Structure of isochorismate synthase DhbC fromBacillus anthracis

Proceedings of The National Academy of Sciences, 1999

Anthranilate synthase catalyzes the synthesis of anthranilate from chorismate and glutamine and is feedback-inhibited by tryptophan. The enzyme of the hyperthermophile Sulfolobus solfataricus has been crystallized in the absence of physiological ligands, and its three-dimensional structure has been determined at 2.5-Å resolution with x-ray crystallography. It is a heterotetramer of anthranilate synthase (TrpE) and glutamine amidotransferase (TrpG) subunits, in which two TrpG:TrpE protomers associate mainly via the TrpG subunits. The small TrpG subunit (195 residues) has the known ''triad'' glutamine amidotransferase fold. The large TrpE subunit (421 residues) has a novel fold. It displays a cleft between two domains, the tips of which contact the TrpG subunit across its active site. Clusters of catalytically essential residues are located inside the cleft, spatially separated from clustered residues involved in feedback inhibition. The structure suggests a model in which chorismate binding triggers a relative movement of the two domain tips of the TrpE subunit, activating the TrpG subunit and creating a channel for passage of ammonia toward the active site of the TrpE subunit. Tryptophan presumably blocks this rearrangement, thus stabilizing the inactive states of both subunits. The structure of the TrpE subunit is a likely prototype for the related enzymes 4-amino 4-deoxychorismate synthase and isochorismate synthase.

Bioorganic & Medicinal Chemistry Letters, 2006

An α-glycoside of the tetrasaccharide sequence β-Ant-(1→3)-α-l-Rhap-(1→3)-α-l-Rhap-(1→2)-α-l-Rhap whose aglycon allows conjugation to suitable carriers was synthesized. The NMR characteristics of the compound are virtually identical with those of the α-anomer of the tetrasaccharide isolated from the major glycoprotein of the Bacillus anthracis exosporium. Thus, the correct structure of the natural product has been proven by chemical synthesis.

Biochemistry, 2007

Menaquinone biosynthesis is initiated by the conversion of chorismate to isochorismate, a reaction that is catalyzed by the menaquinone-specific isochorismate synthase, MenF. The catalytic mechanism of MenF has been probed using a combination of structural and biochemical studies, including the 2.5 A structure of the enzyme, and Lys190 has been identified as the base that activates water for nucleophilic attack at the chorismate C2 carbon. MenF is a member of a larger family of Mg2+ dependent chorismate binding enzymes catalyzing distinct chorismate transformations. The studies reported here extend the mechanism recently proposed for this enzyme family by He et al.: He, Z., Stigers Lavoie, K. D., Bartlett, P. A., and Toney, M. D. (2004) J. Am. Chem. Soc. 126, 2378-85.

Proceedings of the National Academy of Sciences, 2008

Petrobactin, a virulence-associated siderophore produced by Bacillus anthracis, chelates ferric iron through the rare 3,4-isomer of dihydroxybenzoic acid (3,4-DHBA). Most catechol siderophores, including bacillibactin and enterobactin, use 2,3-DHBA as a biosynthetic subunit. Significantly, siderocalin, a factor involved in human innate immunity, sequesters ferric siderophores bearing the more typical 2,3-DHBA moiety, thereby impeding uptake of iron by the pathogenic bacterial cell. In contrast, the unusual 3,4-DHBA component of petrobactin renders the siderocalin system incapable of obstructing bacterial iron uptake. Although recent genetic and biochemical studies have revealed selected early steps in petrobactin biosynthesis, the origin of 3,4-DHBA as well as the function of the protein encoded by the final gene in the B. anthracis siderophore biosynthetic (asb) operon, asbF (BA1986), has remained unclear. In this study we demonstrate that 3,4-DHBA is produced through conversion of the common bacterial metabolite 3-dehydroshikimate (3-DHS) by AsbF-a 3-DHS dehydratase. Elucidation of the cocrystal structure of AsbF with 3,4-DHBA, in conjunction with a series of biochemical studies, supports a mechanism in which an enolate intermediate is formed through the action of this 3-DHS dehydratase metalloenzyme. Structural and functional parallels are evident between AsbF and other enzymes within the xylose isomerase TIM-barrel family. Overall, these data indicate that microbial species shown to possess homologs of AsbF may, like B. anthracis, also rely on production of the unique 3,4-DHBA metabolite to achieve full viability in the environment or virulence within the host.

Acta Crystallographica Section F-structural Biology and Crystallization Communications, 2006

Bacillus anthracis nucleoside diphosphate kinase (BaNdk) is an enzyme whose primary function is to maintain deoxynucleotide triphosphate (dNTP) pools by converting deoxynucleotide diphosphates to triphosphates using ATP as the major phosphate donor. Although the structures of Ndks from a variety of organisms have been elucidated, the enzyme from sporulating bacteria has not been structurally characterized to date. Crystals of the B. anthracis enzyme were grown using the vapour-diffusion method from a hanging drop consisting of 2 ml 10 mg ml À1 protein in 50 mM Tris-HCl pH 8.0, 50 mM NaCl, 5 mM EDTA equilibrated against 500 ml reservoir solution consisting of 2.25 M ammonium formate and 0.1 M HEPES buffer pH 7.25. Diffraction data extending to 2.0 Å were collected at room temperature from a single crystal with unit-cell parameters a = b = 107.53, c = 52.3 Å . The crystals are hexagonal in shape and belong to space group P6 3 22. The crystals contain a monomer in the asymmetric unit, which corresponds to a Matthews coefficient (V M ) of 2.1 Å 3 Da À1 and a solvent content of about 36.9%.

Carbohydrate Research, 2005

Synthesis of the b anomer of the spacer-equipped tetrasaccharide side chain of the major glycoprotein of the Bacillus anthracis exosporium I Abstract-The b glycoside of the tetrasaccharide sequence b-Ant-(1!3)-a-L L-Rhap-(1!3)-a-L L-Rhap-(1!2)-L L-Rhap, whose aglycon allows conjugation to proteins, was synthesized for the first time. A stepwise synthetic approach was applied with thioglycosides as glycosyl donors, and the b anomer of the compound was obtained equipped with a spacer group whose further transformation allows conjugation to suitable carriers. To synthesize the b-anthrosyl linkage with high stereoselectivity, a linker-equipped rhamnotriose derivative was glycosylated with ethyl 4-azido-3-O-benzyl-2-O-bromoacetyl-4,6-dideoxy-1-thio-b-D D-glucopyranoside. Further functionalization of the tetrasaccharide thus obtained, followed by deprotection, gave the target substance.

Journal of Biological Chemistry, 2008

Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step of the lysine biosynthetic pathway. The tetrameric structure of DHDPS is thought to be essential for enzymatic activity, as isolated dimeric mutants of Escherichia coli DHDPS possess less than 2.5% that of the activity of the wildtype tetramer. It has recently been proposed that the dimeric form lacks activity due to increased dynamics. Tetramerization, by buttressing two dimers together, reduces dynamics in the dimeric unit and explains why all active bacterial DHDPS enzymes to date have been shown to be homo-tetrameric. However, in this study we demonstrate for the first time that DHDPS from methicillin-resistant Staphylococcus aureus (MRSA) exists in a monomer-dimer equilibrium in solution. Fluorescence-detected analytical ultracentrifugation was employed to show that the dimerization dissociation constant of MRSA-DHDPS is 33 nM in the absence of substrates and 29 nM in the presence of (S)-aspartate semialdehyde (ASA), but is 20-fold tighter in the presence of the substrate pyruvate (1.6 nM). The MRSA-DHDPS dimer exhibits a ping-pong kinetic mechanism (k cat ‫؍‬ 70 ؎ 2 s ؊1 , K m Pyruvate ‫؍‬ 0.11 ؎ 0.01 mM, and K m ASA ‫؍‬ 0.22 ؎ 0.02 mM) and shows ASA substrate inhibition with a K si ASA of 2.7 ؎ 0.9 mM. We also demonstrate that unlike the E. coli tetramer, the MRSA-DHDPS dimer is insensitive to lysine inhibition. The near atomic resolution (1.45 A ˚) crystal structure confirms the dimeric quaternary structure and reveals that the dimerization interface of the MRSA enzyme is more extensive in buried surface area and noncovalent contacts than the equivalent interface in tetrameric DHDPS enzymes from other bacterial species. These data provide a detailed mechanistic insight into DHDPS catalysis and the evolution of quaternary structure of this important bacterial enzyme.

FEBS Letters, 1996

The pivotal step in enterobactin and menaquinone biosynthesis is the conversion of ehorismate to isochorismate. Circumstantial evidence pointed to Escherichia coil isochorismate hydroxymutase isogenes being responsbible for this conversion.

Journal of Organic Chemistry, 2008

The biosynthesis of the 3,4-dihydroxybenzoate moieties of the siderophore petrobactin, produced by B. anthracis str. Sterne, was probed by isotopic feeding experiments in iron-deficient media with a mixture of unlabeled and D-[ 13 C 6 ]glucose at a ratio of 5:1 (w/w). After isolation of the labeled siderophore, analysis of the isotopomers was conducted via one-dimensional 1 H and 13 C NMR spectroscopy, as well as 13 C-13 C DQFCOSY spectroscopy. Isotopic enrichment and 13 C-13 C coupling constants in the aromatic ring of the isolated siderophore suggested the predominant route for the construction of the carbon backbone of 3,4-DHB (1) involved phosphoenol pyruvate and erythrose-4-phosphate as ultimate precursors. This observation is consistent with that expected if the shikimate pathway is involved in the biosynthesis of these moieties. Enrichment attributable to phosphoenol pyruvate precursors was observed at C1 and C6 of the aromatic ring, as well as into the carboxylate group, while scrambling of the label into C2 was not. This pattern suggests 1 was biosynthesized from early intermediates of the shikimate pathway and not through later shikimate intermediates or aromatic amino acid precursors.

Journal of Biological …, 2004

Spores of Bacillus anthracis, the causative agent of anthrax, are enclosed by a prominent loose fitting layer called the exosporium. The exosporium consists of a basal layer and an external hairlike nap. The filaments of the nap are composed of a highly immunogenic glycoprotein called BclA, which has a long, central collagen-like region with multiple XXG repeats. Most of the triplet repeats are PTG, and nearly all of the triplet repeats contain a threonine residue, providing multiple potential sites for O-glycosylation. In this study, we demonstrated that two O-linked oligosaccharides, a 715-Da tetrasaccharide and a 324-Da disaccharide, are released from spore-and exosporium-associated BclA by hydrazinolysis. Each oligosaccharide is probably attached to BclA through a GalNAc linker, which was lost during oligosaccharide release. We found that multiple copies of the tetrasaccharide are linked to the collagen-like region of BclA, whereas the disaccharide may be attached outside of this region. Using NMR, mass spectrometry, and other analytical techniques, we determined that the structure of the tetrasaccharide is 2-O-methyl-4-(3-hydroxy-3-methylbutamido)-4,6-dideoxy-␤-D-glucopyranosyl-(133)-␣-Lrhamnopyranosyl-(133)-␣-L-rhamnopyranosyl-(132)-L -rhamnopyranose. The previously undescribed nonreducing terminal sugar (i.e. 2-O-methyl-4-(3hydroxy-3-methylbutamido)-4,6-dideoxy-D-glucose) was given the trivial name anthrose. Anthrose was not found in spores of either Bacillus cereus or Bacillus thuringiensis, two species that are the most phylogenetically similar to B. anthracis. Thus, anthrose may be useful for species-specific detection of B. anthracis spores or as a new target for therapeutic intervention.