Publications

• The missing link in petrobactin biosynthesis: asbF encodes a (-)-3-dehydroshikimate dehydratase.

  Fox D.T., Hotta K., Kim C.Y., Koppisch A.T.

  The siderophore petrobactin harbors unique 3,4-dihydroxybenzoyl iron-liganding groups. These moieties are known to be synthesized from shikimate pathway precursors, but no reports of the biosynthetic enzymes responsible for this conversion have been published. The gene encoding AsbF from Bacillus ... >> More

  The siderophore petrobactin harbors unique 3,4-dihydroxybenzoyl iron-liganding groups. These moieties are known to be synthesized from shikimate pathway precursors, but no reports of the biosynthetic enzymes responsible for this conversion have been published. The gene encoding AsbF from Bacillus thuringiensis 97-27 was overexpressed in an Escherichia coli host. AsbF rapidly and efficiently transforms (-)-3-dehydroshikimate (DHS) into 3,4-dihydroxybenzoate (k(cat)(DHS) = 217 +/- 10 min(-1); K(m)(DHS) = 125 +/-14 microM) at 37 degrees C and has an absolute requirement for divalent metal. Finally, the pH versus k(cat)(DHS) profile revealed two ionizable groups (pK(a1) = 7.9 +/- 0.1, and pK(a2) = 9.3 +/- 0.1). << Less

  Biochemistry 47:12251-12253(2008) [PubMed] [EuropePMC]

• Structural and functional analysis of AsbF: origin of the stealth 3,4-dihydroxybenzoic acid subunit for petrobactin biosynthesis.

  Pfleger B.F., Kim Y., Nusca T.D., Maltseva N., Lee J.Y., Rath C.M., Scaglione J.B., Janes B.K., Anderson E.C., Bergman N.H., Hanna P.C., Joachimiak A., Sherman D.H.

  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, sideroc ... >> More

  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. << Less

  Proc. Natl. Acad. Sci. U.S.A. 105:17133-17138(2008) [PubMed] [EuropePMC]

• Unusual ancestry of dehydratases associated with quinate catabolism in Acinetobacter calcoaceticus.

  Elsemore D.A., Ornston L.N.

  Catabolism of quinate to protocatechuate requires the consecutive action of quinate dehydrogenase (QuiA), dehydroquinate dehydratase (QuiB), and dehydroshikimate dehyratase (QuiC), Genes for catabolism of protocatechuate are encoded by the pca operon in the Acinetobacter calcoaceticus chromosome. ... >> More

  Catabolism of quinate to protocatechuate requires the consecutive action of quinate dehydrogenase (QuiA), dehydroquinate dehydratase (QuiB), and dehydroshikimate dehyratase (QuiC), Genes for catabolism of protocatechuate are encoded by the pca operon in the Acinetobacter calcoaceticus chromosome. Observations reported here demonstrate that A. calcoaceticus qui genes are clustered in the order quiBCXA directly downstream from the pca operon. Sequence comparisons indicate that quiX encodes a porin, but the specific function of this protein has not been clearly established. Properties of mutants created by insertion of omega elements show that quiBC is expressed as part of a single transcript, but there is also an independent transcriptional initiation site directly upstream of quiA. The deduced amino acid sequence of QuiC does not resemble any other known sequence. A. calcoaceticus QuiB is most directly related to a family of enzymes with identical catalytic activity and biosynthetic AroD function in coliform bacteria. Evolution of A. calcoaceticus quiB appears to have been accompanied by fusion of a leader sequence for transport of the encoded protein into the inner membrane, and the location of reactions catalyzed by the mature enzyme may account for the failure of A. calcoaceticus aroD to achieve effective complementation of null mutations in quiB. Analysis of a genetic site where a DNA segment encoding a leader sequence was transposed adds to evidence suggesting horizontal transfer of nucleotide sequences within genes during evolution. << Less

  J. Bacteriol. 177:5971-5978(1995) [PubMed] [EuropePMC]