Cyclic AMP-dependent protein lysine acylation in mycobacteria regulates fatty acid and propionate metabolism

Acetylation of lysine residues is a posttranslational modification that is used by both eukaryotes and prokaryotes to regulate a variety of biological processes. Here we identify multiple substrates for the cAMP-dependent protein lysine acetyltransferase from Mycobacterium tuberculosis (KATmt). We demonstrate that a catalytically important lysine residue in a number of FadD (fatty acyl CoA synthetase) enzymes is acetylated by KATmt in a cAMP-dependent manner and that acetylation inhibits the activity of FadD enzymes. A sirtuin-like enzyme can deacetylate multiple FadDs, thus completing the regulatory cycle. Using a strain deleted for the KATmt ortholog in Mycobacterium bovis Bacillus Calmette-Guerin (BCG), we show for the first time that acetylation is dependent on intracellular cAMP levels. KATmt can utilize propionyl CoA as a substrate and, therefore, plays a critical role in alleviating propionyl CoA toxicity in mycobacteria by inactivating acyl CoA synthetase (ACS). The precision by which mycobacteria can regulate the metabolism of fatty acids in a cAMP-dependent manner appears to be unparalleled in other biological organisms and is ideally suited to adapt to the complex environment that pathogenic mycobacteria experience in the host.

Synthesis and crystal structure of copper (II) uracil ternary polymeric complex with 1,10-phenanthroline along with the Hirshfeld surface analysis of the metal binding sites for the uracil ligand

The study of models for ``metal-enzyme-substrate'' interaction has been a proactive area of research owing to its biological and pharmacological importance. In this regard the ternary copper uracil complex with 1,10-phenanthroline represents metal-enzyme-substrate system for DNA binding enzymes. The synthesis of the complex, followed by slow evaporation of the reaction mixture forms two concomitant solvatomorph crystals viz., {Cu(phen)(mu-ura)(H2O)](n)center dot H2O (1a)} and {Cu(phen)(mu-ura)(H2O)](n)center dot CH3OH (1b)}. Both complexes are structurally characterized, while elemental analysis, IR and EPR spectra were recorded for 1b (major product). In both complexes, uracil coordinates uniquely via N1 and N3 nitrogen atom acting as a bidentate bridging ligand forming a 1-D polymer. The two solvatomorphs were quantitatively analyzed for the differences with the aid of Hirshfeld surface analysis. (C) 2014 Elsevier B.V. All rights reserved.

Stimulation by adenosine 3′,5′-cyclic monophosphate of protein synthesis by adenohypophyseal polyribosomes

Addition of dibutyryl 3′,5′-cyclic AMP to slices of bovine pituitary stimulated incorporation of [3H]leucine into protein, whether or not actinomycin D was present; therefore the influence of 3′,5′-cyclic AMP on protein synthesis by bovine pituitary polysomes was studied. If the cyclic nucleotide was added to the complete protein-synthesizing system (including pH 5.0 enzyme), stimulation of [3H]leucine incorporation occurred only with pH 5.0 enzyme from rat liver; there was no stimulation when homologous enzyme, i.e., from bovine pituitary, was used. Addition of 3′,5′-cyclic AMP to the polysomes, before addition of pH 5.0 enzyme, resulted in stimulation of protein synthesis with either source of enzyme, but stimulation was facilitated to a greater degree, over the range 0.5-2 mM 3′,5′-cyclic AMP, when rat liver was the source. The stimulation of protein synthesis was prevented by the addition of cycloheximide. With rat liver pH 5.0 enzyme the product of hydrolysis of 3′,5′-cyclic AMP was mainly 5′-AMP whereas with pituitary pH 5.0 enzyme there was also dephosphorylation and deamination resulting in production of hypoxanthine and other bases. However, using either source of pH 5.0 enzyme and the complete protein-synthesizing system (i.e., including an ATP-regenerating mechanism) most of the 3H from hydrolysis of [3H]3′,5′-cyclic AMP was incorporated into ATP. The data are seen as compatible with a stimulation by 3′,5′-cyclic AMP of translation by pituitary polysomes; the significance of the importance of the source of pH 5.0 enzyme used in the system is obscure.

Crystal Structure of Escherichia coli Diaminopropionate Ammonia-lyase Reveals Mechanism of Enzyme Activation and Catalysis

Pyridoxal 5'-phosphate (PLP)-dependent enzymes utilize the unique chemistry of a pyridine ring to carry out diverse reactions involving amino acids. Diaminopropionate (DAP) ammonia-lyase (DAPAL) is a prokaryotic PLP-dependent enzyme that catalyzes the degradation of D-and L-forms of DAP to pyruvate and ammonia. Here, we report the first crystal structure of DAPAL from Escherichia coli (EcDAPAL) in tetragonal and monoclinic forms at 2.0 and 2.2 angstrom resolutions, respectively. Structures of EcDAPAL soaked with substrates were also determined. EcDAPAL has a typical fold type II PLP-dependent enzyme topology consisting of a large and a small domain with the active site at the interface of the two domains. The enzyme is a homodimer with a unique biological interface not observed earlier. Structure of the enzyme in the tetragonal form had PLP bound at the active site, whereas the monoclinic structure was in the apo-form. Analysis of the apo and holo structures revealed that the region around the active site undergoes transition from a disordered to ordered state and assumes a conformation suitable for catalysis only upon PLP binding. A novel disulfide was found to occur near a channel that is likely to regulate entry of ligands to the active site. EcDAPAL soaked with DL-DAP revealed density at the active site appropriate for the reaction intermediate aminoacrylate, which is consistent with the observation that EcDAPAL has low activity under crystallization conditions. Based on the analysis of the structure and results of site-directed mutagenesis, a two-base mechanism of catalysis involving Asp(120) and Lys(77) is suggested.

Cyclic AMP in Mycobacteria: the second messenger comes first

Cyclic AMP (cAMP) has emerged as a pivotal molecule for signalling in all life forms. Mycobacterial genomes have been found to encode for numerous proteins that are involved in cAMP generation, degradation and utilization. Many of these proteins have domain organizations unique to mycobacteria. This review summarizes recent advances in mechanisms of cAMP synthesis and degradation, focusing on the processes by which cAMP modulates mycobacterial signalling. We explore its impact on the physiology of the organism and on the discourse between M. tuberculosis and its host.

Sequence-dependent conformation of an A-DNA double helix: The crystal structure of the octamer d(G-G-T-A-T-A-C-C)

The crystal structures of the synthetic self-complementary octamer d(G-G-T-A-T-A-C-C) and its 5-bromouracil-containing analogue have been refined to R values of 20% and 14% at resolutions of 1·8 and 2·25 Å, respectively. The molecules adopt an A-DNA type double-helical conformation, which is minimally affected by crystal forces. A detailed analysis of the structure shows a considerable influence of the nucleotide sequence on the base-pair stacking patterns. In particular, the electrostatic stacking interactions between adjacent guanine and thymine bases produce symmetric bending of the double helix and a major-groove widening. The sugar-phosphate backbone appears to be only slightly affected by the base sequence. The local variations in the base-pair orientation are brought about by correlated adjustments in the backbone torsion angles and the glycosidic orientation. Sequence-dependent conformational variations of the type observed here may contribute to the specificity of certain protein-DNA interactions.

A Mycobacterial Cyclic AMP Phosphodiesterase That Moonlights as a Modifier of Cell Wall Permeability

Mycobacterium tuberculosis utilizes many mechanisms to establish itself within the macrophage, and bacterially derived cAMP is important in modulating the host cellular response. Although the genome of M. tuberculosis is endowed with a number of mammalian-like adenylyl cyclases, only a single cAMP phosphodiesterase has been identified that can decrease levels of cAMP produced by the bacterium. We present the crystal structure of the full-length and sole cAMP phosphodiesterase, Rv0805, found in M. tuberculosis, whose orthologs are present only in /the genomes of slow growing and pathogenic mycobacteria. The dimeric core catalytic domain of Rv0805 adopts a metallophosphoesterase fold, and the C-terminal region builds the active site and contributes to multiple substrate utilization.Localization of Rv0805 to the cell wall is dependent on its C terminus, and expression of either wild type or mutationally inactivated Rv0805 in M. smegmatis alters cell permeability to hydrophobic cytotoxic compounds. Rv0805 may therefore play a key role in the pathogenicity of mycobacteria, not only by hydrolyzing bacterial cAMP, but also by moonlighting as a protein that can alter cell wall functioning.

Synthesis, Crystal Structure, and Magnetic Properties of a Ferromagnetically Coupled Angular Trinuclear Copper(11) Complex [Cu3(02CMe)4(bpy)s(HzO)](PF)62

The synthesis, X-ray crystal structure, and magnetic properties of an angular trinuclear copper(II) complex [Cu3(O2CMC)4(bpy)3(H2O)](PF6)2 (1), obtained from a reaction of Cu2(O2CMe)4(H2O)2 With 2,2'-bipyridine (bpy) and NH4PF6 in ethanol, are reported. Complex 1 crystallizes in triclinic space group P1BAR with a = 11.529(1) angstrom, b = 12.121(2) angstrom, c = 17.153(2) angstrom, alpha = 82.01(1)-degrees, beta = 79.42(1)-degrees, gamma = 89.62(1)-degrees, and Z = 2. A total of 6928 data with I > 2.5sigma(I) were refined to R = 0.0441 and R(w) = 0.0557. The structure consists of a trinuclear core bridged by four acetate ligands showing different bonding modes. The coordination geometry at each copper is distorted square-pyramidal with a CuN2O2...O chromophore. The Cu...Cu distances are 3.198(1) angstrom, 4.568(1) angstrom, and 6.277(1) angstrom. There are two monoatomic acetate bridges showing Cu-O-Cu angles of 93.1(1) and 97.5(1)-degrees. Magnetic studies in the temperature range 39-297 K show the presence of a strong ferromagnetically coupled dicopper(II) unit (2J = +158 cm-1) and an essentially isolated copper(II) center (2J' = -0.4 cm-1) in 1. The EPR spectra display an axial spectrum giving g(parallel-to) = 2.28 (A(parallel-to) = 160 X 10(-4) cm-1) and g(perpendicular-to) = 2.06 (A(perpendicular-to) = 12 X 10(-4) cm-1) for the normal copper and two intense isotropic signals with g values 2.70 and 1.74 for the strongly coupled copper pair. The structural features of 1 compare well with the first generation models for ascorbate oxidase.

Cyclic AMP-induced Conformational Changes in Mycobacterial Protein Acetyltransferases

The activities of a number of proteins are regulated by the binding of cAMP and cGMP to cyclic nucleotide binding (CNB) domains that are found associated with one or more effector domains with diverse functions. Although the conserved architecture of CNB domains has been extensively studied by x-ray crystallography, the key to unraveling the mechanisms of cAMP action has been protein dynamics analyses. Recently, we have identified a novel cAMP-binding protein from mycobacteria, where cAMP regulates the activity of an associated protein acetyltransferase domain. In the current study, we have monitored the conformational changes that occur upon cAMP binding to the CNB domain in these proteins, using a combination of bioluminescence resonance energy transfer and amide hydrogen/deuterium exchange mass spectrometry. Coupled with mutational analyses, our studies reveal the critical role of the linker region (positioned between the CNB domain and the acetyltransferase domain) in allosteric coupling of cAMP binding to activation of acetyltransferase catalysis. Importantly, major differences in conformational change upon cAMP binding were accompanied by stabilization of the CNB and linker domain alone. This is in contrast to other cAMP-binding proteins, where cyclic nucleotide binding has been shown to involve intricate and parallel allosteric relays. Finally, this powerful convergence of results from bioluminescence resonance energy transfer and hydrogen/deuterium exchange mass spectrometry reaffirms the power of solution biophysical tools in unraveling mechanistic bases of regulation of proteins in the absence of high resolution structural information.

The non-planar peptide unit II. Comparison of theory with crystal structure datastar, open

The theoretical results derived in Part I (Ramachandran, G.N., Lakshminarayan, A.V. and Kolaskar, A.S. (1973) Biochim. Biophys. Acta 303, 8–13) that the three bonds of the peptide unit meeting at N can have a pyramidal structure is confirmed by an analysis of 14 published crystal structures of small peptides. It is shown that the dihedral angles θN and Δω are correlated, while θC, is small and is uncorrelated with Δω, showing that the non-planar distortion at C′ is generally small.

Crystal structure from packing studies: application to the triclinic structure of the cyclic hexapeptide cyclo-(gly-tyr-gly)2

The paper describes a novel method of finding the position and orientation of a relatively rigid molecule in the unit cell from criteria concerning allowed contact distances between atoms. On application to the crystal structure of a hexapeptide, C25H31N6O8.2H2O, it was possible to solve the structure from this starting point, by a series of SFLS refinements with an increasingly larger number of reflexions at successive stages. The packing analysis succeeded, even though the water molecules were not included to start with.

Crystal structure and conformation of the cyclic dipeptide cyclo-(L-histidyl-L-aspartyl) trihydrate

The crystal structure of cyclo-(L-histidyl-L-aspartyl) trihydrate has been determined by x-ray diffraction techniques, and refined to a final R index of 0.056 for 1601 reflections. The molecule is in a folded conformation, with the imidazole ring facing the diketopiperazine ring. However, since the diketopiperazine ring is essentially planar, the interaction between the two rings is not as intimate as in those cyclic dipeptides in which the diketopiperazine ring is in a boat conformation with the side chain occupying an axial, or flagpole, site. Planarity of the diketopiperazine ring may be dictated by steric interactions between the imidazole ring and the aspartyl side chain. The molecule is a zwitterion, a proton having been transferred from the carboxyl group of the aspartyl side chain to the imidazole ring.

Modular design of synthetic protein mimics. Crystal structure of two seven-residue helical peptide segments linked by .epsilon.-aminocaproic acid

Two seven-residue helical segments, Val-Ala-Leu-Aib-Val-Ala-Leu, were linked synthetically with an epsilon-aminocaproic acid (Acp) linker with the intention of making a stable antiparallel helix-helix motif. The crystal structure of the linked peptide Boc-Val-Ala-Leu-Aib-Val-Ala-Leu-Acp-Val-Ala-Leu-Aib-Val-Ala-Leu-OMe (1) shows the two helices displaced laterally from each other by the linker, but the linker has not folded the molecule into a close-packed antiparallel conformation. Two strong intermolecular NH...O = C hydrogen bonds are formed between the top of the lower helix of one molecule and the bottom of the upper helix in a laterally adjacent molecule to give the appearance of an extended single helix. The composite peptide with Boc and OMe end groups, C76H137N15O18.H2O, crystallize in space group P2(1) with a = 8.802 (1) angstrom, b = 20.409 (4) angstrom, c = 26.315 (3) angstrom, and beta = 90.72 (1)degrees; overall agreement R = 7.86% for 5030 observed reflections (\F(o)\ > 3-sigma(F)); resolution = 0.93 angstrom. Limited evidence for a more compact conformation in solution consistent with an antiparallel helix arrangement is obtained by comparison of the HPLC retention times and CD spectra of peptide 1 with well-characterized continuous helices of similar length and sequence.

Crystal structure of peanut lectin, a protein with an unusual quaternary structure

The x-ray crystal structure of the tetrameric T-antigen-binding lectin from peanut, M(r) 110,000, has been determined by using the multiple isomorphous replacement method and refined to an R value of 0.218 for 22,155 reflections within the 10- to 2.95-A resolution range. Each subunit has essentially the same characteristic tertiary fold that is found in other legume lectins. The structure, however, exhibits an unusual quaternary arrangement of subunits. Unlike other well-characterized tetrameric proteins with identical subunits, peanut lectin has neither 222 (D2) nor fourfold (C4) symmetry. A noncrystallographic twofold axis relates two halves of the molecule. The two monomers in each half are related by a local twofold axis. The mutual disposition of the axes is such that they do not lead to a closed point group. Furthermore, the structure of peanut lectin demonstrates that differences in subunit arrangement in legume lectins could be due to factors intrinsic to the protein molecule and, contrary to earlier suggestions, are not necessarily caused by interactions involving covalently linked sugar. The structure provides a useful framework for exploring the structural basis and the functional implications of the variability in the subunit arrangement in legume lectins despite all of them having nearly the same subunit structure, and also for investigating the general problem of "open" quaternary assembly in oligomeric proteins.

Crystal structure of L-lysyl-L-glutamic acid dihydrate

L-Lysyl-L-glutamic acid dihydrate, C11N3O5H21·2H2O, crystallizes in the monoclinic space group P21 with a = 12.474(2), b = 5.020(1), c = 13.157(2) Å, β= 114.69(1)° and Z = 2. The crystal structure was solved by direct methods and refined to an R value of 0.037 using full matrix least-squares method. The molecule exists as a double zwitterion with both the amino and carboxyl groups ionised. The peptide has a folded conformation with its Lys residue trans and Glu residue gauche−gauche+. The side chains of the Lys and Glu residues correspond to all trans and folded (g−g−g−) conformations respectively. The terminal carboxyl group forms hydrogen bonds with the ξ-amino group of the lysine side chain. The head-to-tail interaction often seen in peptide crystals is absent in the present structure. In the extended crystal structure water molecules form channels along the b direction and are enclosed within helically arranged hydrogen bonds formed by the lysine side chain and the peptide backbone.