism for fatty alcohol biosynthesis in a beneficial insect, E. pela.     

Synergistic roles of acyl‐CoA binding protein (ACBP1) and sterol carrier protein 2 (SCP2) in Toxoplasma lipid metabolism

Toxoplasma gondii relies on apicoplast‐localised FASII pathway and endoplasmic reticulum‐associated fatty acid elongation pathway for the synthesis of fatty acids, which flow through lipid metabolism mainly in the form of long‐chain acyl‐CoA (LCACoAs) esters. Functions of Toxoplasma acyl‐CoA transporters in lipid metabolism remain unclear. Here, we investigated the roles of acyl‐CoA‐binding protein (TgACBP1) and a sterol carrier protein‐2 (TgSCP2) as cytosolic acyl‐CoA transporters in lipid metabolism. The fluormetric binding assay and yeast complementation confirmed the acyl‐CoA binding activities of TgACBP1 and TgSCP2, respectively. Disruption of either TgACBP1 or TgSCP2 caused no obviously phenotypic changes, whereas double disruption resulted in defects in intracellular growth and virulence to mice. Gas chromatography coupled with mass spectrometry (GC–MS) results showed that TgACBP1 or TgSCP2 disruption alone led to decreased abundance of C18:1, whereas double disruption resulted in reduced abundance of C18:1, C22:1, and C24:1. ¹³C labelling assay combined with GC–MS showed that double disruption of TgACBP1 and TgSCP2 led to reduced synthesis rates of C18:0, C22:1, and C24:1. Furthermore, high performance liquid chromatography coupled with high resolution mass spectrometry (HPLC‐HRMS) was used for lipidomic analysis of parasites and indicated that loss of TgACBP1 and TgSCP2 caused serious defects in production of glycerides and phospholipids. Collectively, TgACBP1 and TgSCP2 play synergistic roles in lipid metabolism in T. gondii.     

The use of an acetoacetyl‐CoA synthase in place of a β‐ketothiolase enhances poly‐3‐hydroxybutyrate production in sugarcane mesophyll cells

Engineering the production of polyhydroxyalkanoates (PHAs) into high biomass bioenergy crops has the potential to provide a sustainable supply of bioplastics and energy from a single plant feedstock. One of the major challenges in engineering C₄ plants for the production of poly[(R)‐3‐hydroxybutyrate] (PHB) is the significantly lower level of polymer produced in the chloroplasts of mesophyll (M) cells compared to bundle sheath (BS) cells, thereby limiting the full PHB yield‐potential of the plant. In this study, we provide evidence that the access to substrate for PHB synthesis may limit polymer production in M chloroplasts. Production of PHB in M cells of sugarcane is significantly increased by replacing β‐ketothiolase, the first enzyme in the bacterial PHA pathway, with acetoacetyl‐CoA synthase. This novel pathway enabled the production of PHB reaching an average of 6.3% of the dry weight of total leaf biomass, with levels ranging from 3.6 to 11.8% of the dry weight (DW) of individual leaves. These yields are more than twice the level reported in PHB‐producing sugarcane containing the β‐ketothiolase and illustrate the importance of producing polymer in mesophyll plastids to maximize yield. The molecular weight of the polymer produced was greater than 2 × 10⁶ Da. These results are a major step forward in engineering a high biomass C₄ grass for the commercial production of PHB.