Crystal structure of mycothiol synthase (Rv0819) from Mycobacterium tuberculosis shows structural homology to the GNAT family of N-acetyltransferases

Mycothiol is the predominant low-molecular weight thiol produced by actinomycetes, including Mycobacterium tuberculosis. The last reaction in the biosynthetic pathway for mycothiol is catalyzed by mycothiol synthase (MshD), which acetylates the cysteinyl amine of cysteine-glucosamine-inositol (Cys-GlcN-Ins). The crystal structure of MshD was determined in the presence of coenzyme A and acetyl-CoA. MshD consists of two tandem-repeated domains, each exhibiting the Gcn5-related N-acetyltransferase (GNAT) fold. These two domains superimpose with a root-mean-square deviation of 1.7 A over 88 residues, and each was found to bind one molecule of coenzyme, although the binding sites are quite different. The C-terminal domain has a similar active site to many GNAT members in which the acetyl group of the coenzyme is presented to an open active site slot. However, acetyl-CoA bound to the N-terminal domain is buried, and is apparently not positioned to promote acetyl transfer. A modeled substrate complex indicates that Cys-GlcN-Ins would only fill a portion of a negatively charged channel located between the two domains. This is the first structure determined for an enzyme involved in the biosynthesis of mycothiol.     

Catalytic Mechanism of Bleomycin N-Acetyltransferase Proposed on the Basis of Its Crystal Structure

Bleomycin (Bm) N-acetyltransferase, BAT, is a self-resistance determinant in Bm-producing Streptomyces verticillus ATCC15003. In our present study, we crystallized BAT under both a terrestrial and a microgravity environment in the International Space Station. In addition to substrate-free BAT, the crystal structures of BAT in a binary complex with CoA and in a ternary complex with Bm and CoA were determined. BAT forms a dimer structure via interaction of its C-terminal domains in the monomers. However, each N-terminal domain in the dimer is positioned without mutual interaction. The tunnel observed in the N-terminal domain of BAT has two entrances: one that adopts a wide funnel-like structure necessary to accommodate the metal-binding domain of Bm, and another narrow entrance that accommodates acetyl-CoA (AcCoA). A groove formed on the dimer interface of two BAT C-terminal domains accommodates the DNA-binding domain of Bm. In a ternary complex of BAT, BmA₂, and CoA, a thiol group of CoA is positioned near the primary amine of Bm at the midpoint of the tunnel. This proximity ensures efficient transfer of an acetyl group from AcCoA to the primary amine of Bm. Based on the BAT crystal structure and the enzymatic kinetic study, we propose that the catalytic mode of BAT takes an ordered-like mechanism.     

Choline acetyltransferase structure reveals distribution of mutations that cause motor disorders

Choline acetyltransferase (ChAT) synthesizes acetylcholine in neurons and other cell types. Decreases in ChAT activity are associated with a number of disease states, and mutations in ChAT cause congenital neuromuscular disorders. The crystal structure of ChAT reported here shows the enzyme divided into two domains with the active site in a solvent accessible tunnel at the domain interface. A low-resolution view of the complex with one substrate, coenzyme A, defines its binding site and suggests an additional interaction not found in the related carnitine acetyltransferase. Also, the preference for choline over carnitine as an acetyl acceptor is seen to result from both electrostatic and steric blocks to carnitine binding at the active site. While half of the mutations that cause motor disorders are positioned to affect enzyme activity directly, the remaining changes are surprisingly distant from the active site and must exert indirect effects. The structure indicates how ChAT is regulated by phosphorylation and reveals an unusual pattern of basic surface patches that may mediate membrane association or macromolecular interactions.     

Identification of the mycothiol synthase gene (mshD) encoding the acetyltransferase producing mycothiol in actinomycetes

Mycothiol is the predominant thiol in most actinomycetes, including Mycobacterium tuberculosis, and appears to play a role analogous to glutathione, which is not found in these bacteria. The enzymes involved in mycothiol biosynthesis are of interest as potential targets for new drugs directed against tuberculosis. In this work we describe the isolation and characterization of a Tn 5 transposon mutant of Mycobacterium smegmatis that is blocked in the production of mycothiol and accumulates its precursor, 1 D-myo-inosityl 2- L-cysteinylamido-2-deoxy-alpha-D-glucopyranoside (Cys-GlcN-Ins). Cys-GlcN-Ins isolated from this mutant was used to assay for acetyl-CoA:Cys-GlcN-Ins acetyltransferase (mycothiol synthase, MshD) activity, which was found in wild-type cells, but not in the mutant. Sequencing outward of the DNA of the mutant strain from the site of insertion permitted identification of the mshD gene in the M. smegmatis genome, as well as the orthologous gene Rv0819 in the M. tuberculosis genome. Cloning and expression of mshD from M. tuberculosis (Rv0819) in Escherichia coli gave a transformant with MshD activity, demonstrating that Rv0819 is the mshD mycothiol biosynthesis gene.     

Crystal structures of cyanide complexes of P450cam and the oxygenase domain of inducible nitric oxide synthase-structural models of the short-lived oxygen complexes

The crystal structure of the ternary cyanide complex of P450cam and camphor was determined to 1.8A resolution and found to be identical with the structure of the active oxygen complex [I. Schlichting et al., 2000, Science 287, 1615]. Notably, cyanide binds in a bent mode and induces the active conformation that is characterized by the presence of two water molecules and a flip of the carbonyl of the conserved Asp251. The structure of the ternary complex of cyanide, L-arginine, and the oxygenase domain of inducible nitric oxide synthase was determined to 2.4A resolution. Cyanide binds essentially linearly, interacts with L-Arg, and induces the binding of a water molecule at the active site. This water is positioned by backbone interactions, located 2.8A from the nitrogen atom of cyanide, and could provide a proton required for O-O bond scission in the hydroxylation reaction of nitric oxide synthase.     

Substrate binding and catalytic mechanism of human choline acetyltransferase

Choline acetyltransferase (ChAT) catalyzes the synthesis of the neurotransmitter acetylcholine from choline and acetyl-CoA, and its presence is a defining feature of cholinergic neurons. We report the structure of human ChAT to a resolution of 2.2 A along with structures for binary complexes of ChAT with choline, CoA, and a nonhydrolyzable acetyl-CoA analogue, S-(2-oxopropyl)-CoA. The ChAT-choline complex shows which features of choline are important for binding and explains how modifications of the choline trimethylammonium group can be tolerated by the enzyme. A detailed model of the ternary Michaelis complex fully supports the direct transfer of the acetyl group from acetyl-CoA to choline through a mechanism similar to that seen in the serine hydrolases for the formation of an acyl-enzyme intermediate. Domain movements accompany CoA binding, and a surface loop, which is disordered in the unliganded enzyme, becomes localized and binds directly to the phosphates of CoA, stabilizing the complex. Interactions between this surface loop and CoA may function to lower the KM for CoA and could be important for phosphorylation-dependent regulation of ChAT activity.     

Regional and Subcellular Distribution of ATP-Citrate Lyase and Other Enzymes of Acetyl-CoA Metabolism in Rat Brain

The activities of ATP-citrate lyase in frog, guinea pig, mouse, rat, and human brain vary from 18 to 30 μmol/h/g of tissue, being several times higher than choline acetyltransferase activity. Activities of pyruvate dehydrogenase and acetyl coenzyme A synthetase in rat brain are 206 and 18.4 μmol/h/g of tissue, respectively. Over 70% of the activities of both choline acetyltransferase and ATP-citrate lyase in secondary fractions are found in synaptosomes. Their preferential localization in synaptosomes and synaptoplasm is supported by RSA values above 2. Acetyl CoA synthetase activity is located mainly in whole brain mitochondria (RSA, 2.33) and its activity in synaptoplasm is low (RSA, 0.25). The activities of pyruvate dehydrogenase, citrate synthase, and carnitine acetyltransferase are present mainly in fractions C and B_p. No pyruvate dehydrogenase activity is found in synaptoplasm. Striatum, cerebral cortex, and cerebellum contain similar activities of pyruvate dehydrogenase, citrate synthase, carnitine acetyltransferase, fatty acid synthetase, and acetyl-CoA hydrolase. Activities of acetyl CoA synthetase, choline acetyltransferase and ATP-citrate lyase in cerebellum are about 10 and 4 times lower, respectively, than in other parts of the brain. These data indicate preferential localization of ATP-citrate lyase in cholinergic nerve endings, and indicate that this enzyme is not a rate limiting step in the synthesis of the acetyl moiety of ACh in brain.     

Evidence from Fourier transform infrared spectroscopy for polarization of the carbonyl of oxaloacetate in the active site of citrate synthase

The infrared spectrum of oxaloacetate bound in the active site of citrate synthase has been measured in the binary complex and in the ternary complex with the acetyl coenzyme A (CoA) enolate analogue carboxymethyl-CoA. The carbonyl stretching frequency of oxaloacetate in binary and ternary complexes is found at 1697 cm-1, a shift of 21 cm-1 to lower frequency relative to that of the free ligand. The line widths of the carbonyl absorption in enzyme complexes differ from that of the free ligand, decreasing from a value of 20 cm-1 for the free ligand to 10 cm-1 in the binary complex and 7 cm-1 in the ternary complex with carboxymethyl-CoA. The integrated absorbance of the carbonyl absorption in these enzyme complexes is significantly increased over that of the free ligand at the same concentration, increasing approximately 2-fold in the binary complex and approximately 3-fold in the ternary complex. These results indicate strong polarization of the carbonyl bond in the enzyme-substrate complexes and suggest that ground-state destabilization is a major catalytic strategy of citrate synthase.     

Structure of the E. coli bifunctional GlmU acetyltransferase active site with substrates and products

The biosynthesis of UDP-GlcNAc in bacteria is carried out by GlmU, an essential bifunctional uridyltransferase that catalyzes the CoA-dependent acetylation of GlcN-1-PO(4) to form GlcNAc-1-PO(4) and its subsequent condensation with UTP to form pyrophosphate and UDP-GlcNAc. As a metabolite, UDP-GlcNAc is situated at a branch point leading to the biosynthesis of lipopolysaccharide and peptidoglycan. Consequently, GlmU is regarded as an important target for potential antibacterial agents. The crystal structure of the Escherichia coli GlmU acetyltransferase active site has been determined in complexes with acetyl-CoA, CoA/GlcN-1-PO(4), and desulpho-CoA/GlcNAc-1-PO(4). These structures reveal the enzyme groups responsible for binding the substrates. A superposition of these complex structures suggests that the 2-amino group of GlcN-1-PO(4) is positioned in proximity to the acetyl-CoA to facilitate direct attack on its thioester by a ternary complex mechanism.     

The structural basis of ordered substrate binding by serotonin N-acetyltransferase: enzyme complex at 1.8 A resolution with a bisubstrate analog.

Serotonin N-acetyltransferase, a member of the GNAT acetyltransferase superfamily, is the penultimate enzyme in the conversion of serotonin to melatonin, the circadian neurohormone. Comparison of the structures of the substrate-free enzyme and the complex with a bisubstrate analog, coenzyme A-S-acetyltryptamine, demonstrates that acetyl coenzyme A (AcCoA) binding is accompanied by a large conformational change that in turn leads to the formation of the serotonin-binding site. The structure of the complex also provides insight into how the enzyme may facilitate acetyl transfer. A water-filled channel leading from the active site to the surface provides a pathway for proton removal following amine deprotonation. Furthermore, structural and mutagenesis results indicate an important role for Tyr-168 in catalysis.     

Aminoglycoside 2'-N-acetyltransferase from Mycobacterium tuberculosis in complex with coenzyme A and aminoglycoside substrates

AAC(2')-Ic catalyzes the coenzyme A (CoA)-dependent acetylation of the 2' hydroxyl or amino group of a broad spectrum of aminoglycosides. The crystal structure of the AAC(2')-Ic from Mycobacterium tuberculosis has been determined in the apo enzyme form and in ternary complexes with CoA and either tobramycin, kanamycin A or ribostamycin, representing the first structures of an aminoglycoside acetyltransferase bound to a drug. The overall fold of AAC(2')-Ic places it in the GCN5-related N-acetyltransferase (GNAT) superfamily. Although the physiological function of AAC(2')-Ic is uncertain, a structural analysis of these high-affinity aminoglycoside complexes suggests that the enzyme may acetylate a key biosynthetic intermediate of mycothiol, the major reducing agent in mycobacteria, and participate in the regulation of cellular redox potential.     

The Molecular Structure of Ornithine Acetyltransferase from Mycobacterium tuberculosis Bound to Ornithine, a Competitive Inhibitor

Mycobacterium tuberculosis ornithine acetyltransferase (Mtb OAT; E.C. 2.3.1.35) is a key enzyme of the acetyl recycling pathway during arginine biosynthesis. It reversibly catalyzes the transfer of the acetyl group from N-acetylornithine (NAORN) to l-glutamate. Mtb OAT is a member of the N-terminal nucleophile fold family of enzymes. The crystal structures of Mtb OAT in native form and in its complex with ornithine (ORN) have been determined at 1.7 and 2.4 Å resolutions, respectively. ORN is a competitive inhibitor of this enzyme against l-glutamate as substrate. Although the acyl-enzyme complex of Streptomyces clavuligerus ornithine acetyltransferase has been determined, ours is the first crystal structure to be reported of an ornithine acetyltransferase in complex with an inhibitor. ORN binding does not alter the structure of Mtb OAT globally. However, its presence stabilizes the three C-terminal residues that are disordered and not observed in the native structure. Also, stabilization of the C-terminal residues by ORN reduces the size of the active-site pocket volume in the structure of the ORN complex. The interactions of ORN and the protein residues of Mtb OAT unambiguously delineate the active-site residues of this enzyme in Mtb. Moreover, modeling studies carried out with NAORN based on the structure of the ORN-Mtb OAT complex reveal important interactions of the carbonyl oxygen of the acetyl group of NAORN with the main-chain nitrogen atom of Gly128 and with the side-chain oxygen of Thr127. These interactions likely help in the stabilization of oxyanion formation during enzymatic reaction and also will polarize the carbonyl carbon-oxygen bond, thereby enabling the side-chain atom O^γ1 of Thr200 to launch a nucleophilic attack on the carbonyl-carbon atom of the acetyl group of NAORN.     

Crystal structure of lactaldehyde dehydrogenase from Escherichia coli and inferences regarding substrate and cofactor specificity

Aldehyde dehydrogenases catalyze the oxidation of aldehyde substrates to the corresponding carboxylic acids. Lactaldehyde dehydrogenase from Escherichia coli (aldA gene product, P25553) is an NAD(+)-dependent enzyme implicated in the metabolism of l-fucose and l-rhamnose. During the heterologous expression and purification of taxadiene synthase from the Pacific yew, lactaldehyde dehydrogenase from E. coli was identified as a minor (相似文献   

A structural model for the ternary cleavable complex formed between human topoisomerase I, DNA, and camptothecin

Using the X-ray crystal structure of the human topoisomerase I (TOP1)-DNA cleavable complex, we have developed a general model for the ternary drug-DNA-TOP1 cleavable complex formed with camptothecin (CPT) and its analogues. This model has the drug intercalated between the -1 and +1 base pairs, with the E-ring pointing into the minor groove and the A-ring directed toward the major groove. The ternary complex is stabilized by an array of hydrogen bonding and hydrophobic interactions between the drug and both the enzyme and the DNA. Significantly, the proposed model is consistent with the current body of experimental mutation, cross-linking, and structure-activity data. In addition, the model reveals potential sites of interaction that can provide a rational basis for the design of next generation compounds as well as for de novo drug design.     

Structure of a photoactive rhodium complex intercalated into DNA

Intercalating complexes of rhodium(III) are strong photo-oxidants that promote DNA strand cleavage or electron transfer through the double helix. The 1.2 A resolution crystal structure of a sequence-specific rhodium intercalator bound to a DNA helix provides a rationale for the sequence specificity of rhodium intercalators. It also explains how intercalation in the center of an oligonucleotide modifies DNA conformation. The rhodium complex intercalates via the major groove where specific contacts are formed with the edges of the bases at the target site. The phi ligand is deeply inserted into the DNA base pair stack. The primary conformational change of the DNA is a doubling of the rise per residue, with no change in sugar pucker from B-form DNA. Based upon the five crystallographically independent views of an intercalated DNA helix observed in this structure, the intercalator may be considered as an additional base pair with specific functional groups positioned in the major groove.     

Synthesis of mycothiol, 1D-1-O-(2-[N-acetyl-L-cysteinyl]amino-2-deoxy-alpha-D-glucopyranosyl)-myo-inositol, principal low molecular mass thiol in the actinomycetes

Members of the actinomycetes produce 1D-1-O-(2-[N-acetyl-L-cysteinyl]amino-2-deoxy-alpha-D-glucopyranosyl)-myo-inositol or mycothiol 1 as principal low molecular mass thiol. Chemical synthesis of a biosynthetic precursor of mycothiol, the pseudodisaccharide 1D-1-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-myo-inositol 13 was achieved by the following steps: (1) Enantioselective synthesis gave the glycosyl acceptors (-)-2,3,4,5,6-penta-O-acetyl-D-myo-inositol D-7 and the corresponding L-isomer L-7. (2) Condensation of D-7 and L-7 with the glycosyl donor 3,4,6-tri-O-acetyl-2-deoxy-2-(2,4-dinitrophenylamino)-alpha-D-glucopyranosylbromide afforded the corresponding alpha and beta anomeric products, which could be resolved by silica gel chromatography. (3) Deprotection of these by hydrolysis using an anion exchange resin gave 1D- and 1L-1-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-myo-inositol 13 and 15 and the corresponding beta-coupled anomers 14 and 16. Only 13, and to a much lesser extent 15, were used by enzymes present in an ammonium sulphate fraction of a cellfree extract of Mycobacterium smegmatis for the enzymatic synthesis of mycothiol. In the absence of acetyl-SCoA, the immediate biosynthetic precursor of 1, desacetylmycothiol, was the major product.     

Crystal structure of an aminoglycoside 6'-N-acetyltransferase: defining the GCN5-related N-acetyltransferase superfamily fold.

BACKGROUND: The predominant mechanism of antibiotic resistance employed by pathogenic bacteria against the clinically used aminoglycosides is chemical modification of the drug. The detoxification reactions are catalyzed by enzymes that promote either the phosphorylation, adenylation or acetylation of aminoglycosides. Structural studies of these aminoglycoside-modifying enzymes may assist in the development of therapeutic agents that could circumvent antibiotic resistance. In addition, such studies may shed light on the development of antibiotic resistance and the evolution of different enzyme classes. RESULTS: The crystal structure of the aminoglycoside-modifying enzyme aminoglycoside 6'-N-acetyltransferase type li (AAC(6')-li) in complex with the cofactor acetyl coenzyme A has been determined at 2.7 A resolution. The structure establishes that this acetyltransferase belongs to the GCN5-related N-acetyltransferase superfamily, which includes such enzymes as the histone acetyltransferases GCN5 and Hat1. CONCLUSIONS: Comparison of the AAC(6')-li structure with the crystal structures of two other members of this superfamily, Serratia marcescens aminoglycoside 3-N-acetyltransferase and yeast histone acetyltransferase Hat1, reveals that of the 84 residues that are structurally similar, only three are conserved and none can be implicated as catalytic residues. Despite the negligible sequence identity, functional studies show that AAC(6')-li possesses protein acetylation activity. Thus, AAC(6')-li is both a structural and functional homolog of the GCN5-related histone acetyltransferases.     

Crystal structure of two ternary complexes of phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Bacillus stearothermophilus with NAD and D-glyceraldehyde 3-phosphate

The crystal structure of the phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPDH) from Bacillus stearothermophilus was solved in complex with its cofactor, NAD, and its physiological substrate, D-glyceraldehyde 3-phosphate (D-G3P). To isolate a stable ternary complex, the nucleophilic residue of the active site, Cys(149), was substituted with alanine or serine. The C149A and C149S GAPDH ternary complexes were obtained by soaking the crystals of the corresponding binary complexes (enzyme.NAD) in a solution containing G3P. The structures of the two binary and the two ternary complexes are presented. The D-G3P adopts the same conformation in the two ternary complexes. It is bound in a non-covalent way, in the free aldehyde form, its C-3 phosphate group being positioned in the P(s) site and not in the P(i) site. Its C-1 carbonyl oxygen points toward the essential His(176), which supports the role proposed for this residue along the two steps of the catalytic pathway. Arguments are provided that the structures reported here are representative of a productive enzyme.NAD.D-G3P complex in the ground state (Michaelis complex).     

1.9-A structures of ternary complexes of citrate synthase with D- and L-malate: mechanistic implications

The structures of four isomorphous crystals of ternary complexes of chicken heart citrate synthase with D- or L-malate and acetyl coenzyme A or carboxymethyl coenzyme A have been determined by X-ray crystallography and fully refined at 1.9-A resolution. The structures show that both L-malate and D-malate bind in a very similar way in the presence of acetylCoA and that the enzyme conformation is "closed". Hydrogen bond geometry is suggested to account for the difference in binding constants of the two stereoisomers. The structures suggest that steric hindrance can account for the observation that proton exchange of acetyl coenzyme A with solvent is catalyzed by citrate synthase in the presence of L-malate but not D-malate. The ternary complexes with malate reveal the mode of binding of the substrate acetylCoA in the ground state. The carbonyl oxygen of the acetyl group is hydrogen bonded to a water molecule and to histidine 274, allowing unambiguous identification of the orientation of this group. The structures support the hypothesis that carboxymethyl coenzyme A is a transition-state analogue for the enolization step of the reaction (Bayer et al., 1981) and additionally support proposed mechanisms for the condensation reaction (Karpusas et al., 1990; Alter et al., 1990).     

Subunit structure of dihydrolipoamide acetyltransferase component of pyruvate dehydrogenase complex from bovine kidney

The pyruvate dehydrogenase complex has been isolated from bovine kidney mitochondria under special anti-proteolytic conditions yielding preparations with a specific activity of up to 20 U/mg protein. Dihydrolipoamide acetyltransferase resolved from the complex was subjected to limited proteolysis resulting in the formation of two major fragments with apparent molecular weights of 36000 and 28000. The fragments were isolated by extraction from dodecyl sulfate polyacrylamide gels and were both shown to possess enzymatic activity for acetyl transfer. Acetylation studies indicated that each fragment contains one protein-bound lipoyl group. It is concluded that the kidney dihydrolipoamide acetyltransferase subunit consists of two homologous if not identical domains. A model is suggested where the acetyltransferase core of the mammalian pyruvate dehydrogenase complex is made up of 30 polypeptide chains whose 60 domains could be arranged in pentagonal dodecahedron symmetry quite similar as proposed for the 60 subunit structure of the acetyltransferase core.