Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:6.4.1.2 (acetyl-CoA carboxylase)
2,876 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Biotin carboxyl carrier protein (BCCP) is the small biotinylated subunit of Escherichia coli acetyl-CoA carboxylase (ACC), the enzyme that catalyzes the first committed step of fatty acid synthesis. Similar proteins are found in other bacteria and in chloroplasts. E. coli BCCP is a member of a large family of protein domains modified by covalent attachment of biotin to a specific lysine residue. However, the BCCP biotinyl domain differs from many of these proteins in that an eight-amino acid residue insertion is present upstream of the biotinylated lysine. X-ray crystallographic and multidimensional NMR studies show that these residues constitute a structure that has the appearance of an extended thumb that protrudes from the otherwise highly symmetrical domain structure. I report that expression of two mutant BCCPs lacking the thumb residues fails to restore growth and fatty acid synthesis to a temperature-sensitive E. coli strain that lacks BCCP when grown at nonpermissive temperature. Alignment of BCCPs from various organisms shows that only two of the eight thumb residues are strictly conserved, and amino acid substitution of either residue results in proteins giving only weak growth of the temperature-sensitive E. coli strain. Therefore, the thumb structure is essential for the function of BCCP in the ACC reaction and provides a useful motif for distinguishing the biotinylated proteins of multisubunit ACCs from those of enzymes catalyzing other biotin-dependent reactions. An unexpected result was that expression of a mutant BCCP in which the biotinylated lysine residue was substituted with cysteine was able to partially restore growth and fatty acid synthesis to the temperature-sensitive E. coli strain. This complementation was shown to be specific to BCCPs having native structure (excepting the biotinylated lysine) and is interpreted in terms of dimerization of the BCCP biotinyl domain during the ACC reaction.
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PMID:The biotinyl domain of Escherichia coli acetyl-CoA carboxylase. Evidence that the "thumb" structure id essential and that the domain functions as a dimer. 1149 22

Biotin protein ligase of Escherichia coli, the BirA protein, catalyses the covalent attachment of the biotin prosthetic group to a specific lysine of the biotin carboxyl carrier protein (BCCP) subunit of acetyl-CoA carboxylase. BirA also functions to repress the biotin biosynthetic operon and synthesizes its own corepressor, biotinyl-5'-AMP, the catalytic intermediate in the biotinylation reaction. We have previously identified two charge substitution mutants in BCCP, E119K, and E147K that are poorly biotinylated by BirA. Here we used site-directed mutagenesis to investigate residues in BirA that may interact with E119 or E147 in BCCP. None of the complementary charge substitution mutations at selected residues in BirA restored activity to wild-type levels when assayed with our BCCP mutant substrates. However, a BirA variant, in which K277 of the C-terminal domain was substituted with Glu, had significantly higher activity with E119K BCCP than did wild-type BirA. No function has been identified previously for the BirA C-terminal domain, which is distinct from the central domain thought to contain the ATP binding site and is known to contain the biotin binding site. Kinetic analysis of several purified mutant enzymes indicated that a single amino acid substitution within the C-terminal domain (R317E) and located some distance from the presumptive ATP binding site resulted in a 25-fold decrease in the affinity for ATP. Our data indicate that the C-terminal domain of BirA is essential for the catalytic activity of the enzyme and contributes to the interaction with ATP and the protein substrate, the BCCP biotin domain.
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PMID:The C-terminal domain of biotin protein ligase from E. coli is required for catalytic activity. 1171 29

The key event for antimicrobial action begins when streptomycin binds to the 30S subunit (S12 protein) of a ribosome. Lysine 42 and lysine 87 are involved. It is proposed that antagonism of acetyl coenzyme A carboxylase by streptomycin results in faulty fatty acids, lipids and derivatives marked exclusively for cell membrane synthesis. Streptomycin-sensitive growing cells are fatally wounded when defective membranes leak K(+) ions, then amino acids, nucleotides, oligonucleotides and proteins as increasing amounts of streptomycin enter the cell.
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PMID:Streptomycin revisited: molecular action in the microbial cell. 1186 97

Biotin carboxyl carrier protein (BCCP) is the small biotinylated subunit of Escherichia coli acetyl-CoA carboxylase, the enzyme that catalyzes the first committed step of fatty acid synthesis. E. coli BCCP is a member of a large family of protein domains modified by covalent attachment of biotin. In most biotinylated proteins, the biotin moiety is attached to a lysine residue located about 35 residues from the carboxyl terminus of the protein, which lies in the center of a strongly conserved sequence that forms a tightly folded anti-parallel beta-barrel structure. Located upstream of the conserved biotinoyl domain sequence are proline/alanine-rich sequences of varying lengths, which have been proposed to act as flexible linkers. In E. coli BCCP, this putative linker extends for about 42 residues with over half of the residues being proline or alanine. I report that deletion of the 30 linker residues located adjacent to the biotinoyl domain resulted in a BCCP species that was defective in function in vivo, although it was efficiently biotinylated. Expression of this BCCP species failed to restore normal growth and fatty acid synthesis to a temperature-sensitive E. coli strain that lacks BCCP when grown at nonpermissive temperatures. In contrast, replacement of the deleted BCCP linker with a linker derived from E. coli pyruvate dehydrogenase gave a chimeric BCCP species that had normal in vivo function. Expression of BCCPs having deletions of various segments of the linker region of the chimeric protein showed that some deletions of up to 24 residues had significant or full biological activity, whereas others had very weak or no activity. The inactive deletion proteins all lacked an APAAAAA sequence located adjacent to the tightly folded biotinyl domain, whereas deletions that removed only upstream linker sequences remained active. Deletions within the linker of the wild type BCCP protein also showed that the residues adjacent to the tightly folded domain play an essential role in protein function, although in this case some proteins with deletions within this region retained activity. Retention of activity was due to fusion of the domain to upstream sequences. These data provide new evidence for the functional and structural similarities of biotinylated and lipoylated proteins and strongly support a common evolutionary origin of these enzyme subunits.
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PMID:Interchangeable enzyme modules. Functional replacement of the essential linker of the biotinylated subunit of acetyl-CoA carboxylase with a linker from the lipoylated subunit of pyruvate dehydrogenase. 1195 2

The Escherichia coli biotin repressor functions in biotin retention and regulation of biotin biosynthesis. Biotin retention is accomplished via the two-step biotinylation of the biotin-dependent enzyme, acetyl-CoA carboxylase. In the first step of this reaction the substrates biotin and ATP are utilized in synthesis of the activated biotin, biotinyl-5'-AMP, while in the second step this activated biotin is transferred to a unique lysine residue of the biotin carboxyl carrier protein subunit of the carboxylase. Regulation of biotin biosynthesis is accomplished through binding of the repressor to the transcription control region of the biotin biosynthetic operon. The adenylated or activated biotin functions as the corepressor in this DNA binding process. The activated biotin is a mixed anhydride and thus labile. In efforts to develop tools for structural and thermodynamic studies of the biotin regulatory interactions, two analogs of the adenylate, a sulfamoyl derivative and an ester derivative, have been synthesized and functionally characterized. Results of fluorescence measurements indicate that both analogs bind with high affinity to the repressor and that both are inactive in biotin transfer to the acceptor protein. Functional studies of their corepressor properties indicate that while the sulfamoyl is a weak allosteric activator, the ester closely mimics the physiological corepressor in activation of assembly of the transcription repression complex. Results of these studies also provide further insight into the allosteric mechanism of the biotin repressor.
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PMID:The biotin repressor: modulation of allostery by corepressor analogs. 1503 56

Biotin-protein ligase is an enzyme that catalyzes the ATP-dependent biotinylation of a specific lysine residue in acetyl-CoA carboxylase. The biotin-protein ligase from Pyrococcus horikoshii OT3 has been cloned, overexpressed and purified. Crystallization was performed by the microbatch method or the vapour-diffusion method using PEG 2000 as a precipitant at 295 K. X-ray diffraction data have been collected to 1.6 A resolution from a native crystal and to 1.55 A resolution from a selenomethionine-derivative crystal for multiple anomalous dispersion phasing using synchrotron radiation at 100 K. The native crystal belongs to the monoclinic space group P2(1), with unit-cell parameters a = 38.601, b = 78.264, c = 70.147 A, beta = 101.48 degrees. Assuming a homodimer per asymmetric unit gives a VM value of 2.14 A3 Da(-1) and a solvent content of 42.5%. Cocrystals with biotin, ADP and biotinyl-5'-AMP were prepared and diffraction data sets were collected to 1.6, 1.6 and 1.45 A resolution, respectively.
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PMID:Purification, crystallization and preliminary crystallographic analysis of the biotin-protein ligase from Pyrococcus horikoshii OT3. 1651 Sep 91

The unrelenting emergence of antibiotic-resistant bacterial pathogens demands the investigation of antibiotics with new modes of action. The pseudopeptide antibiotic andrimid is a nanomolar inhibitor of the bacterial acetyl-CoA carboxylase that catalyses the first committed step in prokaryotic fatty acid biosynthesis. Recently, the andrimid (adm) biosynthetic gene cluster was isolated and heterologously expressed in Escherichia coli. This establishes a heterologous biological host in which to rapidly probe features of andrimid formation and to use biosynthetic engineering to make unnatural variants of this important and promising new class of antibiotics. Bioinformatic analysis of the adm cluster revealed a dissociated biosynthetic assembly system lacking canonical amide synthases between the first three carrier protein domains. Here we report that AdmF, a transglutaminase (TGase) homologue, catalyses the formation of the first amide bond, an N-acyl-beta-peptide link, in andrimid biosynthesis. Hence, AdmF is a newly discovered biosynthetic enzyme that acts as a stand-alone amide synthase between protein-bound, thiotemplated substrates in an antibiotic enzymatic assembly line. TGases (enzyme class (EC) 2.3.2.13) normally catalyse the cross-linking of (poly)peptides by creating isopeptidic bonds between the gamma-carboxamide group of a glutamine side chain of one protein and various amine donors, including lysine side chains. To the best of our knowledge, the present study constitutes the first report of a TGase-like enzyme recruited for the assembly of an antibiotic. Moreover, genome mining using the AdmF sequence yielded additional TGases in unassigned natural product biosynthetic pathways. With many more microbial genomes being sequenced, such a strategy could potentially unearth biosynthetic pathways producing new classes of antibiotics.
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PMID:A transglutaminase homologue as a condensation catalyst in antibiotic assembly lines. 1770 Jun 81

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.
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PMID:Biotinoyl domain of human acetyl-CoA carboxylase: Structural insights into the carboxyl transfer mechanism. 1824 44

AMP-activated protein kinase (AMPK) serves as an energy sensor and is considered a promising drug target for treatment of type II diabetes and obesity. A previous report has shown that mammalian AMPK alpha1 catalytic subunit including autoinhibitory domain was inactive. To test the hypothesis that small molecules can activate AMPK through antagonizing the autoinhibition in alpha subunits, we screened a chemical library with inactive human alpha1(394) (alpha1, residues 1-394) and found a novel small-molecule activator, PT1, which dose-dependently activated AMPK alpha1(394), alpha1(335), alpha2(398), and even heterotrimer alpha1beta1gamma1. Based on PT1-docked AMPK alpha1 subunit structure model and different mutations, we found PT1 might interact with Glu-96 and Lys-156 residues near the autoinhibitory domain and directly relieve autoinhibition. Further studies using L6 myotubes showed that the phosphorylation of AMPK and its downstream substrate, acetyl-CoA carboxylase, were dose-dependently and time-dependently increased by PT1 with-out an increase in cellular AMP:ATP ratio. Moreover, in HeLa cells deficient in LKB1, PT1 enhanced AMPK phosphorylation, which can be inhibited by the calcium/calmodulin-dependent protein kinase kinases inhibitor STO-609 and AMPK inhibitor compound C. PT1 also lowered hepatic lipid content in a dose-dependent manner through AMPK activation in HepG2 cells, and this effect was diminished by compound C. Taken together, these data indicate that this small-molecule activator may directly activate AMPK via antagonizing the autoinhibition in vitro and in cells. This compound highlights the effort to discover novel AMPK activators and can be a useful tool for elucidating the mechanism responsible for conformational change and autoinhibitory regulation of AMPK.
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PMID:Small molecule antagonizes autoinhibition and activates AMP-activated protein kinase in cells. 1832 58

SIRT1, a histone/protein deacetylase, and AMP-activated protein kinase (AMPK) are key enzymes responsible for longevity and energy homeostasis. We examined whether a mechanistic connection exists between these molecules that involves the major AMPK kinase LKB1. Initial studies demonstrated that LKB1 is acetylated in cultured (HEK293T) cells, mouse white adipose tissue, and rat liver. In the 293T cells, SIRT1 overexpression diminished lysine acetylation of LKB1 and concurrently increased its activity, cytoplasmic/nuclear ratio, and association with the LKB1 activator STRAD. In contrast, short hairpin RNA for SIRT1, where studied, had opposite effects on these parameters. Mass spectrometric analysis established that acetylation of LKB1 occurs on multiple, but specific, lysine residues; however, only mutation of lysine 48 to arginine, which mimics deacetylation, reproduced all of the effects of activated SIRT1. SIRT1 also affected downstream targets of LKB1. Thus its overexpression increased AMPK and acetyl-CoA carboxylase phosphorylation, and conversely, RNA interference-mediated SIRT1 knockdown reduced AMPK phosphorylation and that of another LKB1 target MARK1. Consistent with the results in cultured cells, total LKB1 lysine acetylation was decreased by 60% in the liver of 48-h starved rats compared with starved-refed rats, and this was associated with modest but significant increases in both LKB1 and AMPK activities. These results suggest that LKB1 deacetylation is regulated by SIRT1 and that this in turn influences its intracellular localization, association with STRAD, kinase activity, and ability to activate AMPK.
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PMID:SIRT1 modulation of the acetylation status, cytosolic localization, and activity of LKB1. Possible role in AMP-activated protein kinase activation. 1868 77


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