Sharon Ackerman

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Structure, mechanism and evolution of KDO8P synthase

Domenico L. Gatti

1 March 2004 - 29 February 2008
NATIONAL INSTITUTE OF GENERAL MEDICAL SCIENCES
Total Funding: $ 1,125,991

The lipopolysaccharide (LPS) of Gram negative bacteria, also known as endotoxin, is responsible for the pathophysiological phenomena of the shock syndrome associated with Gram negative sepsis. 3-Deoxy-Dmanno-octulosonate (KDO) is an essential constituent of the LPS of all Gram negative bacteria. This eight carbon sugar is the first component of the oligosaccharide core region that links the lipid A moiety of LPS to the O-antigen. The combination of lipid A and two KDO units is the LPS component where the endotoxin activity is located. The first step in the synthesis of KDO is the condensation of arabinose 5-phosphate (A5P) and phosphoenolpyruvate (PEP) to form KDO 8-phosphate (KDO8P), the phosphorylated precursor of KDO, catalyzed by KDO8P synthase (KDO8PS). Since LPS is essential for bacterial growth, this enzyme is a potential target for new antimicrobial drugs. There are two classes of highly homologous KDO8PS's differing primarily in the requirement, or lack thereof, of a metal ion for activity. We have determined the structure of one member of the metal-free class, the Escherichia coil enzyme, and of one member of the metal-requiring class, the enzyme from the hyperthermophile Aquifex aeolicus. These studies have revealed that KDO8PS is a homotetramer with two active sites located on each face of the enzyme. There is alternation of catalysis between the two faces of the enzyme, such that when PEP and A5P bind and react in the active sites located on one face, only PEP binds at the active sites located on the other face. Despite the wealth of information derived from the initial structural and biochemical studies, the mechanism of the reaction catalyzed by KDO8PS is not clear yet. The current project will employ methods of kinetic analysis, spectroscopy, rational mutagenesis, directed evolution and X-ray diffraction to investigate (a) the function of individual residues and active site water in catalysis, and the mechanistic differences between metallo and non-metallo synthases, (b) the molecular mechanism of alternating face catalysis.

15 Resulting Publications

• 1.

  2013

  Sharon H Ackerman; Domenico L Gatti

  Biapenem inactivation by B2 metallo β-lactamases: energy landscape of the hydrolysis reaction.

  PloS one 2013;8(1):e55136.

• 2.

  2012

  Domenico L Gatti

  Biapenem inactivation by B2 metallo β-lactamases: energy landscape of the post-hydrolysis reactions.

  PloS one 2012;7(1):e30079.

• 3.

  2012

  Sharon H Ackerman; Elisabeth R Tillier; Domenico L Gatti

  Accurate simulation and detection of coevolution signals in multiple sequence alignments.

  PloS one 2012;7(10):e47108.

• 4.

  2011

  Rebeca Acin-Perez; Domenico L Gatti; Yidong Bai; Giovanni Manfredi

  Protein phosphorylation and prevention of cytochrome oxidase inhibition by ATP: coupled mechanisms of energy metabolism regulation.

  Cell metabolism 2011;13(6):712-9.

• 5.

  2011

  Sharon H Ackerman; Domenico L Gatti

  The contribution of coevolving residues to the stability of KDO8P synthase.

  PloS one 2011;6(3):e17459.

• 6.

  2010

  Peng Tao; H Bernhard Schlegel; Domenico L Gatti

  Common basis for the mechanism of metallo and non-metallo KDO8P synthases.

  Journal of inorganic biochemistry 2010;104(12):1267-75.

• 7.

  2010

  Ann Meulemans; Sara Seneca; Thomas Pribyl; Joel Smet; Valerie Alderweirldt; Anouk Waeytens; Willy Lissens; Rudy Van Coster; Linda De Meirleir; Jean-Paul di Rago; et al.

  Defining the pathogenesis of the human Atp12p W94R mutation using a Saccharomyces cerevisiae yeast model.

  The Journal of biological chemistry 2010;285(6):4099-109.

• 8.

  2009

  Peng Tao; Domenico L Gatti; H Bernhard Schlegel

  The energy landscape of 3-deoxy-D-manno-octulosonate 8-phosphate synthase.

  Biochemistry 2009;48(49):11706-14.

• 9.

  2009

  Anthony Ludlam; Joseph Brunzelle; Thomas Pribyl; Xingjue Xu; Domenico L Gatti; Sharon H Ackerman

  Chaperones of F1-ATPase.

  The Journal of biological chemistry 2009;284(25):17138-46.

• 10.

  2009

  Fathima Kona; Peng Tao; Philip Martin; Xingjue Xu; Domenico L Gatti

  Electronic structure of the metal center in the Cd(2+), Zn(2+), and Cu(2+) substituted forms of KDO8P synthase: implications for catalysis.

  Biochemistry 2009;48(16):3610-30.

• 11.

  2007

  Fathima Kona; Xingjue Xu; Philip Martin; Petr Kuzmic; Domenico L Gatti

  Structural and mechanistic changes along an engineered path from metallo to nonmetallo 3-deoxy-D-manno-octulosonate 8-phosphate synthases.

  Biochemistry 2007;46(15):4532-44.

• 12.

  2005

  Xingjue Xu; Fathima Kona; Jian Wang; Jinshuang Lu; Timothy Stemmler; Domenico L Gatti

  The catalytic and conformational cycle of Aquifex aeolicus KDO8P synthase: role of the L7 loop.

  Biochemistry 2005;44(37):12434-44.

• 13.

  2005

  Lindsay J Cole; Domenico L Gatti; Barrie Entsch; David P Ballou

  Removal of a methyl group causes global changes in p-hydroxybenzoate hydroxylase.

  Biochemistry 2005;44(22):8047-58.

• 14.

  2004

  Ayana Hinton; Domenico L Gatti; Sharon H Ackerman

  The molecular chaperone, Atp12p, from Homo sapiens. In vitro studies with purified wild type and mutant (E240K) proteins.

  The Journal of biological chemistry 2004;279(10):9016-22.

• 15.

  2003

  Xingjue Xu; Jian Wang; Claude Grison; Sylvian Petek; Philippe Coutrot; Matthew R Birck; Ronald W Woodard; Domenico L Gatti

  Structure-based design of novel inhibitors of 3-deoxy-D-manno-octulosonate 8-phosphate synthase.

  Drug design and discovery 2003;18(2-3):91-9.

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