Structure-guided inhibitor design for human acetyl-coenzyme A carboxylase by interspecies active site conversion

Inhibition of acetyl-CoA carboxylases (ACCs), a crucial enzyme for fatty acid metabolism, has been shown to promote fatty acid oxidation and reduce body fat in animal models. Therefore, ACCs are attractive targets for structure-based inhibitor design, particularly the carboxyltransferase (CT) domain, which is the primary site for inhibitor interaction. We have cloned, expressed, and purified the CT domain of human ACC2 using baculovirus-mediated insect cell expression system. However, attempts to crystallize the human ACC2 CT domain have not been successful in our hands. Hence, we have been using the available crystal structure of yeast CT domain to design human ACC inhibitors. Unfortunately, as the selectivity of the lead series has increased against the full-length human enzyme, the potency against the yeast enzyme has decreased significantly. This loss of potency against the yeast enzyme correlated with a complete lack of binding of the human-specific compounds to crystals of the yeast CT domain. Here, we address this problem by converting nine key active site residues of the yeast CT domain to the corresponding human residues. The resulting humanized yeast ACC-CT (yCT-H9) protein exhibits biochemical and biophysical properties closer to the human CT domain and binding to human specific compounds. We report high resolution crystal structures of yCT-H9 complexed with inhibitors that show a preference for the human CT domain. These structures offer insights that explain the species selectivity of ACC inhibitors and may guide future drug design programs.     

Biotinoyl domain of human acetyl-CoA carboxylase: Structural insights into the carboxyl transfer mechanism

Acetyl-CoA carboxylase (ACC) catalyzes the first step in fatty acid biosynthesis: the synthesis of malonyl-CoA from acetyl-CoA. As essential regulators of fatty acid biosynthesis and metabolism, ACCs are regarded as therapeutic targets for the treatment of metabolic diseases such as obesity. In ACC, the biotinoyl domain performs a critical function by transferring an activated carboxyl group from the biotin carboxylase domain to the carboxyl transferase domain, followed by carboxyl transfer to malonyl-CoA. Despite the intensive research on this enzyme, only the bacterial and yeast ACC structures are currently available. To explore the mechanism of ACC holoenzyme function, we determined the structure of the biotinoyl domain of human ACC2 and analyzed its characteristics and interaction with the biotin ligase, BirA using NMR spectroscopy. The 3D structure of the hACC2 biotinoyl domain has a similar folding topology to the earlier determined domains from E. coli and P. shermanii. However, the local structures near the biotinylation sites have notable differences that include the geometry of the consensus "Met-Lys-Met" (MKM) motif and the absence of "thumb" structure in the hACC2 biotinoyl domain. Observations of the NMR signals upon the biotinylation indicate that the biotin group of hACC2 does not affect the structure of the biotinoyl domain, while the biotin group for E. coli ACC interacts directly with the thumb residues that are not present in the hACC2 structure. These results imply that, in the E. coli ACC reaction, the biotin moiety carrying the carboxyl group from BC to CT can pause at the thumb of the BCCP domain. The human biotinoyl domain, however, lacks the thumb structure and does not have additional noncovalent interactions with the biotin moiety; thus, the flexible motion of the biotinylated lysine residue must underlie the "swinging arm" motion. The chemical shift perturbation and the cross saturation experiments of the human ACC2 holo-biotinoyl upon the addition of the biotin ligase (BirA) showed the interaction surface near the MKM motif, the two glutamic acids (Glu 926, Glu 953), and the positively charged residues (several lysine and arginine residues). This study provides insight into the mechanism of ACC holoenzyme function and supports the swinging arm model in human ACCs.     

Structure,function and selective inhibition of bacterial acetyl-coa carboxylase

Acetyl-CoA carboxylase (ACC) catalyses the first committed step in fatty acid biosynthesis: a metabolic pathway required for several important biological processes including the synthesis and maintenance of cellular membranes. ACC employs a covalently attached biotin moiety to bind a carboxyl anion and then transfer it to acetyl-CoA, yielding malonyl-CoA. These activities occur at two different subsites: the biotin carboxylase (BC) and carboxyltransferase (CT). Structural biology, together with small molecule inhibitor studies, has provided new insights into the molecular mechanisms that govern ACC catalysis, specifically the BC and CT subunits. Here, we review these recent findings and highlight key differences between the bacterial and eukaryotic isozymes with a view to establish those features that provide an opportunity for selective inhibition. Especially important are examples of highly selective small molecule inhibitors capable of differentiating between ACCs from different phyla. The implications for early stage antibiotic discovery projects, stemming from these studies, are discussed.     

Multi-subunit acetyl-CoA carboxylases

Acetyl-CoA carboxylase (ACC) catalyses the first committed step of fatty acid synthesis, the carboxylation of acetyl-CoA to malonyl-CoA. Two physically distinct types of enzymes are found in nature. Bacterial and most plant chloroplasts contain a multi-subunit ACC (MS-ACC) enzyme that is readily dissociated into its component proteins. Mammals, fungi, and plant cytosols contain the second type of ACC, a single large multifunctional polypeptide. This review will focus on the structures, regulation, and enzymatic mechanisms of the bacterial and plant MS-ACCs.     

A colorimetric assay of 1-aminocyclopropane-1-carboxylate (ACC) based on ninhydrin reaction for rapid screening of bacteria containing ACC deaminase

Aims: 1‐Aminocyclopropane‐1‐carboxylate (ACC) deaminase activity is an efficient marker for bacteria to promote plant growth by lowering ethylene levels in plants. We aim to develop a method for rapidly screening bacteria containing ACC deaminase, based on a colorimetric ninhydrin assay of ACC. Methods and Results: A reliable colorimetric ninhydrin assay was developed to quantify ACC using heat‐resistant polypropylene chimney‐top 96‐well PCR plates, having the wells evenly heated in boiling water, preventing accidental contamination from boiling water and limiting evaporation. With this method to measure bacterial consumption of ACC, 44 ACC‐utilizing bacterial isolates were rapidly screened out from 311 bacterial isolates that were able to grow on minimal media containing ACC as the sole nitrogen source. The 44 ACC‐utilizing bacterial isolates showed ACC deaminase activities and belonged to the genus Burkholderia, Pseudomonas or Herbaspirillum. Conclusions: Determination of bacterial ACC consumption by the PCR‐plate ninhydrin–ACC assay is a rapid and efficient method for screening bacteria containing ACC deaminase from a large number of bacterial isolates. Significance and Impact of the Study: The PCR‐plate ninhydrin–ACC assay extends the utility of the ninhydrin reaction and enables a rapid screening of bacteria containing ACC deaminase from large numbers of bacterial isolates.     

Manipulation of Ethylene Synthesis in Roots Through Bacterial ACC Deaminase for Improving Nodulation in Legumes

Symbiotic association between rhizobia and legumes results in the development of unique structures on roots, called nodules. Nodulation is a very complex process involving a variety of genes that control NOD factors (bacterial signaling molecules), which are essential for the establishment, maintenance and regulation of this process and development of root nodules. Ethylene is an established potent plant hormone that is also known for its negative role in nodulation. Ethylene is produced endogenously in all plant tissues, particularly in response to both biotic and abiotic stresses. Exogenous application of ethylene and ethylene-releasing compounds are known to inhibit the formation and functioning of nodules. While inhibitors of ethylene synthesis or its physiological action enhance nodulation in legumes, some rhizobial strains also nodulate the host plant intensively, most likely by lowering endogenous ethylene levels in roots through their 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity. Co-inoculation with ACC deaminase containing plant growth promoting rhizobacteria plus rhizobia has been shown to further promote nodulation compared to rhizobia alone. Transgenic rhizobia or legume plants with expression of bacterial ACC deaminase could be another viable option to alleviate the negative effects of ethylene on nodulation. Several studies have well documented the role of ethylene and bacterial ACC deaminase in development of nodules on legume roots and will be the primary focus of this critical review.     

The Bacterial signal transduction protein GlnB regulates the committed step in fatty acid biosynthesis by acting as a dissociable regulatory subunit of acetyl‐CoA carboxylase

Biosynthesis of fatty acids is one of the most fundamental biochemical pathways in nature. In bacteria and plant chloroplasts, the committed and rate‐limiting step in fatty acid biosynthesis is catalyzed by a multi‐subunit form of the acetyl‐CoA carboxylase enzyme (ACC). This enzyme carboxylates acetyl‐CoA to produce malonyl‐CoA, which in turn acts as the building block for fatty acid elongation. In Escherichia coli, ACC is comprised of three functional modules: the biotin carboxylase (BC), the biotin carboxyl carrier protein (BCCP) and the carboxyl transferase (CT). Previous data showed that both bacterial and plant BCCP interact with signal transduction proteins belonging to the P_II family. Here we show that the GlnB paralogues of the P_II proteins from E. coli and Azospirillum brasiliense, but not the GlnK paralogues, can specifically form a ternary complex with the BC‐BCCP components of ACC. This interaction results in ACC inhibition by decreasing the enzyme turnover number. Both the BC‐BCCP‐GlnB interaction and ACC inhibition were relieved by 2‐oxoglutarate and by GlnB uridylylation. We propose that the GlnB protein acts as a 2‐oxoglutarate‐sensitive dissociable regulatory subunit of ACC in Bacteria.     

Methods for isolating and characterizing ACC deaminase-containing plant growth-promoting rhizobacteria

One of the major mechanisms utilized by plant growth-promoting rhizobacteria (PGPR) to facilitate plant growth and development is the lowering of ethylene levels by deamination of 1-aminocyclopropane-1-carboxylic acid (ACC) the immediate precursor of ethylene in plants. The enzyme catalysing this reaction, ACC deaminase, hydrolyses ACC to α -ketobutyrate and ammonia. Several bacterial strains that can utilize ACC as a sole source of nitrogen have been isolated from rhizosphere soil samples. All of these strains are considered to be PGPR based on the ability to promote canola seedling root elongation under gnotobiotic conditions. The treatment of plant seeds or roots with these bacteria reduces the amount of ACC in plants, thereby lowering the concentration of ethylene. Here, a rapid procedure for the isolation of ACC deaminase-containing bacteria, a root elongation assay for evaluating the effects of selected bacteria on root growth, and a method of assessing bacterial ACC deaminase activity are described in detail. This should allow researchers to readily isolate new PGPR strains adapted to specific environments.     

粘红酵母乙酰辅酶A羧化酶（ACC）基因的克隆及CT功能域基因的原核表达

利用简并PCR结合染色体步移法首次克隆获得粘红酵母乙酰辅酶A羧化酶（ACC）基因的全长序列信息。序列分析表明,该基因包含2个内含子,分别位于42～147 bp和315～677 bp处,编码区域总长为6 801bp,推导的氨基酸序列进行二级结构分析具备乙酰辅酶A羧化酶典型的3个功能域：生物素羧化酶（BC）、生物素羧基载体蛋白（BCCP）和羧基转移酶（CT）。克隆该基因的CT功能域基因,连接到原核表达载体pET-28a上,在Escherichia coli BL21（DE3）中成功表达,利用Ni-NTA树脂柱纯化获得CT的可溶性重组蛋白,浓度为1.8mg/mL,为研究ACC的功能和针对CT作用的除草剂机理研究提供了有价值的材料。     

Perspectives of bacterial ACC deaminase in phytoremediation

Phytoremediation of contaminated soil and water environments is regulated and coordinated by the plant root system, yet root growth is often inhibited by pollutant-induced stress. Prolific root growth could maximize rates of hyperaccumulation of inorganic contaminants or rhizodegradation of organic pollutants, and thus accelerate phytoremediation. Accelerated ethylene production in response to stress induced by contaminants is known to inhibit root growth and is considered as a major limitation in improving phytoremediation efficiency. Recent work shows that bacterial 1-aminocyclopropane-1-carboxylate (ACC) deaminase regulates ethylene levels in plants by metabolizing its precursor ACC into alpha-ketobutyric acid and ammonia. Plants inoculated with ACC deaminase bacteria or transgenic plants that express bacterial ACC deaminase genes can regulate their ethylene levels and consequently contribute to a more extensive root system. Such proliferation of roots in contaminated soil can lead to enhanced uptake of heavy metals or rhizodegradation of xenobiotics.     

Determinants of the localization, magnitude, and duration of a specific mucosal IgA plasma cell response in enterically immunized rats

The origin and fate of specific IgA plasma cells in intestinal lamina propria were studied in rats immunized enterically with cholera toxin (CT). Our major goal was to define how an anti-CT response is focused and sustained at the site of antigen challenge. To distinguish antigen-dependent from antigen-independent mechanisms, CT exposure was restricted to defined portions of intestine and, in some studies, the distribution of antitoxin-containing plasma cells (ACC) was examined in nonimmune adoptive recipients of post-challenge thoracic duct lymphocytes. After enteric priming and challenge, ACC appeared throughout the gut, but were most numerous at the challenged site. About 25% of ACC appearing at the site of jejunal challenge were due to antigen-driven proliferation of memory cells within the lamina propria; the remainder arose elsewhere, apparently in mucosal follicles or mesenteric lymph nodes, and migrated systemically as antitoxin-containing plasmablasts before homing to the lamina propria. The homing of these migrating ACC precursors was not affected by mucosal exposure to CT, nor did they undergo appreciable antigen-driven division after arrival in gut lamina propria. However, homing was specific for the organ from which they arose, i.e., precursors arising from duodenal challenge homed selectively to jejunum, whereas those from colonic challenge homed to the colon. The organ specificity of homing was determined during the challenge response and was independent of the origin of memory cells participating in the response. The survival of migrating ACC precursors did not differ in segments of gut exposed or nonexposed to CT. However, CT exposure at the time of their migration evoked another secondary-type response, due to stimulation of comigrating memory cells, thus sustaining the secondary response at a high level. These results and those in a previous report identify important mechanisms that affect the localization, magnitude, and duration of a specific IgA response, at least in the intestine. These include: 1) organ-specific homing of migrating IgA plasmablasts, 2) antigen-driven generation of IgA plasma cells from memory cells within the lamina propria, 3) enhanced memory at the site of mucosal priming compared to that a distant mucosae, and 4) regeneration of memory cells during the secondary response.     

Tolerance of transgenic canola expressing 1-aminocyclopropane-1-carboxylic acid deaminase to growth inhibition by nickel.

Plant growth-promoting bacteria are useful to phytoremediation strategies in that they confer advantages to plants in contaminated soil. When plant growth-promoting bacteria contain the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase, the bacterial cell acts as a sink for ACC, the immediate biosynthetic precursor of the plant growth regulator ethylene thereby lowering plant ethylene levels and decreasing the negative effects of various environmental stresses. In an effort to gain the advantages provided by bacterial ACC deaminase in the phytoremediation of metals from the environment two transgenic canola lines with the gene for this enzyme were generated and tested. In these transgenic canola plants, expression of the ACC deaminase gene is driven by either tandem constitutive cauliflower mosaic virus (CaMV) 35S promoters or the root specific rolD promoter from Agrobacterium rhizogenes. Following the growth of transgenic and non-transformed canola in nickel contaminated soil, it was observed that the rolD plants demonstrate significantly increased tolerance to nickel compared to the non-transformed control plants.     

Effect of a Nickel-Tolerant ACC Deaminase-Producing Pseudomonas Strain on Growth of Nontransformed and Transgenic Canola Plants

Four bacterial strains were isolated from soils at nickel-contaminated sites based on their ability to utilize 1-aminocyclopropane-1-carboxylate (ACC) as a sole source of nitrogen. The four isolates were all identified as Pseudomonas putida Biovar B, and subsequent testing revealed that they all exhibited traits previously associated with plant growth promotion (i.e., indoleacetic acid and siderophore production and ACC deaminase activity). These four strains were also tolerant of nickel concentrations of up to 13.2 mM in the culture medium. The strain, HS-2, selected for further characterization, was used in pot experiments to inoculate both nontransformed and transgenic canola plants (expressing a bacterial ACC deaminase gene in its roots). Plants inoculated with the HS-2 strain produced an increase in plant biomass as well as in nickel (Ni) uptake by shoots and roots. The results suggest that this strain is a potential candidate to be used as an inoculant in both phytoremediation protocols and in plant growth promotion.     

Pseudomonas brassicacearum strain Am3 containing 1-aminocyclopropane-1-carboxylate deaminase can show both pathogenic and growth-promoting properties in its interaction with tomato

The role of bacterial 1-aminocyclopropane-1-carboxylate (ACC) deaminase activity in the interaction between tomato (Lycopersicon esculentum=Solanum lycopersicum) and Pseudomonas brassicacearum was studied in different strains. The phytopathogenic strain 520-1 possesses ACC deaminase activity, an important trait of plant growth-promoting rhizobacteria (PGPR) that stimulates root growth. The ACC-utilizing PGPR strain Am3 increased in vitro root elongation and root biomass of soil-grown tomato cv. Ailsa Craig at low bacterial concentrations (10(6) cells ml-1 in vitro and 10(6) cells g-1 soil) but had negative effects on in vitro root elongation at higher bacterial concentrations. A mutant strain of Am3 (designated T8-1) that was engineered to be ACC deaminase deficient failed to promote tomato root growth in vitro and in soil. Although strains T8-1 and 520-1 inhibited root growth in vitro at higher bacterial concentrations (>10(6) cells ml-1), they did not cause disease symptoms in vitro after seed inoculation, or in soil supplemented with bacteria. All the P. brassicacearum strains studied caused pith necrosis when stems or fruits were inoculated with a bacterial suspension, as did the causal organism of this disease (P. corrugata 176), but the non-pathogenic strain Pseudomonas sp. Dp2 did not. Strains Am3 and T8-1 were marked with antibiotic resistance and fluorescence to show that bacteria introduced to the nutrient solution or on seeds in vitro, or in soil were capable of colonizing the root surface, but were not detected inside root tissues. Both strains showed similar colonization ability either on root surfaces or in wounded stems. The results suggest that bacterial ACC deaminase of P. brassicacearum Am3 can promote growth in tomato by masking the phytopathogenic properties of this bacterium.     

Bacteria with ACC deaminase can promote plant growth and help to feed the world

To feed all of the world's people, it is necessary to sustainably increase agricultural productivity. One way to do this is through the increased use of plant growth-promoting bacteria; recently, scientists have developed a more profound understanding of the mechanisms employed by these bacteria to facilitate plant growth. Here, it is argued that the ability of plant growth-promoting bacteria that produce 1-aminocyclopropane-1-carboxylate (ACC) deaminase to lower plant ethylene levels, often a result of various stresses, is a key component in the efficacious functioning of these bacteria. The optimal functioning of these bacteria includes the synergistic interaction between ACC deaminase and both plant and bacterial auxin, indole-3-acetic acid (IAA). These bacteria not only directly promote plant growth, they also protect plants against flooding, drought, salt, flower wilting, metals, organic contaminants, and both bacterial and fungal pathogens. While a considerable amount of both basic and applied work remains to be done before ACC deaminase-producing plant growth-promoting bacteria become a mainstay of plant agriculture, the evidence indicates that with the expected shift from chemicals to soil bacteria, the world is on the verge of a major paradigm shift in plant agriculture.